U.S. patent application number 10/486306 was filed with the patent office on 2006-11-09 for sphks as modifiers of the p53 pathway and methods of use.
Invention is credited to Michael B. Costa, Lori Friedman, Roel P. Funke, Tak Hung, Danxi Li, Gregory D. Plowman.
Application Number | 20060252035 10/486306 |
Document ID | / |
Family ID | 26977360 |
Filed Date | 2006-11-09 |
United States Patent
Application |
20060252035 |
Kind Code |
A1 |
Friedman; Lori ; et
al. |
November 9, 2006 |
Sphks as modifiers of the p53 pathway and methods of use
Abstract
Human SPHK 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
SPHK are provided.
Inventors: |
Friedman; Lori; (San Carlos,
CA) ; Plowman; Gregory D.; (San Carlos, CA) ;
Costa; Michael B.; (San Francisco, CA) ; Li;
Danxi; (Zionsville, IN) ; Funke; Roel P.;
(Brisbane, CA) ; Hung; Tak; (Roster City,
CA) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF LLP
300 S. WACKER DRIVE
32ND FLOOR
CHICAGO
IL
60606
US
|
Family ID: |
26977360 |
Appl. No.: |
10/486306 |
Filed: |
August 2, 2002 |
PCT Filed: |
August 2, 2002 |
PCT NO: |
PCT/US02/24623 |
371 Date: |
April 11, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60310362 |
Aug 6, 2001 |
|
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60357501 |
Feb 15, 2002 |
|
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Current U.S.
Class: |
435/6.16 ;
435/15; 435/7.23 |
Current CPC
Class: |
G01N 33/57415 20130101;
G01N 33/57419 20130101; A61P 9/00 20180101; G01N 33/6875 20130101;
G01N 2510/00 20130101; G01N 33/5011 20130101; G01N 33/5091
20130101; G01N 33/5041 20130101; G01N 2500/00 20130101; G01N
33/57449 20130101; G01N 33/57423 20130101; G01N 33/68 20130101;
C12Q 1/485 20130101; G01N 33/6842 20130101; A61P 35/00 20180101;
G01N 33/5008 20130101; A61P 43/00 20180101 |
Class at
Publication: |
435/006 ;
435/007.23; 435/015 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/574 20060101 G01N033/574; C12Q 1/48 20060101
C12Q001/48 |
Claims
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 SPHK 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 SPHK 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 SPHK 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 SPHK 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 SPHK 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 SPHK polypeptide comprising
an amino acid sequence selected from group consisting of SEQ ID
NOs:10, 11, 12, and 13, 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 SPHK, (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 SPHK 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 SPHK 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
application 60/310,362 filed Aug. 6, 2001, and 60/357,501 filed
Feb. 15, 2002. The contents of the prior applications are hereby
incorporated in their entirety.
BACKGROUND OF THE INVENTION
[0002] The p53 gene is mutated in over 50 different types of human
cancers, including familial and spontaneous cancers, and is
believed to be the most commonly mutated gene in human cancer
(Zambetti and Levine, FASEB (1993) 7:855-865; Hollstein, et al.,
Nucleic Acids Res. (1994) 22:3551-3555). Greater than 90% of
mutations in the p53 gene are missense mutations that alter a
single amino acid that inactivates p53 function. Aberrant forms of
human p53 are associated with poor prognosis, more aggressive
tumors, metastasis, and short survival rates (Mitsudomi et al.,
Clin Cancer Res 2000 October; 6(10):4055-63; Koshland, Science
(1993) 262:1953).
[0003] The human p53 protein normally functions as a central
integrator of signals including DNA damage, hypoxia, nucleotide
deprivation, and oncogene activation (Prives, Cell (1998) 95:5-8).
In response to these signals, p53 protein levels are greatly
increased with the result that the accumulated p53 activates cell
cycle arrest or apoptosis depending on the nature and strength of
these signals. Indeed, multiple lines of experimental evidence have
pointed to a key role for p53 as a tumor suppressor (Levine, Cell
(1997) 88:323-331). For example, homozygous p53 "knockout" mice are
developmentally normal but exhibit nearly 100% incidence of
neoplasia in the first year of life (Donehower et al., Nature
(1992) 356:215-221).
[0004] The biochemical mechanisms and pathways through which p53
functions in normal and cancerous cells are not fully understood,
but one clearly important aspect of p53 function is its activity as
a gene-specific transcriptional activator. Among the genes with
known p53-response elements are several with well-characterized
roles in either regulation of the cell cycle or apoptosis,
including GADD45, p21/Waf1/Cip1, cyclin G, Bax, IGF-BP3, and MDM2
(Levine, Cell (1997) 88:323-331).
[0005] Sphingosine-1-phosphate (SPP) is a lipid messenger
containing both intracellular and extracellular functions.
Intracellularly, it mediates proliferation and survival, and
extracellularly, it is a ligand for EDG1 (Meyer zu Heringdorf, D.
et al. (2001) Eur J Pharmacol 414, 145-54; Ancellin, N. et al.
(2002) J Biol Chem 277, 6667-75). A variety of stimuli can increase
cellular levels of SPP by activating sphingosine kinase (SPHK), the
enzyme that initiates the phosphorylation of sphingosine.
Inhibitors of SPHK can block formation of SPP and inhibit cellular
proliferation induced by a variety of factors, including
platelet-derived growth factor and serum (Banno, Y. et al. (1998)
Biochem J 335, 3014).
[0006] Sphingosine kinase-1 (SPHK1) phosphorylates sphingosine to
form sphingosine 1-phosphate, a lipid messenger mediating signaling
pathways involved in diverse functions like regulating
intracellular Ca.sup.2+ mobilization and cell proliferation and
cell survival (Meyer zu Heringdorf, D. et al. (1998) Embo Journal
17, 2830-7; Nava, V. et al. (2000) FEBS Lett 473, 814).
[0007] Sphingosine kinase 2 (SPHK2) phosphorylates sphingosine to
form sphingosine 1-phosphate, which functions as a second messenger
regulating proliferation and survival, as well as a ligand for
selected G protein-coupled receptors (Meyer zu Heringdorf, D. et
al. (1998) supra; Nava, V. et al. (2000) supra; Liu, H. et al.
(2000) J Biol Chem; 275(26): 19513-20). Expression of SPHK2 in BEK
293 cells elevates SPP levels (Liu, H. et al. (2000) supra).
[0008] SPHK sequences are highly conserved among evolutionarily
diverse organisms such as human, Caenorhabditis elegans, and yeast
(Kohama, T. et al. (1998) J. Biol. Chem. 273: 23722-23728).
[0009] The ability to manipulate the genomes of model organisms
such as C. elegans provides a powerful means to analyze biochemical
processes that, due to significant evolutionary conservation, have
direct relevance to more complex vertebrate organisms. Due to a
high level of gene and pathway conservation, the strong similarity
of cellular processes, and the functional conservation of genes
between these model organisms and mammals, identification of the
involvement of novel genes in particular pathways and their
functions in such model organisms can directly contribute to the
understanding of the correlative pathways and methods of modulating
them in mammals (see, for example, Dulubova I, et al, J Neurochem
2001 April; 77(1):229-38; Cai T, et al., Diabetologia 2001 January;
44(1):81-8; Pasquinelli A E, et al., Nature. 2000 Nov. 2;
408(6808):37-8; Ivanov I P, et al., EMBO J 2000 Apr. 17;
19(8):1907-17; Vajo Z et al., Mamm Genome 1999 October;
10(10):10004). 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 C.
elegans, and identified their human orthologs, hereinafter referred
to as SPHK. The invention provides methods for utilizing these p53
modifier genes and polypeptides to identify SPHK-modulating agents
that are candidate therapeutic agents that can be used in the
treatment of disorders associated with defective or impaired p53
function and/or SPHK function. Preferred SPHK-modulating agents
specifically bind to SPHK polypeptides and restore p53 function.
Other preferred SPHK-modulating agents are nucleic acid modulators
such as antisense oligomers and RNAi that repress SPHK gene
expression or product activity by, for example, binding to and
inhibiting the respective nucleic acid (i.e. DNA or mRNA).
[0012] SPHK modulating agents may be evaluated by any convenient in
vitro or in vivo assay for molecular interaction with an SPHK
polypeptide or nucleic acid. In one embodiment, candidate SPHK
modulating agents are tested with an assay system comprising a SPHK
polypeptide or nucleic acid. 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. SPHK-modulating agents include SPHK related proteins
(e.g. dominant negative mutants, and biotherapeutics);
SPHK-specific antibodies; SPHK-specific antisense oligomers and
other nucleic acid modulators; and chemical agents that
specifically bind to or interact with SPHK or compete with SPHK
binding partner (e.g. by binding to an SPHK binding partner). In
one specific embodiment, a small molecule modulator is identified
using a kinase assay. In specific embodiments, the screening assay
system is selected from a binding assay, an apoptosis assay, a cell
proliferation assay, an angiogenesis assay, and a hypoxic induction
assay.
[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
SPHK function and/or the p53 pathway in a mammalian cell by
contacting the mammalian cell with an agent that specifically binds
a SPHK 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 C. elegans, where a homozygous p53 deletion mutant
was used. Various specific genes were silenced by RNA inhibition
(RNAi). Methods for using RNAi to silence genes in C. elegans are
known in the art (Fire A, et al., 1998 Nature 391:806-811; Fire, A.
Trends Genet. 15, 358-363 (1999); WO9932619). Genes causing altered
phenotypes in the worms were identified as modifiers of the p53
pathway. A modifier of particular interest, C34C6.5 was identified
followed by identification of its human orthologs. Accordingly,
vertebrate orthologs of these modifiers, and preferably the human
orthologs, sphingosine kinase (SPHK) 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 SPHK function are
provided herein. Modulation of the SPHK 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. SPHK-modulating agents that act by inhibiting or
enhancing SPHK expression, directly or indirectly, for example, by
affecting an SPHK function such as enzymatic (e.g., catalytic) or
binding activity, can be identified using methods provided herein.
SPHK modulating agents are useful in diagnosis, therapy and
pharmaceutical development.
Nucleic Acids and Polypeptides of the Invention
[0017] Sequences related to SPHK nucleic acids and polypeptides
that can be used in the invention are disclosed in Genbank
(referenced by Genbank identifier (GI) number) as GI#s 11464966
(SEQ ID NO:1), 9910561 (SEQ ID NO:3), 19923819 (SEQ ID NO:4),
12052923 (SEQ ID NO:6), 13359166 (SEQ ID NO:7), 18594429 (SEQ ID
NO:8), and 12232440 (SEQ ID NO:9) for nucleic acid, and GI#s
11464967 (SEQ ID NO:10), 9910562 (SEQ ID NO:11), 13359167 (SEQ ID
NO:12), and 20336726 (SEQ ID NO:13) for polypeptides. Additionally,
sequences of clones N18H08 (SEQ ID NO:2) and N31F03 (SEQ D NO:5)
can also be used in the methods of invention.
[0018] SPHKs are kinase proteins with kinase domains. The term
"SPHK polypeptide" refers to a full-length SPHK protein or a
functionally active fragment or derivative thereof. A "functionally
active" SPHK fragment or derivative exhibits one or more functional
activities associated with a full-length, wild-type SPHK protein,
such as antigenic or immunogenic activity, enzymatic activity,
ability to bind natural cellular substrates, etc. The functional
activity of SPHK 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, New Jersey) and as further
discussed below. For purposes herein, functionally active fragments
also include those fragments that comprise one or more structural
domains of an SPHK, such as a kinase domain or a binding domain.
Protein domains can be identified using the PFAM program (Bateman
A., et al., Nucleic Acids Res, 1999, 27:260-2). For example, the
kinase domains of SPHKs from GI#s 11461967, 9910562, and 20336726
(SEQ ID NOs:10, 11, and 13, respectively) are located respectively
at approximately amino acid residues 16-153, 146 to 283, and 132 to
278. Methods for obtaining SPHK 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:10, 11, 12, or 13 (an SPHK). In
further preferred embodiments, the fragment comprises the entire
kinase (functionally active) domain.
[0019] The term "SPHK nucleic acid" refers to a DNA or RNA molecule
that encodes a SPHK polypeptide. Preferably, the SPHK 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 SPHK. Normally, orthologs in different species retain the same
function, due to presence of one or more protein motifs and/or
3-dimensional structures. Orthologs are generally identified by
sequence homology analysis, such as BLAST analysis, usually using
protein bait sequences. Sequences are assigned as a potential
ortholog if the best hit sequence from the forward BLAST result
retrieves the original query sequence in the reverse BLAST (Huynen
M A and Bork P, Proc Natl Acad Sci (1998) 95:5849-5856; Huynen M A
et al., Genome Research (2000) 10: 1204-1210). Programs for
multiple sequence alignment, such as CLUSTAL (Thompson J D et al,
1994, Nucleic Acids Res 22:4673-4680) may be used to highlight
conserved regions and/or residues of orthologous proteins and to
generate phylogenetic trees. In a phylogenetic tree representing
multiple homologous sequences from diverse species (e.g., retrieved
through BLAST analysis), orthologous sequences from two species
generally appear closest on the tree with respect to all other
sequences from these two species. Structural threading or other
analysis of protein folding (e.g., using software by ProCeryon,
Biosciences, Salzburg, Austria) may also identify potential
orthologs. In evolution, when a gene duplication event follows
speciation, a single gene in one species, such as C. elegans, may
correspond to multiple genes (paralogs) in another, such as human.
As used herein, the term "orthologs" encompasses paralogs. As used
herein, "percent (%) sequence identity" with respect to a subject
sequence, or a specified portion of a subject sequence, is defined
as the percentage of nucleotides or amino acids in the candidate
derivative sequence identical with the nucleotides or amino acids
in the subject sequence (or specified portion thereof), after
aligning the sequences and introducing gaps, if necessary to
achieve the maximum percent sequence identity, as generated by the
program WU-BLAST-2.0a19 (Altschul et al., J. Mol. Biol. (1997)
215:403-410) with all the search parameters set to default values.
The HSP S and HSP S2 parameters are dynamic values and are
established by the program itself depending upon the composition of
the particular sequence and composition of the particular database
against which the sequence of interest is being searched. A %
identity value is determined by the number of matching identical
nucleotides or amino acids divided by the sequence length for which
the percent identity is being reported. "Percent (%) amino acid
sequence similarity" is determined by doing the same calculation as
for determining % amino acid sequence identity, but including
conservative amino acid substitutions in addition to identical
amino acids in the computation.
[0020] 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.
[0021] Alternatively, an alignment for nucleic acid sequences is
provided by the local homology algorithm of Smith and Waterman
(Smith and Waterman, 1981, Advances in Applied Mathematics
2:482-489; database: European Bioinformatics Institute; Smith and
Waterman, 1981, J. of Molec. Biol., 147:195-197; Nicholas et al.,
1998, "A Tutorial on Searching Sequence Databases and Sequence
Scoring Methods" (www.psc.edu) and references cited therein; W. R.
Pearson, 1991, Genomics 11:635-650). This algorithm can be applied
to amino acid sequences by using the scoring matrix developed by
Dayhoff (Dayhoff: Atlas of Protein Sequences and Structure, M. O.
Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research
Foundation, Washington, D.C., USA), and normalized by Gribskov
(Gribskov 1986 Nucl. Acids Res. 14(6):6745-6763). The
Smith-Waterman algorithm may be employed where default parameters
are used for scoring (for example, gap open penalty of 12, gap
extension penalty of two). From the data generated, the "Match"
value reflects "sequence identity."
[0022] Derivative nucleic acid molecules of the subject nucleic
acid molecules include sequences that hybridize to the nucleic acid
sequence of SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, or 9. 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, 7, 8, or 9 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).
[0023] 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.
[0024] 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.
Isolation, Production, Expression, and Mis-expression of SPHK
Nucleic Acids and Polypeptides
[0025] SPHK nucleic acids and polypeptides, useful for identifying
and testing agents that modulate SPHK function and for other
applications related to the involvement of SPHK in the p53 pathway.
SPHK 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 an SPHK protein for assays used to assess SPHK
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 SPHK is
expressed in a cell line known to have defective p53 function (e.g.
SAOS-2 osteoblasts, H1299 lung cancer cells, C33A and HT3 cervical
cancer cells, HT-29 and DLD-1 colon cancer cells, among others,
available from American Type Culture Collection (ATCC), Manassas,
Va.). The recombinant cells are used in cell-based screening assay
systems of the invention, as described further below.
[0026] The nucleotide sequence encoding an SPHK polypeptide can be
inserted into any appropriate expression vector. The necessary
transcriptional and translational signals, including
promoter/enhancer element, can derive from the native SPHK gene
and/or its flanking regions or can be heterologous. A variety of
host-vector expression systems may be utilized, such as mammalian
cell systems infected with virus (e.g. vaccinia virus, adenovirus,
etc.); insect cell systems infected with virus (e.g. baculovirus);
microorganisms such as yeast containing yeast vectors, or bacteria
transformed with bacteriophage, plasmid, or cosmid DNA. A host cell
strain that modulates the expression of, modifies, and/or
specifically processes the gene product may be used.
[0027] To detect expression of the SPHK gene product, the
expression vector can comprise a promoter operably linked to an
SPHK 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 SPHK gene product based on the physical or functional
properties of the SPHK protein in in vitro assay systems (e.g.
immunoassays).
[0028] The SPHK protein, fragment, or derivative may be optionally
expressed as a fusion, or chimeric protein product (i.e. it is
joined via a peptide bond to a heterologous protein sequence of a
different protein), for example to facilitate purification or
detection. A chimeric product can be made by ligating the
appropriate nucleic acid sequences encoding the desired amino acid
sequences to each other using standard methods and expressing the
chimeric product. A chimeric product may also be made by protein
synthetic techniques, e.g. by use of a peptide synthesizer
(Hunkapiller et al., Nature (1984) 310:105-111).
[0029] Once a recombinant cell that expresses the SPHK gene
sequence is identified, the gene product can be isolated and
purified using standard methods (e.g. ion exchange, affinity, and
gel exclusion chromatography; centrifugation; differential
solubility; electrophoresis). Alternatively, native SPHK proteins
can be purified from natural sources, by standard methods (e.g.
immunoaffinity purification). Once a protein is obtained, it may be
quantified and its activity measured by appropriate methods, such
as immunoassay, bioassay, or other measurements of physical
properties, such as crystallography.
[0030] The methods of this invention may also use cells that have
been engineered for altered expression (mis-expression) of SPHK 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).
Genetically Modified Animals
[0031] Animal models that have been genetically modified to alter
SPHK 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 SPHK in a p53 pathway process such as apoptosis or cell
proliferation. Preferably, the altered SPHK expression results in a
detectable phenotype, such as decreased or increased levels of cell
proliferation, angiogenesis, or apoptosis compared to control
animals having normal SPHK 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.
[0032] 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).
[0033] In one embodiment, the transgenic animal is a "knock-out"
animal having a heterozygous or homozygous alteration in the
sequence of an endogenous SPHK gene that results in a decrease of
SPHK function, preferably such that SPHK 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 SPHK gene is used to construct a
homologous recombination vector suitable for altering an endogenous
SPHK gene in the mouse genome. Detailed methodologies for
homologous recombination in mice are available (see Capecchi,
Science (1989) 244:1288-1292; Joyner et al., Nature (1989)
338:153-156). Procedures for the production of non-rodent
transgenic mammals and other animals are also available (Houdebine
and Chourrout, supra; Pursel et al., Science (1989) 244:1281-1288;
Simms et al., Bio/Technology (1988) 6:179-183). In a preferred
embodiment, knock-out animals, such as mice harboring a knockout of
a specific gene, may be used to produce antibodies against the
human counterpart of the gene that has been knocked out (Claesson M
H et al., (1994) Scan J Immunol 40:257-264; Declerck P J et al.,
(1995) J Biol Chem. 270:8397-400).
[0034] 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 SPHK gene, e.g., by introduction of additional
copies of SPHK, or by operatively inserting a regulatory sequence
that provides for altered expression of an endogenous copy of the
SPHK gene. Such regulatory sequences include inducible,
tissue-specific, and constitutive promoters and enhancer elements.
The knock-in can be homozygous or heterozygous.
[0035] 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).
[0036] 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 SPHK function and phenotypic changes are compared with
appropriate control animals such as genetically modified animals
that receive placebo treatment, and/or animals with unaltered SPHK
expression that receive candidate therapeutic agent.
[0037] In addition to the above-described genetically modified
animals having altered SPHK function, animal models having
defective p53 function (and otherwise normal SPHK function), can be
used in the methods of the present invention. For example, a p53
knockout mouse can be used to assess, in vivo, the activity of a
candidate p53 modulating agent identified in one of the in vitro
assays described below. p53 knockout mice are described in the
literature (Jacks et al., Nature 2001; 410:1111-1116, 1043-1044;
Donehower et al., supra). Preferably, the candidate p53 modulating
agent when administered to a model system with cells defective in
p53 function, produces a detectable phenotypic change in the model
system indicating that the p53 function is restored, i.e., the
cells exhibit normal cell cycle progression.
Modulating Agents
[0038] The invention provides methods to identify agents that
interact with and/or modulate the function of SPHK and/or the p53
pathway. Modulating agents identified by the methods are also part
of the invention. Such agents are useful in a variety of diagnostic
and therapeutic applications associated with the p53 pathway, as
well as in further analysis of the SPHK 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 SPHK activity by administering a
SPHK-interacting or -modulating agent.
[0039] As used herein, an "SPHK-modulating agent" is any agent that
modulated SPHK function, for example, an agent that interacts with
SPHK to inhibit or enhance SPHK activity or otherwise affect normal
SPHK function. SPHK function can be affected at any level,
including transcription, protein expression, protein localization,
and cellular or extra-cellular activity. In a preferred embodiment,
the SPHK--modulating agent specifically modulates the function of
the SPHK. The phrases "specific modulating agent", "specifically
modulates", etc., are used herein to refer to modulating agents
that directly bind to the SPHK polypeptide or nucleic acid, and
preferably inhibit, enhance, or otherwise alter, the function of
the SPHK. These phrases also encompasses modulating agents that
alter the interaction of the SPHK with a binding partner,
substrate, or cofactor (e.g. by binding to a binding partner of an
SPHK, or to a protein/binding partner complex, and altering SPHK
function). In a further preferred embodiment, the SPHK-modulating
agent is a modulator of the p53 pathway (e.g. it restores and/or
upregulates p53 function) and thus is also a p53-modulating
agent.
[0040] Preferred SPHK-modulating agents include small molecule
compounds; SPHK-interacting proteins, including antibodies and
other biotherapeutics; and nucleic acid modulators such as
antisense and RNA inhibitors. The modulating agents may be
formulated in pharmaceutical compositions, for example, as
compositions that may comprise other active ingredients, as in
combination therapy, and/or suitable carriers or excipients.
Techniques for formulation and administration of the compounds may
be found in "Remington's Pharmaceutical Sciences" Mack Publishing
Co., Easton, Pa., 19.sup.th edition.
[0041] Small Molecule Modulators
[0042] Small molecules, are often preferred to modulate function of
proteins with enzymatic function, and/or containing protein
interaction domains. Chemical agents, referred to in the art as
"small molecule" compounds are typically organic, non-peptide
molecules, having a molecular weight 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 SPHK 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 SPHK-modulating activity. Methods
for generating and obtaining compounds are well known in the art
(Schreiber S L, Science (2000) 151: 1964-1969; Radmann J and
Gunther J, Science (2000) 151:1947-1948).
[0043] Small molecule modulators identified from screening assays,
as described below, can be used as lead compounds from which
candidate clinical compounds may be designed, optimized, and
synthesized. Such clinical compounds may have utility in treating
pathologies associated with the 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.
[0044] Protein Modulators
[0045] Specific SPHK-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 SPHK-modulating agents. In a preferred embodiment,
SPHK-interacting proteins affect normal SPHK function, including
transcription, protein expression, protein localization, and
cellular or extra-cellular activity. In another embodiment,
SPHK-interacting proteins are useful in detecting and providing
information about the function of SPHK proteins, as is relevant to
p53 related disorders, such as cancer (e.g., for diagnostic
means).
[0046] An SPHK-interacting protein may be endogenous, i.e. one that
naturally interacts genetically or biochemically with an SPHK, such
as a member of the SPHK pathway that modulates SPHK expression,
localization, and/or activity. SPHK-modulators include dominant
negative forms of SPHK-interacting proteins and of SPHK proteins
themselves. Yeast two-hybrid and variant screens offer preferred
methods for identifying endogenous SPHK-interacting proteins
(Finley, R. L. et al. (1996) in DNA Cloning-Expression Systems: A
Practical Approach, eds. Glover D. & Hames B. D (Oxford
University Press, Oxford, England), pp. 169-203; Fashema S F et
al., Gene (2000) 250:1-14; Drees B L Curr Opin Chem Biol (1999)
3:64-70; Vidal M and Legrain P Nucleic Acids Res (1999) 27:919-29;
and U.S. Pat. No. 5,928,868). Mass spectrometry is an alternative
preferred method for the elucidation of protein complexes (reviewed
in, e.g., Pandley A and Mann M, Nature (2000) 405:837-846; Yates J
R 3.sup.rd, Trends Genet (2000) 16:5-8).
[0047] An SPHK-interacting protein may be an exogenous protein,
such as an SPHK-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). SPHK antibodies are further
discussed below.
[0048] In preferred embodiments, an SPHK-interacting protein
specifically binds an SPHK protein. In alternative preferred
embodiments, an SPHK-modulating agent binds an SPHK substrate,
binding partner, or cofactor.
[0049] Antibodies
[0050] In another embodiment, the protein modulator is an SPHK
specific antibody agonist or antagonist. The antibodies have
therapeutic and diagnostic utilities, and can be used in screening
assays to identify SPHK modulators. The antibodies can also be used
in dissecting the portions of the SPHK pathway responsible for
various cellular responses and in the general processing and
maturation of the SPHK.
[0051] Antibodies that specifically bind SPHK polypeptides can be
generated using known methods. Preferably the antibody is specific
to a mammalian ortholog of SPHK polypeptide, and more preferably,
to human SPHK. 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 SPHK
which are particularly antigenic can be selected, for example, by
routine screening of SPHK 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:10, 11, 12, or 13. 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 SPHK or substantially purified fragments thereof. If SPHK
fragments are used, they preferably comprise at least 10, and more
preferably, at least 20 contiguous amino acids of an SPHK protein.
In a particular embodiment, SPHK-specific antigens and/or
immunogens are coupled to carrier proteins that stimulate the
immune response. For example, the subject polypeptides are
covalently coupled to the keyhole limpet hemocyanin (KLH) carrier,
and the conjugate is emulsified in Freund's complete adjuvant,
which enhances the immune response. An appropriate immune system
such as a laboratory rabbit or mouse is immunized according to
conventional protocols.
[0052] The presence of SPHK-specific antibodies is assayed by an
appropriate assay such as a solid phase enzyme-linked immunosorbant
assay (ELISA) using immobilized corresponding SPHK polypeptides.
Other assays, such as radioimmunoassays or fluorescent assays might
also be used.
[0053] Chimeric antibodies specific to SPHK polypeptides can be
made that contain different portions from different animal species.
For instance, a human immunoglobulin constant region may be linked
to a variable region of a murine mAb, such that the antibody
derives its biological activity from the human antibody, and its
binding specificity from the murine fragment. Chimeric antibodies
are produced by splicing together genes that encode the appropriate
regions from each species (Morrison et al., Proc. Natl. Acad. Sci.
(1984) 81:6851-6855; Neuberger et al., Nature (1984) 312:604-608;
Takeda et al., Nature (1985) 31:452-454). Humanized antibodies,
which are a form of chimeric antibodies, can be generated by
grafting complementary-determining regions (CDRs) (Carlos, T. M.,
J. M. Harlan. 1994. Blood 84:2068-2101) of mouse antibodies into a
background of human framework regions and constant regions by
recombinant DNA technology (Riechmann L M, et al., 1988 Nature 323:
323-327). Humanized antibodies contain .about.10% murine sequences
and .about.90% human sequences, and thus further reduce or
eliminate immunogenicity, while retaining the antibody
specificities (Co M S, and Queen C. 1991 Nature 351: 501-501;
Morrison S L. 1992 Ann. Rev. Immun. 10:239-265). Humanized
antibodies and methods of their production are well-known in the
art (U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,762, and
6,180,370).
[0054] SPHK-specific single chain antibodies which are recombinant,
single chain polypeptides formed by linking the heavy and light
chain fragments of the Fv regions via an amino acid bridge, can be
produced by methods known in the art (U.S. Pat. No. 4,946,778;
Bird, Science (1988) 242:423-426; Huston et al., Proc. Natl. Acad.
Sci. USA (1988) 85:5879-5883; and Ward et al., Nature (1989)
334:544-546).
[0055] Other suitable techniques for antibody production involve in
vitro exposure of lymphocytes to the antigenic polypeptides or
alternatively to selection of libraries of antibodies in phage or
similar vectors (Huse et al., Science (1989) 246:1275-1281). As
used herein, T-cell antigen receptors are included within the scope
of antibody modulators (Harlow and Lane, 1988, supra).
[0056] The polypeptides and antibodies of the present invention may
be used with or without modification. Frequently, antibodies will
be labeled by joining, either covalently or non-covalently, a
substance that provides for a detectable signal, or that is toxic
to cells that express the targeted protein (Menard S, et al., Int
J. Biol Markers (1989) 4:131-134). A wide variety of labels and
conjugation techniques are known and are reported extensively in
both the scientific and patent literature. Suitable labels include
radionuclides, enzymes, substrates, cofactors, inhibitors,
fluorescent moieties, fluorescent emitting lanthanide metals,
chemiluminescent moieties, bioluminescent moieties, magnetic
particles, and the like (U.S. Pat. Nos. 3,817,837; 3,850,752;
3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241). Also,
recombinant immunoglobulins may be produced (U.S. Pat. No.
4,816,567). Antibodies to cytoplasmic polypeptides may be delivered
and reach their targets by conjugation with membrane-penetrating
toxin proteins (U.S. Pat. No. 6,086,900).
[0057] When used therapeutically in a patient, the antibodies of
the subject invention are typically administered parenterally, when
possible at the target site, or intravenously. The therapeutically
effective dose and dosage regimen is determined by clinical
studies. Typically, the amount of antibody administered is in the
range of about 0.1 mg/kg to about 10 mg/kg of patient weight. For
parenteral administration, the antibodies are formulated in a unit
dosage injectable form (e.g., solution, suspension, emulsion) in
association with a pharmaceutically acceptable vehicle. Such
vehicles are inherently nontoxic and non-therapeutic. Examples are
water, saline, Ringer's solution, dextrose solution, and 5% human
serum albumin. Nonaqueous vehicles such as fixed oils, ethyl
oleate, or liposome carriers may also be used. The vehicle may
contain minor amounts of additives, such as buffers and
preservatives, which enhance isotonicity and chemical stability or
otherwise enhance therapeutic potential. The antibodies'
concentrations in such vehicles are typically in the range of about
1 mg/ml to about 10 mg/ml. Immunotherapeutic methods are further
described in the literature (U.S. Pat. No. 5,859,206;
WO0073469).
[0058] Nucleic Acid Modulators
[0059] Other preferred SPHK-modulating agents comprise nucleic acid
molecules, such as antisense oligomers or double stranded RNA
(dsRNA), which generally inhibit SPHK activity. Preferred nucleic
acid modulators interfere with the function of the SPHK nucleic
acid such as DNA replication, transcription, translocation of the
SPHK RNA to the site of protein translation, translation of protein
from the SPHK RNA, splicing of the SPHK RNA to yield one or more
mRNA species, or catalytic activity which may be engaged in or
facilitated by the SPHK RNA.
[0060] In one embodiment, the antisense oligomer is an
oligonucleotide that is sufficiently complementary to an SPHK mRNA
to bind to and prevent translation, preferably by binding to the 5'
untranslated region. SPHK-specific antisense oligonucleotides,
preferably range from at least 6 to about 200 nucleotides. In some
embodiments the oligonucleotide is preferably at least 10, 15, or
20 nucleotides in length. In other embodiments, the oligonucleotide
is preferably less than 50, 40, or 30 nucleotides in length. The
oligonucleotide can be DNA or RNA or a chimeric mixture or
derivatives or modified versions thereof, single-stranded or
double-stranded. The oligonucleotide can be modified at the base
moiety, sugar moiety, or phosphate backbone. The oligonucleotide
may include other appending groups such as peptides, agents that
facilitate transport across the cell membrane,
hybridization-triggered cleavage agents, and intercalating
agents.
[0061] In another embodiment, the antisense oligomer is a
phosphothioate morpholino oligomer (PMO). PMOs are assembled from
four different morpholino subunits, each of which contain one of
four genetic bases (A, C, G, or T) linked to a six-membered
morpholine ring. Polymers of these subunits are joined by non-ionic
phosphodiamidate intersubunit linkages. Details of how to make and
use PMOs and other antisense oligomers are well known in the art
(e.g. see WO99/18193; Probst J C, Antisense Oligodeoxynucleotide
and Ribozyme Design, Methods. (2000) 22(3):271-281; Summerton J,
and Weller D. 1997 Antisense Nucleic Acid Drug Dev.:7:187-95; U.S.
Pat. No. 5,235,033; and U.S. Pat. No. 5,378,841).
[0062] Alternative preferred SPHK nucleic acid modulators are
double-stranded RNA species mediating RNA interference (RNAi). RNAi
is the process of sequence-specific, post-transcriptional gene
silencing in animals and plants, initiated by double-stranded RNA
(dsRNA) that is homologous in sequence to the silenced gene.
Methods relating to the use of RNAi to silence genes in C. elegans,
Drosophila, plants, and humans are known in the art (Fire A, et
al., 1998 Nature 391:806-811; Fire, A. Trends Genet. 15, 358-363
(1999); Sharp, P. A. RNA interference 2001. Genes Dev. 15, 485-490
(2001); Hammond, S. M., et al., Nature Rev. Genet. 2, 110-1119
(2001); Tuschl, T. Chem. Biochem. 2, 239-245 (2001); Hamilton, A.
et al., Science 286, 950-952 (1999); Hammond, S. M., et al., Nature
404, 293-296 (2000); Zamore, P. D., et al., Cell 101, 25-33 (2000);
Bernstein, E., et al., Nature 409, 363-366 (2001); Elbashir, S. M.,
et al., Genes Dev. 15, 188-200 (2001); WO0129058; WO9932619;
Elbashir S M, et al., 2001 Nature 411:494-498).
[0063] Nucleic acid modulators are commonly used as research
reagents, diagnostics, and therapeutics. For example, antisense
oligonucleotides, which are able to inhibit gene expression with
exquisite specificity, are often used to elucidate the function of
particular genes (see, for example, U.S. Pat. No. 6,165,790).
Nucleic acid modulators are also used, for example, to distinguish
between functions of various members of a biological pathway. For
example, antisense oligomers have been employed as therapeutic
moieties in the treatment of disease states in animals and man and
have been demonstrated in numerous clinical trials to be safe and
effective (Milligan J F, et al, Current Concepts in Antisense Drug
Design, J Med Chem. (1993) 36:1923-1937; Tonkinson J L et al.,
Antisense Oligodeoxynucleotides as Clinical Therapeutic Agents,
Cancer Invest. (1996) 14:54-65). Accordingly, in one aspect of the
invention, an SPHK-specific nucleic acid modulator is used in an
assay to further elucidate the role of the SPHK in the p53 pathway,
and/or its relationship to other members of the pathway. In another
aspect of the invention, an SPHK-specific antisense oligomer is
used as a therapeutic agent for treatment of p53-related disease
states.
Assay Systems
[0064] The invention provides assay systems and screening methods
for identifying specific modulators of SPHK 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 SPHK nucleic acid or protein.
In general, secondary assays further assess the activity of a SPHK
modulating agent identified by a primary assay and may confirm that
the modulating agent affects SPHK in a manner relevant to the p53
pathway. In some cases, SPHK modulators will be directly tested in
a secondary assay.
[0065] In a preferred embodiment, the screening method comprises
contacting a suitable assay system comprising an SPHK polypeptide
or nucleic acid with a candidate agent under conditions whereby,
but for the presence of the agent, the system provides a reference
activity (e.g. kinase activity), which is based on the particular
molecular event the screening method detects. A statistically
significant difference between the agent-biased activity and the
reference activity indicates that the candidate agent modulates
SPHK activity, and hence the p53 pathway. The SPHK polypeptide or
nucleic acid used in the assay may comprise any of the nucleic
acids or polypeptides described above.
[0066] Primary Assays
[0067] The type of modulator tested generally determines the type
of primary assay.
[0068] Primary Assays for Small Molecule Modulators
[0069] 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,
colorimetric, spectrophotometric, and amperometric methods, to
provide a read-out for the particular molecular event detected.
[0070] Cell-based screening assays usually require systems for
recombinant expression of SPHK 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
SPHK-interacting proteins are used in screens to identify small
molecule modulators, the binding specificity of the interacting
protein to the SPHK protein may be assayed by various known methods
such as substrate processing (e.g. ability of the candidate
SPHK-specific binding agents to function as negative effectors in
SPHK-expressing cells), binding equilibrium constants (usually at
least about 10.sup.7 M.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 SPHK 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.
[0071] The screening assay may measure a candidate agent's ability
to specifically bind to or modulate activity of a SPHK polypeptide,
a fusion protein thereof, or to cells or membranes bearing the
polypeptide or fusion protein. The SPHK polypeptide can be full
length or a fragment thereof that retains functional SPHK activity.
The SPHK polypeptide may be fused to another polypeptide, such as a
peptide tag for detection or anchoring, or to another tag. The SPHK
polypeptide is preferably human SPHK, or is an ortholog or
derivative thereof as described above. In a preferred embodiment,
the screening assay detects candidate agent-based modulation of
SPHK interaction with a binding target, such as an endogenous or
exogenous protein or other substrate that has SPHK-specific binding
activity, and can be used to assess normal SPHK gene function.
[0072] Suitable assay formats that may be adapted to screen for
SPHK modulators are known in the art. Preferred screening assays
are high throughput or ultra high throughput and thus provide
automated, cost-effective means of screening compound libraries for
lead compounds (Fernandes P B, Curr Opin Chem Biol (1998)
2:597-603; Sundberg S A, Curr Opin Biotechnol 2000, 11:47-53). In
one preferred embodiment, screening assays uses fluorescence
technologies, including fluorescence polarization, time-resolved
fluorescence, and fluorescence resonance energy transfer. These
systems offer means to monitor protein-protein or DNA-protein
interactions in which the intensity of the signal emitted from
dye-labeled molecules depends upon their interactions with partner
molecules (e.g., Selvin P R, Nat Struct Biol (2000) 7:730-4;
Fernandes P B, supra; Hertzberg R P and Pope A J, Curr Opin Chem
Biol (2000) 4:445-451).
[0073] A variety of suitable assay systems may be used to identify
candidate SPHK 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); U.S. Pat. No. 6,020,135 (p53 modulation), among
others). Specific preferred assays are described in more detail
below.
[0074] Kinase assays. In some preferred embodiments the screening
assay detects the ability of the test agent to modulate the kinase
activity of an SPHK 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 (lck) kinase activity monitors the transfer of the gamma
phosphate from gamma-.sup.33P ATP to a biotinylated peptide
substrate; the substrate is captured on a streptavidin coated bead
that transmits the signal (Beveridge M et al., J Biomol Screen
(2000) 5:205-212). This assay uses the scintillation proximity
assay (SPA), in which only radio-ligand bound to receptors tethered
to the surface of an SPA bead are detected by the scintillant
immobilized within it, allowing binding to be measured without
separation of bound from free ligand.
[0075] Various sphingosine kinase assays, appropriate for
identifying modulators of hSPHK, have been described in the
literature (Meyer zu Heringsdorf et al. (1998) EMBO J. 17, 2830-7;
Edsal and Spiegel, Analytical Biochemistry 272, 80-86 (1999); and
Tolan et al., Cell. Signal. Vol. 11, No. 5, pp. 349-354, 1999))
[0076] Other assays for protein kinase activity may use antibodies
that specifically recognize phosphorylated substrates. For
instance, the kinase receptor activation (KIRA) assay measures
receptor tyrosine kinase activity by ligand stimulating the intact
receptor in cultured cells, then capturing solubilized receptor
with specific antibodies and quantifying phosphorylation via
phosphotyrosine ELISA (Sadick M D, Dev Biol Stand (1999)
97:121-133).
[0077] Another example of antibody based assays for protein kinase
activity is TRF (time-resolved fluorometry). This method utilizes
europium chelate-labeled anti-phosphotyrosine antibodies to detect
phosphate transfer to a polymeric substrate coated onto microtiter
plate wells. The amount of phosphorylation is then detected using
time-resolved, dissociation-enhanced fluorescence (Braunwalder A F,
et al., Anal Biochem 1996 Jul. 1; 238(2):159-64).
[0078] 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 an
SPHK, 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 SPHK function plays a direct
role in apoptosis. For example, an apoptosis assay may be performed
on cells that over- or under-express SPHK relative to wild type
cells. Differences in apoptotic response compared to wild type
cells suggests that the SPHK plays a direct role in the apoptotic
response. Apoptosis assays are described further in U.S. Pat. No.
6,133,437.
[0079] Cell proliferation and cell cycle assays. 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.
[0080] 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).
[0081] 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 SPHK are seeded
in soft agar plates, and colonies are measured and counted after
two weeks incubation.
[0082] 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 an SPHK may be
stained with propidium iodide and evaluated in a flow cytometer
(available from Becton Dickinson).
[0083] Accordingly, a cell proliferation or cell cycle assay system
may comprise a cell that expresses an SPHK, 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 SPHK 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 SPHK relative to wild type cells. Differences in
proliferation or cell cycle compared to wild type cells suggests
that the SPHK plays a direct role in cell proliferation or cell
cycle.
[0084] 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 an
SPHK, 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 SPHK function plays a direct
role in cell proliferation. For example, an angiogenesis assay may
be performed on cells that over- or under-express SPHK relative to
wild type cells. Differences in angiogenesis compared to wild type
cells suggests that the SPHK plays a direct role in
angiogenesis.
[0085] 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 SPHK 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 an SPHK, 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 SPHK 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 SPHK relative to wild type
cells. Differences in hypoxic response compared to wild type cells
suggests that the SPHK plays a direct role in hypoxic
induction.
[0086] 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.
[0087] 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.
[0088] High-throughput cell adhesion assays have also been
described. In one such assay, small molecule ligands and peptides
are bound to the surface of microscope slides using a microarray
spotter, intact cells are then contacted with the slides, and
unbound cells are washed off. In this assay, not only the binding
specificity of the peptides and modulators against cell lines are
determined, but also the functional cell signaling of attached
cells using immunofluorescence techniques in situ on the microchip
is measured (Falsey J R et al., Bioconjug Chem. 2001 May-June;
12(3):346-53).
[0089] Cell Migration. An invasion/migration assay (also called a
migration assay) tests the ability of cells to overcome a physical
barrier and to migrate towards pro-angiogenic signals. Migration
assays are known in the art (e.g., Paik J H et al., 2001, J Biol
Chem 276:11830-11837). In a typical experimental set-up, cultured
endothelial cells are seeded onto a matrix-coated porous lamina,
with pore sizes generally smaller than typical cell size. The
matrix generally simulates the environment of the extracellular
matrix, as described above. The lamina is typically a membrane,
such as the transwell polycarbonate membrane (Corning Costar
Corporation, Cambridge, Mass.), and is generally part of an upper
chamber that is in fluid contact with a lower chamber containing
pro-angiogenic stimuli. Migration is generally assayed after an
overnight incubation with stimuli, but longer or shorter time
frames may also be used. Migration is assessed as the number of
cells that crossed the lamina, and may be detected by staining
cells with hemotoxylin solution (VWR Scientific, South San
Francisco, Calif.), or by any other method for determining cell
number. In another exemplary set up, cells are fluorescently
labeled and migration is detected using fluorescent readings, for
instance using the Falcon HTS FluoroBlok (Becton Dickinson). While
some migration is observed in the absence of stimulus, migration is
greatly increased in response to pro-angiogenic factors. As
described above, a preferred assay system for migration/invasion
assays comprises testing an SPHK's response to a variety of
pro-angiogenic factors, including tumor angiogenic and inflammatory
angiogenic agents, and culturing the cells in serum free
medium.
[0090] Sprouting assay. A sprouting assay is a three-dimensional in
vitro angiogenesis assay that uses a cell-number defined spheroid
aggregation of endothelial cells ("spheroid"), embedded in a
collagen gel-based matrix. The spheroid can serve as a starting
point for the sprouting of capillary-like structures by invasion
into the extracellular matrix (termed "cell sprouting") and the
subsequent formation of complex anastomosing networks (Korff and
Augustin, 1999, J Cell Sci 112:3249-58). In an exemplary
experimental set-up, spheroids are prepared by pipetting 400 human
umbilical vein endothelial cells into individual wells of a
nonadhesive 96-well plates to allow overnight spheroidal
aggregation (Korff and Augustin: J Cell Biol 143: 1341-52, 1998).
Spheroids are harvested and seeded in 900 .mu.l of
methocel-collagen solution and pipetted into individual wells of a
24 well plate to allow collagen gel polymerization. Test agents are
added after 30 min by pipetting 100 .mu.l of 10-fold concentrated
working dilution of the test substances on top of the gel. Plates
are incubated at 37.degree. C. for 24 h. Dishes are fixed at the
end of the experimental incubation period by addition of
paraformaldehyde. Sprouting intensity of endothelial cells can be
quantitated by an automated image analysis system to determine the
cumulative sprout length per spheroid.
Primary Assays for Antibody Modulators
[0091] For antibody modulators, appropriate primary assays test is
a binding assay that tests the antibody's affinity to and
specificity for the SPHK 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 SPHK-specific
antibodies; others include FACS assays, radioimmunoassays, and
fluorescent assays.
[0092] Primary Assays for Nucleic Acid Modulators
[0093] For nucleic acid modulators, primary assays may test the
ability of the nucleic acid modulator to inhibit or enhance SPHK
gene expression, preferably mRNA expression. In general, expression
analysis comprises comparing SPHK expression in like populations of
cells (e.g., two pools of cells that endogenously or recombinantly
express SPHK) 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 SPHK 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 SPHK 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).
[0094] Secondary Assays
[0095] Secondary assays may be used to further assess the activity
of SPHK-modulating agent identified by any of the above methods to
confirm that the modulating agent affects SPHK in a manner relevant
to the p53 pathway. As used herein, SPHK-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 SPHK.
[0096] Secondary assays generally compare like populations of cells
or animals (e.g., two pools of cells or animals that endogenously
or recombinantly express SPHK) in the presence and absence of the
candidate modulator. In general, such assays test whether treatment
of cells or animals with a candidate SPHK-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.
[0097] Cell-Based Assays
[0098] 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.
[0099] Animal Assays
[0100] A variety of non-human animal models of normal or defective
p53 pathway may be used to test candidate SPHK 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.
[0101] 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 SPHK 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 SPHK. 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.
[0102] In another preferred embodiment, the effect of the candidate
modulator on SPHK 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 SPHK 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.
Diagnostic and Therapeutic Uses
[0103] Specific SPHK-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 or impaired p53 function (e.g. due to overexpression,
underexpression, or misexpression of p53, or due to gene
mutations), comprising the step of administering an agent to the
cell that specifically modulates SPHK activity. Preferably, the
modulating agent produces a detectable phenotypic change in the
cell indicating that the p53 function is restored. The phrase
"function is restored", and equivalents, as used herein, means that
the desired phenotype is achieved, or is brought closer to normal
compared to untreated cells. For example, with restored p53
function, cell proliferation and/or progression through cell cycle
may normalize, or be brought closer to normal relative to untreated
cells. The invention also provides methods for treating disorders
or disease associated with impaired p53 function by administering a
therapeutically effective amount of an SPHK-modulating agent that
modulates the p53 pathway. The invention further provides methods
for modulating SPHK function in a cell, preferably a cell
pre-determined to have defective or impaired SPHK function, by
administering an SPHK-modulating agent. Additionally, the invention
provides a method for treating disorders or disease associated with
impaired SPHK function by administering a therapeutically effective
amount of an SPHK-modulating agent.
[0104] The discovery that SPHK 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.
[0105] Various expression analysis methods can be used to diagnose
whether SPHK 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 an SPHK, are identified as
amenable to treatment with an SPHK modulating agent. In a preferred
application, the p53 defective tissue overexpresses an SPHK
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
SPHK cDNA sequences as probes, can determine whether particular
tumors express or overexpress SPHK. Alternatively, the TaqMan.RTM.
is used for quantitative RT-PCR analysis of SPHK expression in cell
lines, normal tissues and tumor samples (PE Applied
Biosystems).
[0106] Various other diagnostic methods may be performed, for
example, utilizing reagents such as the SPHK oligonucleotides, and
antibodies directed against an SPHK, as described above for: (1)
the detection of the presence of SPHK gene mutations, or the
detection of either over- or under-expression of SPHK mRNA relative
to the non-disorder state; (2) the detection of either an over- or
an under-abundance of SPHK gene product relative to the
non-disorder state; and (3) the detection of perturbations or
abnormalities in the signal transduction pathway mediated by
SPHK.
[0107] Thus, in a specific embodiment, the invention is drawn to a
method for diagnosing a disease or disorder in a patient that is
associated with alterations in SPHK expression, the method
comprising: a) obtaining a biological sample from the patient; b)
contacting the sample with a probe for SPHK expression; c)
comparing results from step (b) with a control; and d) determining
whether step (c) indicates a likelihood of the disease or disorder.
Preferably, the disease is cancer, most preferably a cancer as
shown in TABLE 1. The probe may be either DNA or protein, including
an antibody.
EXAMPLES
[0108] The following experimental section and examples are offered
by way of illustration and not by way of limitation.
[0109] I. C. elegans P53 Screen
[0110] A systematic RNAi of various genes was carried out in worms
homozygous for p53 deletion. p53 (-/-) worms have a normal
phenotype, but are defective in germline apoptotic response to
ionizing radiation as p53 is involved in the DNA damage response.
After silencing of each gene by RNAi, worms were subject to
gamma-irradiation, and phenotypes were scored.
[0111] Worms subjected to SPHK RNAi had an increase in germline
apoptosis in p53 (-/-) mutants compared to non-RNAi control
animals, as visualized by acridine orange staining. C34C6.5 was
thus a suppressor of the p53 (-/-) phenotype. Human orthologs of
the modifiers are referred to herein as SPHK.
[0112] BLAST analysis (Altschul et al., supra) was employed to
identify Targets from C. elegans modifiers. For example,
representative sequences from SPHK, GI#s 11464967, 9910562, and
20336726 (SEQ ID NOs:10, 11, and 13, respectively), share 28%, 32%,
and 28% amino acid identity, respectively, with the C. elegans
C34C6.5.
[0113] Various domains, signals, and functional subunits in
proteins were analyzed using the PSORT (Nakai K., and Horton P.,
Trends Biochem Sci, 1999, 24:34-6; Kenta Nakai, Protein sorting
signals and prediction of subcellular localization, Adv. Protein
Chem. 54, 277-344 (2000)), PFAM (Bateman A., et al., Nucleic Acids
Res, 1999, 27:260-2), SMART (Ponting C P, et al., SMART:
identification and annotation of domains from signaling and
extracellular protein sequences. Nucleic Acids Res. 1999 Jan. 1;
27(1):229-32), TM-HMM (Erik L. L. Sonnhammer, Gunnar von Heijne,
and Anders Krogh: A hidden Markov model for predicting
transmembrane helices in protein sequences. In Proc. of Sixth Int.
Conf. on Intelligent Systems for Molecular Biology, p 175-182 Ed J.
Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C.
Sensen Menlo Park, Calif.: AAAI Press, 1998), and clust (Remm M,
and Sonnhammer E. Classification of transmembrane protein families
in the Caenorhabditis elegans genome and identification of human
orthologs. Genome Res. 2000 November; 10(11): 1679-89) programs.
For example, the kinase domains of SPHKs from GL#s 11461967,
9910562, and 20336726 (SEQ ID NOs:10, 11, and 13, respectively) are
located respectively at approximately amino acid residues 16-153,
146 to 283, and 132 to 278
[0114] II. High-Throughput In Vitro Fluorescence Polarization
Assay
[0115] Fluorescently-labeled SPHK peptide/substrate are added to
each well of a 96-well microtiter plate, along with a test agent in
a test buffer (10 mM HEPES, 10 mM NaCl, 6 mM magnesium chloride, pH
7.6). Changes in fluorescence polarization, determined by using a
Fluorolite FPM-2 Fluorescence Polarization Microtiter System
(Dynatech Laboratories, Inc), relative to control values indicates
the test compound is a candidate modifier of SPHK activity.
[0116] III. High-Throughout In Vitro Binding Assay.
[0117] .sup.33P-labeled SPHK 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.
[0118] IV. Immunoprecipitations and Immunoblotting
[0119] For coprecipitation of transfected proteins,
3.times.10.sup.6 appropriate recombinant cells containing the SPHK
proteins are plated on 10-cm dishes and transfected on the
following day with expression constructs. The total amount of DNA
is kept constant in each transfection by adding empty vector. After
24 h, cells are collected, washed once with phosphate-buffered
saline and lysed for 20 min on ice in 1 ml of lysis buffer
containing 50 mM Hepes, pH 7.9, 250 mM NaCl, 20
mM-glycerophosphate, 1 mM sodium orthovanadate, 5 mM p-nitrophenyl
phosphate, 2 mM dithiothreitol, protease inhibitors (complete,
Roche Molecular Biochemicals), and 1% Nonidet P-40. Cellular debris
is removed by centrifugation twice at 15,000.times.g for 15 min.
The cell lysate is incubated with 25 .mu.l of M2 beads (Sigma) for
2 h at 4.degree. C. with gentle rocking.
[0120] 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).
[0121] V. Kinase Assay
[0122] A purified or partially purified SPHK 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 (Mg.sup.2+ or
Mn.sup.2+) required for enzymatic activity. Following the
incubation, the enzyme reaction is quenched using EDTA. Samples of
the reaction are transferred to a 96-well glass fiber filter plate
(MultiScreen, Millipore). The filters are subsequently washed with
phosphate-buffered saline, dilute phosphoric acid (0.5%) or other
suitable medium to remove excess radiolabeled ATP. Scintillation
cocktail is added to the filter plate and the incorporated
radioactivity is quantitated by scintillation counting
(Wallac/Perkin Elmer). Activity is defined by the amount of
radioactivity detected following subtraction of the negative
control reaction value (EDTA quench).
[0123] VI. Expression Analysis
[0124] All cell lines used in the following experiments are NCI
(National Cancer Institute) lines, and are available from ATCC
(American Type Culture Collection, Manassas, Va. 20110-2209).
Normal and tumor tissues were obtained from Impath, U C Davis,
Clontech, Stratagene, and Ambion.
[0125] TaqMan analysis was used to assess expression levels of the
disclosed genes in various samples.
[0126] 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.).
[0127] 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.
[0128] 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).
[0129] 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)).
[0130] Results are shown in Table 1. 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.
TABLE-US-00001 TABLE 1 SEQ ID NO NA_GI# breast colon lung ovary 8
18594429 2 11 4 30 1 13 1 7 1 11464966 4 11 4 30 6 14 1 7 4 9910561
2 11 7 25 4 11 0 3
[0131]
Sequence CWU 1
1
13 1 1783 DNA Homo sapiens 1 ggagggagcg aggccgggga gtccgctcca
gcggggcgct ccagtccctc agacgtgggc 60 tgagcttggg acgagctgcg
ttccgcccca ggccactgta gggaacggcg gtggcgcctc 120 cccagcaaac
cggaccgact gggtccagcc gccgcaggga atgacaccgg tgctcctaca 180
gccacggctc cgggcgggga aggcgagccc cacagccggc cctgcgacgc ccgcctgggc
240 agcaccgata aggagctgaa ggcaggagcc gccgccacgg gcagcgcccc
cacagcgcca 300 gggaccccct ggcagcggga gccgcgggtc gaggttatgg
atccagcggg cggcccccgg 360 ggcgtgctcc cgcggccctg ccgcgtgctg
gtgctgctga acccgcgcgg cggcaagggc 420 aaggccttgc agctcttccg
gagtcacgtg cagccccttt tggctgaggc tgaaatctcc 480 ttcacgctga
tgctcactga gcggcggaac cacgcgcggg agctggtgcg gtcggaggag 540
ctgggccgct gggacgctct ggtggtcatg tctggagacg ggctgatgca cgaggtggtg
600 aacgggctca tggagcggcc tgactgggag accgccatcc agaagcccct
gtgtagcctc 660 ccagcaggct ctggcaacgc gctggcagct tccttgaacc
attatgctgg ctatgagcag 720 gtcaccaatg aagacctcct gaccaactgc
acgctattgc tgtgccgccg gctgctgtca 780 cccatgaacc tgctgtctct
gcacacggct tcggggctgc gcctcttctc tgtgctcagc 840 ctggcctggg
gcttcattgc tgatgtggac ctagagagtg agaagtatcg gcgtctgggg 900
gagatgcgct tcactctggg caccttcctg cgtctggcag ccctgcgcac ctaccgcggc
960 cgactggcct acctccctgt aggaagagtg ggttccaaga cacctgcctc
ccccgttgtg 1020 gtccagcagg gcccggtaga tgcacacctt gtgccactgg
aggagccagt gccctctcac 1080 tggacaatgg tgcccgacga ggactttgtg
ctaatcctgg cactgctgca ctcgcacctg 1140 ggcagtgaga tgtttgctgc
acccatgggc cgctgtgcag ctggcgtcat gcatctgttc 1200 tacgtgcggg
cgggagtgtc tcgtgccatg ctgctgcgct tcttcctggc catggagaag 1260
ggcaggcata tggagtatga atgcccctac ttggtatatg tgcccgtggt cgccttccgc
1320 ttggagccca aggatgggaa aggtgtgttt gcagtggatg gggaattgat
ggttagcgag 1380 gccgtgcagg gccaggtgca cccaaactac ttctggatgg
tcagtggttg cgtggagccc 1440 ccgcccagct ggaagcccca gcagatgcca
ccgccagaag agcccttatg acccctgggc 1500 cacgctgtgc cttagtgtct
acttgcagga cccttcctcc ttccctaggg ctgcagggcc 1560 tgtccacagt
tcctgtgggg gtggaggaga ctcctctgga gaagggtgag aaggtggagg 1620
ttatgctttg gggggacagg ccagaatgaa gtcctgggtc aggagcccag ctggctgggc
1680 ccagctgcct atgtaaggcc ttctagtttg ttctgagacc cccaccccac
gaaccaaatc 1740 caaataaagt gacattccca gcctgaaaaa aaaaaaaaaa aaa
1783 2 1272 DNA Homo sapiens 2 cctgggcagc accgataagg agctgaaggc
aggagccgcc gccacgggca gcgcccccac 60 agcgccaggg accccctggc
agcgggagcc gcgggtcgag gttatggatc cagcgggcgg 120 cccccggggc
gtgctcccgc ggccctgccg cgtgctggtg ctgctgaacc cgcgcggcgg 180
caagggcaag gccttgcagc tcttccggag tcacgtgcag ccccttttgg ctgaggctga
240 aatctccttc acgctgatgc tcactgagcg gcggaaccac gcgcgggagc
tggtgcggtc 300 ggaggagctg ggccgctggg acgctctggt ggtcatgtct
ggagacgggc tgatgcacga 360 ggtggtgaac gggctcatgg agcggcctga
ctgggagacc gccatccaga agcccctgtg 420 tagcctccca gcaggctctg
gcaacgcgct ggcagcttcc ttgaaccatt atgctggcta 480 tgagcaggtc
accaatgaag acctcctgac caactgcacg ctattgctgt gccgccggct 540
gctgtcaccc atgaacctgc tgtctctgca cacggcttcg gggctgcgcc tcttctctgt
600 gctcagcctg gcctggggct tcattgctga tgtggaccta gagagtgaga
agtatcggcg 660 tctgggggag atgcgcttca ctctgggcac cttcctgcgt
ctggcagccc tgcgcaccta 720 ccgcggccga ctggcctacc tccctgtagg
aagagtgggt tccaagacac ctgcctcccc 780 cgttgtggtc cagcagggcc
cggtagatgc acaccttgtg ccactggagg agccagtgcc 840 ctctcactgg
acagtggtgc ccgacgagga ctttgtgcta gtcctggcac tgctgcactc 900
gcacctgggc agtgagatgt ttgctgcacc catgggccgc tgtgcagctg gcgtcatgca
960 tctgttctac gtgcgggcgg gagtgtctcg tgccatgctg ctgcgcctct
tcctggccat 1020 ggagaagggc aggcatatgg agtatgaatg cccctacttg
gtatatgtgc ccgtggtcgc 1080 cttccgcttg gagcccaagg atgggaaagg
tgtgtttgca gtggatgggg aattgatggt 1140 tagcgaggcc gtgcagggcc
aggtgcaccc aaactacttc tggatggtca gcggttgcgt 1200 ggagcccccg
cccagctgga agccccagca gatgccaccg ccagaagagc ccttatgacc 1260
cctgggccac gc 1272 3 1857 DNA Homo sapiens 3 atggccccgc ccccaccgcc
actggctgcc agcaccccgc tcctccatgg cgagtttggc 60 tcctacccag
cccgaggccc acgctttgcc ctcaccctta catcgcaggc cctgcacata 120
cagcggctgc gccccaaacc tgaagccagg ccccggggtg gcctggtccc gttggccgag
180 gtctcaggct gctgcaccct gcgaagccgc agcccctcag actcagcggc
ctacttctgc 240 atctacacct accctcgggg ccggcgcggg gcccggcgca
gagccactcg caccttccgg 300 gcagatgggg ccgccaccta cgaagagaac
cgtgccgagg cccagcgctg ggccactgcc 360 ctcacctgtc tgctccgagg
actgccactg cccggggatg gggagatcac ccctgacctg 420 ctacctcggc
cgccccggtt gcttctattg gtcaatccct ttgggggtcg gggcctggcc 480
tggcagtggt gtaagaacca cgtgcttccc atgatctctg aagctgggct gtccttcaac
540 ctcatccaga cagaacgaca gaaccacgcc cgggagctgg tccaggggct
gagcctgagt 600 gagtgggatg gcatcgtcac ggtctcggga gacgggctgc
tccatgaggt gctgaacggg 660 ctcctagatc gccctgactg ggaggaagct
gtgaagatgc ctgtgggcat cctcccctgc 720 ggctcgggca acgcgctggc
cggagcagtg aaccagcacg ggggatttga gccagccctg 780 ggcctcgacc
tgttgctcaa ctgctcactg ttgctgtgcc ggggtggtgg ccacccactg 840
gacctgctct ccgtgacgct ggcctcgggc tcccgctgtt tctccttcct gtctgtggcc
900 tggggcttcg tgtcagatgt ggatatccag agcgagcgct tcagggcctt
gggcagtgcc 960 cgcttcacac tgggcacggt gctgggcctc gccacactgc
acacctaccg cggacgcctc 1020 tcctacctcc ccgccactgt ggaacctgcc
tcgcccaccc ctgcccatag cctgcctcgt 1080 gccaagtcgg agctgaccct
aaccccagac ccagccccgc ccatggccca ctcacccctg 1140 catcgttctg
tgtctgacct gcctcttccc ctgccccagc ctgccctggc ctctcctggc 1200
tcgccagaac ccctgcccat cctgtccctc aacggtgggg gcccagagct ggctggggac
1260 tggggtgggg ctggggatgc tccgctgtcc ccggacccac tgctgtcttc
acctcctggc 1320 tctcccaagg cagctctaca ctcacccgtc tccgaagggg
cccccgtaat tcccccatcc 1380 tctgggctcc cacttcccac ccctgatgcc
cgggtagggg cctccacctg cggcccgccc 1440 gaccacctgc tgcctccgct
gggcaccccg ctgcccccag actgggtgac gctggagggg 1500 gactttgtgc
tcatgttggc catctcgccc agccacctag gcgctgacct ggtggcagct 1560
ccgcatgcgc gcttcgacga cggcctggtg cacctgtgct gggtgcgtag cggcatctcg
1620 cgggctgcgc tgctgcgcct tttcttggcc atggagcgtg gtagccactt
cagcctgggc 1680 tgtccgcagc tgggctacgc cgcggcccgt gccttccgcc
tagagccgct cacaccacgc 1740 ggcgtgctca cagtggacgg ggagcaggtg
gagtatgggc cgctacaggc acagatgcac 1800 cctggcatcg gtacactgct
cactgggcct cctggctgcc cggggcggga gccctga 1857 4 2731 DNA Homo
sapiens 4 ggggaacaaa ggtgagcgaa aggaggaggc agaatccggg cagagggcag
ggagagggcc 60 tgtggggaag ggacctcagt cctgctccca cccgctccct
ggagagcagg cggccagaca 120 cccagaggcc agaccaggag ctgaccggga
gctggggcca cgggcctagg agcaccctgg 180 tcagggctaa ggccatggcc
ccgcccccac cgccactggc tgccagcacc ccgctcctcc 240 atggcgagtt
tggctcctac ccagcccgag gcccacgctt tgccctcacc cttacatcgc 300
aggccctgca catacagcgg ctgcgcccca aacctgaagc caggccccgg ggtggcctgg
360 tcccgttggc cgaggtctca ggctgctgca ccctgcgaag ccgcagcccc
tcagactcag 420 cggcctactt ctgcatctac acctaccctc ggggccggcg
cggggcccgg cgcagagcca 480 ctcgcacctt ccgggcagat ggggccgcca
cctacgaaga gaaccgtgcc gaggcccagc 540 gctgggccac tgccctcacc
tgtctgctcc gaggactgcc actgcccggg gatggggaga 600 tcacccctga
cctgctacct cggccgcccc ggttgcttct attggtcaat ccctttgggg 660
gtcggggcct ggcctggcag tggtgtaaga accacgtgct tcccatgatc tctgaagctg
720 ggctgtcctt caacctcatc cagacagaac gacagaacca cgcccgggag
ctggtccagg 780 ggctgagcct gagtgagtgg gatggcatcg tcacggtctc
gggagacggg ctgctccatg 840 aggtgctgaa cgggctccta gatcgccctg
actgggagga agctgtgaag atgcctgtgg 900 gcatcctccc ctgcggctcg
ggcaacgcgc tggccggagc agtgaaccag cacgggggat 960 ttgagccagc
cctgggcctc gacctgttgc tcaactgctc actgttgctg tgccggggtg 1020
gtggccaccc actggacctg ctctccgtga cgctggcctc gggctcccgc tgtttctcct
1080 tcctgtctgt ggcctggggc ttcgtgtcag atgtggatat ccagagcgag
cgcttcaggg 1140 ccttgggcag tgcccgcttc acactgggca cggtgctggg
cctcgccaca ctgcacacct 1200 accgcggacg cctctcctac ctccccgcca
ctgtggaacc tgcctcgccc acccctgccc 1260 atagcctgcc tcgtgccaag
tcggagctga ccctaacccc agacccagcc ccgcccatgg 1320 cccactcacc
cctgcatcgt tctgtgtctg acctgcctct tcccctgccc cagcctgccc 1380
tggcctctcc tggctcgcca gaacccctgc ccatcctgtc cctcaacggt gggggcccag
1440 agctggctgg ggactggggt ggggctgggg atgctccgct gtccccggac
ccactgctgt 1500 cttcacctcc tggctctccc aaggcagctc tacactcacc
cgtctccgaa ggggcccccg 1560 taattccccc atcctctggg ctcccacttc
ccacccctga tgcccgggta ggggcctcca 1620 cctgcggccc gcccgaccac
ctgctgcctc cgctgggcac cccgctgccc ccagactggg 1680 tgacgctgga
gggggacttt gtgctcatgt tggccatctc gcccagccac ctaggcgctg 1740
acctggtggc agctccgcat gcgcgcttcg acgacggcct ggtgcacctg tgctgggtgc
1800 gtagcggcat ctcgcgggct gcgctgctgc gccttttctt ggccatggag
cgtggtagcc 1860 acttcagcct gggctgtccg cagctgggct acgccgcggc
ccgtgccttc cgcctagagc 1920 cgctcacacc acgcggcgtg ctcacagtgg
acggggagca ggtggagtat gggccgctac 1980 aggcacagat gcaccctggc
atcggtacac tgctcactgg gcctcctggc tgcccggggc 2040 gggagccctg
aaactaaaca agcttggtac ccgccggggg cggggcctac attccaatgg 2100
ggcggagcct gagctagggg gtgtggcctg gctgctagag ttgtggtggc aggggccctg
2160 gccccgtctc aggattgcgc tcgctttcat gggaccagac gtgatgctgg
aaggtgggcg 2220 tcgtcacggt taaagagaaa tgggctcgtc ccgagggtag
tgcctgatca atgagggcgg 2280 ggcctggcgt ctgatctggg gccgccctta
cggggcaggg ctcagtcctg acgcttgcca 2340 cctgctccta cccggccagg
atggctgagg gcggagtcta ttttacgcgt cgcccaatga 2400 caggacctgg
aatgtactgg ctggggtagg cctcagtgag tcggccggtc agggcccgca 2460
gcctcgcccc atccactccg gtgcctccat ttagctggcc aatcagccca ggaggggcag
2520 gttccccggg gccggcgcta ggatttgcac taatgttcct ctccccgcgg
gtgggggcgg 2580 ggaaattcat atcccctgtt cgtctcatgc gcgtcctccg
tccccaatct aaaaagcaat 2640 tgaaaaggtc tatgcaataa aggcagtcgc
ttcattcctc tcaaaaaaaa aaaaaaaaaa 2700 aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa a 2731 5 1857 DNA Homo sapiens 5 atggccccgc ccccaccgcc
actggctgcc agcaccccgc tcctccatgg cgagtttggc 60 tcctacccag
cccgaggccc acgctttgcc ctcaccctta catcgcaggc cctgcacata 120
cagcggctgc gccccaaacc tgaagccagg ccccggggtg gcctggtccc gttggccgag
180 gtctcaggct gctgcaccct gcgaagccgc agcccctcag actcagcggc
ctacttctgc 240 atctacacct accctcgggg ccggcgcggg gcccggcgca
gagccactcg caccttccgg 300 gcagatgggg ccgccaccta cgaagagaac
cgtgccgagg cccagcgctg ggccactgcc 360 ctcacctgtc tgctccgagg
actgccactg cccggggatg gggagatcac ccctgacctg 420 ctacctcggc
cgccccggtt gcttctattg gtcaatccct ttgggggtcg gggcctggcc 480
tggcagtggt gtaagaacca cgtgcttccc atgatctctg aagctgggct gtccttcaac
540 ctcatccaga cagaacgaca gaaccacgcc cgggagctgg tccaggggct
gagcctgagt 600 gagtgggatg gcatcgtcac ggtctcggga gacgggctgc
tccatgaggt gctgaacggg 660 ctcctagatc gccctgactg ggaggaagct
gtgaagatgc ctgtgggcat cctcccctgc 720 ggctcgggca acgcgctggc
cggagcagtg aaccagcacg ggggatttga gccagccctg 780 ggcctcgacc
tgttgctcaa ctgctcactg ttgctgtgcc ggggtggtgg ccacccactg 840
gacctgctct ccgtgacgct ggcctcgggc tcccgctgtt tctccttcct gtctgtggcc
900 tggggcttcg tgtcagatgt ggatatccag agcgagcgct tcagggcctt
gggcagtgcc 960 cgcttcacac tgggcacggt gctgggcctc gccacactgc
acacctaccg cggacgcctc 1020 tcctacctcc ccgccactgt ggaacctgcc
tcgcccaccc ctgcccatag cctgcctcgt 1080 gccaagtcgg agctgaccct
aaccccagac ccagccccgc ccatggccca ctcacccctg 1140 catcgttctg
tgtctgacct gcctcttccc ctgccccagc ctgccctggc ctctcctggc 1200
tcgccagaac ccctgcccat cctgtccctc aacggtgggg gcccagagct ggctggggac
1260 tggggtgggg ctggggatgc tccgctgtcc ccggacccac tgctgtcttc
acctcctggc 1320 tctcccaagg cagctctaca ctcacccgtc tccgaagggg
cccccgtaat tcccccatcc 1380 tctgggctcc cacttcccac ccctgatgcc
cgggtagggg cctccacctg cggcccgccc 1440 gaccacctgc tgcctccgct
gggcaccccg ctgcccccag actgggtgac gctggagggg 1500 gactttgtgc
tcatgttggc catctcgccc agccacctag gcgctgacct ggtggcagct 1560
ccgcatgcgc gcttcgacga cggcctggtg cacctgtgct gggtgcgtag cggcatctcg
1620 cgggctgcgc tgctgcgcct tttcttggcc atggagcgtg gtagccactt
cagtctgggc 1680 tgtccgcagc tgggctacgc cgcggcccgt gccttccgcc
tagagccgct cacaccacgc 1740 ggcgtgctca cagtggacgg ggagcaggtg
gagtatgggc cgctacaggc acagatgcac 1800 cctggcatcg gtacactgct
cactgggcct cctggctgcc cggggcggga gccctga 1857 6 2875 DNA Homo
sapiens 6 agtgttggag gtgaggaggc ggggctggca gggctagtcg gggcatctgg
aaatttccga 60 ccccacgctt cgggcgtttc cttatcaggt tcaccgctcc
ctgatctcgc gctgcacttc 120 gtaggcgcag ccgctgcttg ggaagtccta
cttaagagct gaaggtcagg ccaggacagt 180 gagacctgac tccttgctcc
taccagccta ctatggctta agacccaggg ccagggtccc 240 gttgatgtaa
cagagcagag gaccagcaga tgaatggaca ccttgaagca gaggagcagc 300
aggaccagag gccagaccag gagctgaccg ggagctgggg ccacgggcct aggagcaccc
360 tggtcagggc taaggccatg gccccgcccc caccgccact ggctgccagc
acctcgctcc 420 tccatggcga gtttggctcc tacccagccc gaggcccacg
ctttgccctc acccttacat 480 cgcaggccct gcacatacag cggctgcgcc
ccaaacctga agccaggccc cggggtggcc 540 tggtcccgtt ggccgaggtc
tcaggctgct gcaccctgcg aagccgcagc ccctcagact 600 cagcggccta
cttctgcatc tacacctacc ctcggggccg gcgcggggcc cggcgcagag 660
ccactcgcac cttccgggca gatggggccg ccacctacga agagaaccgt gccgaggccc
720 agcgctgggc cactgccctc acctgtctgc tccgaggact gccactgccc
ggggatgggg 780 agatcacccc tgacctgcta cctcggccgc cccggttgct
tctattggtc aatccctttg 840 ggggtcgggg cctggcctgg cagtggtgta
agaaccacgt gcttcccatg atctctgaag 900 ctgggctgtc cttcaacctc
atccagacag aacgacagaa ccacgcccgg gagctggtcc 960 aggggctgag
cctgagtgag tgggatggca tcgtcacggt ctcgggagac gggctgctcc 1020
atgaggtgct gaacgggctc ctagatcgcc ctgactggga ggaagctgtg aagatgcctg
1080 tgggcatcct cccctgcggc tcgggcaacg cgctggccgg agcagtgaac
cagcacgggg 1140 gatttgagcc agccctgggc ctcgacctgt tgctcaactg
ctcactgttg ctgtgccggg 1200 gtggtggcca cccactggac ctgctctccg
tgacgctggc ctcgggctcc cgctgtttct 1260 ccttcctgtc tgtggcctgg
ggcttcgtgt cagatgtgga tatccagagc gagcgcttca 1320 gggccttggg
cagtgcccgc ttcacactgg gcacggtgct gggcctcgcc acactgcaca 1380
cctaccgcgg acgcctctcc tacctccccg ccactgtgga acctgcctcg cccacccctg
1440 cccatagcct gcctcgtgcc aagtcggagc tgaccctaac cccagaccca
gccccgccca 1500 tggcccactc acccctgcat cgttctgtgt ctgacctgcc
tcttcccctg ccccagcctg 1560 ccctggcctc tcctggctcg ccagaacccc
tgcccatcct gtccctcaac ggtgggggcc 1620 cagagctggc tggggactgg
ggtggggctg gggatgctcc gctgtccccg gacccactgc 1680 tgtcttcacc
tcctggctct cccaaggcag ctctacactc acccgtctcc gaaggggccc 1740
ccgtaattcc cccatcctct gggctcccac ttcccacccc tgatgcccgg gtaggggcct
1800 ccacctgcgg cccgcccgac cacctgctgc ctccgctagg caccccgctg
cccccagact 1860 gggtgacgct ggagggggac tttgtgctca tgttggccat
ctcgcccagc cacctaggcg 1920 ctgacctggt ggcagctccg catgcgcgct
tcgacgacgg cctggtgcac ctgtgctggg 1980 tgcgtagcgg catctcgcgg
gctgcgctgc tgcgcctttt cttggccatg gagcgtggta 2040 gccacttcag
cctgggctgt ccgcagctgg gctacgccgc ggcccgtgcc ttccgcctag 2100
agccgctcac accacgcggc gtgctcacag tggacgggga gcaggtggag tatgggccgc
2160 tacaggcaca gatgcaccct ggcatcggta cactgctcac tgggcctcct
ggctgcccgg 2220 ggcgggagcc ctgaaactaa acaagcttgg tacccgccgg
gggcggggcc tacattccaa 2280 tggggcggag cctgagctag ggggtgtggc
ctggctgcta gagttgtggt ggcaggggcc 2340 ctggccccgt ctcaggattg
cgctcgcttt catgggacca gacgtgatgc tggaaggtgg 2400 gcgtcgtcac
ggttaaagag aaatgggctc gtcccgaggg tagtgcctga tcaatgaggg 2460
cggggcctgg cgtctgatct ggggccgccc ttacggggca gggctcagtc ctgacgcttg
2520 ccacctgctc ctacccggcc aggatggctg agggcggagt ctattttacg
cgtcgcccaa 2580 tgacaggacc tggaatgtac tggctggggt aggcctcagt
gagtcggccg gtcagggccc 2640 gcagcctcgc cccatccact ccggtgcctc
catttagctg gccaatcagc ccaggagggg 2700 caggttcccc ggggccggcg
ctaggatttg cactaatgtt cctctccccg cgggtggggg 2760 cggggaaatt
catatcccct gttcgtctca tgcgcgtcct ccgtccccaa tctaaaaagc 2820
aattgaaaag gtctatgcaa taaaggcagt cgcttcattc ctctcaaaaa aaaaa 2875 7
4171 DNA Homo sapiens 7 gatcatcgcc gttgaggaaa cagacgttca cgggaaacat
caaggcagtg gaaaatggca 60 gaaaatggaa aagccttacg cttttacagt
tcactgtgta aagagagcac gacggcaccg 120 ctggaagtgg gcgcaggtga
ctttctggtg tccagaggag cagctgtgtc acttgtggct 180 gcagaccctg
cgggagatgc tggagaagct gacgtccaga ccaaagcatt tactggtatt 240
tatcaacccg tttggaggaa aaggacaagg caagcggata tatgaaagaa aagtggcacc
300 actgttcacc ttagcctcca tcaccactga catcatcgtt actgaacatg
ctaatcaggc 360 caaggagact ctgtatgaga ttaacataga caaatacgac
ggcatcgtct gtgtcggcgg 420 agatggtatg ttcagcgagg tgctgcacgg
tctgattggg aggacgcaga ggagcgccgg 480 ggtcgaccag aaccaccccc
gggctgtgct ggtccccagt agcctccgga ttggaatcat 540 tcccgcaggg
tcaacggact gcgtgtgtta ctccaccgtg ggcaccagcg acgcagaaac 600
ctcggcgctg catatcgttg ttggggactc gctggccatg gatgtgtcct cagtccacca
660 caacagcaca ctccttcgct actccgtgtc cctgctgggc tacggcttct
acggggacat 720 catcaaggac agtgagaaga aacggtggtt gggtcttgcc
agatacgact tttcaggttt 780 aaagaccttc ctctcccacc actgctatga
agggacagtg tccttcctcc ctgcacaaca 840 cacggtggga tctccaaggg
ataggaagcc ctgccgggca ggatgctttg tttgcaggca 900 aagcaagcag
cagctggagg aggagcagaa gaaagcactg tatggtttgg aagctgcgga 960
ggacgtggag gagtggcaag tcgtctgtgg gaagtttctg gccatcaatg ccacaaacat
1020 gtcctgtgct tgtcgccgga gccccagggg cctctccccg gctgcccact
tgggagacgg 1080 gtcttctgac ctcatcctca tccggaaatg ctccaggttc
aattttctga gatttctcat 1140 caggcacacc aaccagcagg accagtttga
cttcactttt gttgaagttt atcgcgtcaa 1200 gaaattccag tttacgtcga
agcacatgga ggatgaggac agcgacctca aggagggggg 1260 gaagaagcgc
tttgggcaca tttgcagcag ccacccctcc tgctgctgca ccgtctccaa 1320
cagctcctgg aactgcgacg gggaggtcct gcacagccct gccatcgagg tcagagtcca
1380 ctgccagctg gttcgactct ttgcacgagg aattgaagag aatccgaagc
cagactcaca 1440 cagctgagaa gccggcgtcc tgctctcgaa ctgggaaagt
gtgaaaacta tttaagataa 1500 ttattacaga ccaattatgt tgatatatac
atttaaatgt agaaatttat ttttgatagt 1560 taaatcttga ttttagaaga
aaaccctttt gtcaacaatt ttgtgtacat atttggcatt 1620 ttcagttctg
tacgcatctg cgggttgcag cccacgccgc ttactctcag cggatgcagc 1680
tgctcacttg ggggcactgg cctcttaggt tttaacgatg tcaacagtgt agtttagaaa
1740 atggcccgtt agtggctcta ttgcaataat gttagggaca ttatatgatt
tccacgcagg 1800 tcacaccatc tgggcctgag gtagcagtgg gtcactttga
tccactttgc aggacttatt 1860 ctgtaacggt ttgtggccaa gttttgggaa
gtggttgatt ctctttgcct tcatttcacc 1920 ttcctcttcg tttacggtta
ggacatcgct gcttgatcct tacaatactg tgcaactgca 1980 atgcaacgtg
gccctgcttc aggtgatccg cgggaggggc ctccacgcca gcgccgggaa 2040
ggctgctggg gcctccacac ctgcctcatc acggcggcga ggctacgaca atccggctgg
2100 gagcatgacc ttggcgtctg ttctgggagc acagatgata agctctggaa
gctggcagtg 2160 tgtaaagcac tggcaagttt gttactgtta aaatgtcaaa
taccaatgct ttatatcgac 2220 gcgaagtgct taacacagcc gggcttgggg
gcagtcagga ggaagctggc catccgtgga 2280 ggaggggccg gtcctggact
cccgcaggac tcctctgatg cagggcctga agtctgtaca 2340 cgtggtccag
atttgtcctt gtcttttctt cacactgagt tctctatatt
tattgaacat 2400 cttgtccttt taagccagag tagtgtaaac tgcgtctcgg
atgtctgtct tttgcctcga 2460 agccacgatg gatcgctggt ttcctctgca
gcgcgagggc tccggcgacc agaggattct 2520 tcccggaagg cattcctgcc
gcgctccccg gggcacccct caattgtgta ctacgtcctt 2580 gtttagtgtg
tatccgtgcc cacgtagatg atgtctgtaa cgtagttttg tttgaaatat 2640
gagaatatgc ggcttaaact ttgatctgta aggagcgggg ccgtggccgt ttggagcacg
2700 ctgtagacac cgttcctcat gctgccgggt gggttttgca gaagctccct
tagtgatttc 2760 atgtttaaca ggcagcatcc attttcagaa tttcctggca
ttgatttata ttttgaagca 2820 tacaggaaac ttctcgtttc ctcgtttagc
cccacccaga tcaggtgaaa gggcagcttt 2880 aatggtggtt tttatggacc
acattatcag agagcactgt gcaagccaaa tggttcaata 2940 atgaatgaaa
attctgggtg taaagagtaa atatgccctg gctctttcta ccaatgtttg 3000
ctcctggttg gaaagaaacc aaagatttaa gacgggctgc tcttccagac tggctgtgcc
3060 tgcctgtgcc cagcaacctg tgcagccggc agtgtgcctg gtgtcacgcc
aggaggctgt 3120 ggctgctgtg ggccctctgg aattgtgctc ctcacaaagt
ttccccaaaa ggttcttcta 3180 agcctttatt gtccctggta aatgtttccc
ggctgggcgc ggtggctcac gcctgtaatc 3240 ccagcacttt gggaggccga
ggcgggtgga tcacctaagg tcaggagttt gagatcagcc 3300 tgcccaacat
ggtgaaacct cgtctctact aaaaatacac aacttagcca gtcttgttgg 3360
cgcacgcctg taatctcagc tactagggac gctgaggcag gagaatcgct tgaacccaag
3420 aaagaggtgg aggttgcggt gagccaagat tgcgccactg cactccagcc
tgggcaaaca 3480 gagggagact ccatcgcccc cccccaacaa aaaaaaaagt
ttcccataca ctggcctgcc 3540 ccaaaaccca ctaacaattt tagcaaaaca
gtccaggcca aagaggaagc atttcatgtt 3600 caataagaaa cccagccatt
ccgcatggct ggttcctgag tggctctggt gatactctcc 3660 agccacctgc
tgacattgag aatctcagac ctcgggactg ctgttgcggt accgtgtgtc 3720
tgacacctgc cagcagccct ttgctatctg cgcgcaggat gggggtgact gcccagacat
3780 tcccgctaga taggctctga tttccggggc agcctttcag atgcggcaga
catacaacac 3840 ctgtacttta gagttttaag ggaaaaaaaa tcagaagtgc
tggttagata gtaaaaactt 3900 aggataactt agaaaggcta gttttagctt
cctttgtggc tccctggtgc aaaacaatta 3960 gcagttatgc aatggacctg
attctagttt attctaatta agaagtgagg ccgagtttga 4020 cttcgttcct
gaatacaatc ttgagtaact gggaaagtct gagtgaaagg atggcctcat 4080
tctctttcta atcttgctgg tttcaagatt agaaaatggc attatttgat ctgaaatgtt
4140 tgagaagaca cgaataaagt tacttgggca g 4171 8 4407 DNA Homo
sapiens 8 ggccgctaac ggtccggcgc ccctcggcgt ccgcgcgccc ccagcctggc
ggacgagccc 60 ggcggcggag atgggggcga cgggggcggc ggagccgctg
caatccgtgc tgtgggtgaa 120 gcagcagcgc tgcgccgtga gcctggagcc
cgcgcgggct ctgctgcgct ggtggcggag 180 cccggggccc ggagccggcg
cccccggcgc ggatgcctgc tctgtgcctg tatctgagat 240 catcgccgtt
gaggaaacag acgttcacgg gaaacatcaa ggcagtggaa aatggcagaa 300
aatggaaaag ccttacgctt ttacagttca ctgtgtaaag agagcacgac ggcaccgctg
360 gaagtgggcg caggtgactt tctggtgtcc agaggagcag ctgtgtcact
tgtggctgca 420 gaccctgcgg gagatgctgg agaagctgac gtccagacca
aagcatttac tggtatttat 480 caacccgttt ggaggaaaag gacaaggcaa
gcggatatat gaaagaaaag tggcaccact 540 gttcacctta gcctccatca
ccactgacat catcgttact gaacatgcta atcaggccaa 600 ggagactctg
tatgagatta acatagacaa atacgacggc atcgtctgtg tcggcggaga 660
tggtatgttc agcgaggtgc tgcacggtct gattgggagg acgcagagga gcgccggggt
720 cgaccagaac cacccccggg ctgtgctggt ccccagtagc ctccggattg
gaatcattcc 780 cgcagggtca acggactgcg tgtgttactc caccgtgggc
accagcgacg cagaaacctc 840 ggcgctgcat atcgttgttg gggactcgct
ggccatggat gtgtcctcag tccaccacaa 900 cagcacactc cttcgctact
ccgtgtccct gctgggctac ggcttctacg gggacatcat 960 caaggacagt
gagaagaaac ggtggttggg tcttgccaga tacgactttt caggtttaaa 1020
gaccttcctc tcccaccact gctatgaagg gacagtgtcc ttcctccctg cacaacacac
1080 ggtgggatct ccaagggata ggaagccctg ccgggcagga tgctttgttt
gcaggcaaag 1140 caagcagcag ctggaggagg agcagaagaa agcactgtat
ggtttggaag ctgcggagga 1200 cgtggaggag tggcaagtcg tctgtgggaa
gtttctggcc atcaatgcca caaacatgtc 1260 ctgtgcttgt cgccggagcc
ccaggggcct ctccccggct gcccacttgg gagacgggtc 1320 ttctgacctc
atcctcatcc ggaaatgctc caggttcaat tttctgagat ttctcatcag 1380
gcacaccaac cagcaggacc agtttgactt cacttttgtt gaagtttatc gcgtcaagaa
1440 attccagttt acgtcgaagc acatggagga tgaggacagc gacctcaagg
agggggggaa 1500 gaagcgcttt gggcacattt gcagcagcca cccctcctgc
tgctgcaccg tctccaacag 1560 ctcctggaac tgcgacgggg aggtcctgca
cagccctgcc atcgaggtca gagtccactg 1620 ccagctggtt cgactctttg
cacgaggaat tgaagagaat ccgaagccag actcacacag 1680 ctgagaagcc
ggcgtcctgc tcacaaactg ggaaagtgtg aaaactattt aagataatta 1740
ttacagacca attatgttga tatatacatt taaatgtaga aatttatttt tgatagttaa
1800 atcttgattt tagaagaaaa cccttttgtc aacaattttg tgtacatatt
tggcattttc 1860 agttctgtac gcatctgcgg gttgcagccc acgccgctta
ctctcagcgg atgcagctgc 1920 tcacttgggg gcactggcct cttaggtttt
aacgatgtca acagtgtagt ttagaaaatg 1980 gcccgttagt ggctctattg
caataatgtt agggacatta tatgatttcc acgcaggtca 2040 caccatctgg
gcctgaggta gcagtgggtc actttgatcc actttgcagg acttattctg 2100
taacggtttg tggccaagtt ttgggaagtg gttgattctc tttgccttca tttcaccttc
2160 ctcttcgttt acggttagga catcgctgct tgatccttac aatactgtgc
aactgcaatg 2220 caacgtggcc ctgcttcagg tgatccgcgg gaggggcctc
cacgccagcg ccgggaaggc 2280 tgctggggcc tccacacctg cctcatcacg
gcggcgaggc tacgacaatc cggctgggag 2340 catgaccttg gcgtctgttc
tgggagcacg gatgataagc tctggaagct ggcagtgtgt 2400 aaagcactgg
caagtttgtt actgttaaaa tgtcaaatac caatgcttta tatcgacgcg 2460
aagtgcttaa cacagccggg cttgggggca gtcaggagga agctggccat ccgtggagga
2520 ggggccggtc ctggactccc gcaggactcc tctgaggcag ggcctgaagt
ctgtacacgt 2580 ggtccagatt tgtccttgtc ttttcttcac actgagttct
ctatatttat tgaacatctt 2640 gtccttttaa gccagagtag tgtaaactgc
gtctcggatg tctgtctttt gcctcgaagc 2700 cacgatggat cgctggtttc
ctctgcagcg cgagggctcc ggcgaccaga ggattcttcc 2760 cggaaggcat
tcctgccgcg ctccccgggg cacccctcaa ttgtgtacta cgtccttgtt 2820
tagtgtgtat ccgtgcccac gtagatgatg tctgtaacgt agttttgttt gaaatatgag
2880 aatatgcggc ttaaactttg atctgtaagg agcggggccg tggccgtttg
gagcacgctg 2940 tagacaccgt tcctcatgct gccgggtggg ttttgcagaa
gctcccttag tgatttcatg 3000 tttaacaggc agcatccatt ttcagaattt
cctggcattg atttatattt tgaagcatac 3060 aggaaacttc tcgtttcctc
gtttagcccc acccagatca ggtgaaaggg cagctttaat 3120 ggtggttttt
atggaccaca ttatcagaga gcactgtgca agccaaatgg ttcaataatg 3180
aatgaaaatt ctgggtgtaa agagtaaata tgccctggct ctttctacca atgtttgctc
3240 ctggttggaa agaaaccaaa gatttaagac gggctgctct tccagactgg
ctgtgcctgc 3300 ctgtgcccag caacctgtgc agccggcagt gtgcctggtg
tcacgccagg aggctgtggc 3360 tgctgtgggc cctctggaat tgtgctcctc
acaaagtttc cccaaaaggt tcttctaagc 3420 ctttattgtc cctggtaaat
gtttcccggc tgggcgcggt ggctcacgcc tgtaatccca 3480 gcactttggg
aggccgaggc gggtggatca cctaaggtca ggagtttgag atcagcctgc 3540
ccaacatggt gaaacctcgt ctctactaaa aatacacaac ttagccagtc ttgttggcgc
3600 acgcctgtaa tctcagctac tagggatgct gaggcaggag aatcgcttga
acccaagaaa 3660 gaggtggagg ttgcggtgag ccaagattgc gccactgcac
tccagcctgg gcaaacagag 3720 ggagactcca tcgccccccc caacaaaaaa
aaaagtttcc catacactgg cctgccccaa 3780 aacccactaa caattttagc
aaaacagtcc aggccaaaga ggaagcattt catgttcaat 3840 aagaaaccca
gccattccgc atggctggtt cctgagtggc tctggtgata ctctccagcc 3900
acctgctgac attcagaatc tcagacctcg ggactgctgt tgcggtaccg tgtgtctgac
3960 acctgccagc agccctttgc tatctgcgcg caggatgggg gtgactgccc
agacattccc 4020 gctagatagg ctctgatttc cggggcagcc tttcagatgc
ggcagacata caacacctgt 4080 actttagagt tttaagggaa aaaaaatcag
aagtgctggt tagatagtaa aaacttagga 4140 taacttagaa aggctagttt
tagcttcctt tgtggctccc tggtgcaaaa caattagcag 4200 ttatgcaatg
gacctgattc tagtttattc taattaagaa gtgaggccga gtttgacttc 4260
gttcctgaat acaatcttga gtaactggga aagtctgagt gaaaggatgg cctcattctc
4320 tttctaatct tgctggtttc aagattagaa aatggcatta tttgatctga
aatgtttgag 4380 aagacacgaa taaagttact tgggcag 4407 9 2619 DNA Homo
sapiens 9 gattttagaa gaaaaccctt ttgtcaacaa ttttgtgtac atatttggca
ttttcagttc 60 tgtacgcatc tgcgggttgc agcccacgcc gcttactctc
agcggatgca gctgctcact 120 tgggggcact ggcctcttag gttttaacga
tgtcaacagt gtagtttaga aaatggcccg 180 ttagtggctc tattgcaata
atgttaggga cattatatga tttccacgca ggtcacacca 240 tctgggcctg
aggtagcagt gggtcacttt gatccacttt gcaggactta ttctgtaacg 300
gtttgtggcc aagttttggg aagtggttga ttctctttgc cttcatttca ccttcctctt
360 cgtttacggt taggacatcg ctgcttgatc cttacaatac tgtgcaactg
caatgcaacg 420 tggccctgct tcaggtgatc cgcgggaggg gcctccacgc
cagcgccggg aaggctgctg 480 gggcctccac acctgcctca tcacggcggc
gaggctacga caatccggct gggagcatga 540 ccttggcgtc tgttctggga
gcacggatga taagctctgg aagctggcag tgtgtaaagc 600 actggcaagt
ttgttactgt taaaatgtca aataccaatg ctttatatcg acgcgaagtg 660
cttaacacag ccgggcttgg gggcagtcag gaggaagctg gccatccgtg gaggaggggc
720 cggtcctgga ctcccgcagg actcctctga ggcagggcct gaagtctgta
cacgtggtcc 780 agatttgtcc ttgtcttttc ttcacactga gttctctata
tttattgaac atcttgtcct 840 tttaagccag agtagtgtaa actgcgtctc
ggatgtctgt cttttgcctc gaagccacga 900 tggatcgctg gtttcctctg
cagcgcgagg gctccggcga ccagaggatt cttcccggaa 960 ggcattcctg
ccgcgctccc cggggcaccc ctcaattgtg tactacgtcc ttgtttagtg 1020
tgtatccgtg cccacgtaga tgatgtctgt aacgtagttt tgtttgaaat atgagaatat
1080 gcggcttaaa ctttgatctg taaggagcgg ggccgtggcc gtttggagca
cgctgtagac 1140 accgttcctc atgctgccgg gtgggttttg cagaagctcc
cttagtgatt tcatgtttaa 1200 caggcagcat ccatttccag aatttcctgg
cattgattta tattttgaag catacaggaa 1260 acttctcgtt tcctcgttta
gccccaccca gatcaggtga aagggcagct ttaatggtgg 1320 tttttatgga
ccacattatc agagagcact gtgcaagcca aatggttcaa taatgaatga 1380
aaattctggg tgtaaagagt aaatatgccc tggctctttc taccaatgtt tgctcctggt
1440 tggaaagaaa ccaaagattt aagacgggct gctcttccag actggctgtg
cctgcctgtg 1500 cccagcaacc tgtgcagccg gcagtgtgcc tggtgtcacg
ccaggaggct gtggctgctg 1560 tgggccctct ggaattgtgc tcctcacaaa
gtttccccaa aaggttcttc taagccttta 1620 ttgtccctgg taaatgtttc
ccggctgggc gcggtggctc acgcctgtaa tcccagcact 1680 ttgggaggcc
gaggcgggtg gatcacctaa ggtcaggagt ttgagatcag cctgcccaac 1740
atggtgaaac ctcgtctcta ctaaaaatac acaacttagc cagtcttgtt ggcgcacgcc
1800 tgtaatctca gctactaggg atgctgaggc aggagaatcg cttgaaccca
agaaagaggt 1860 ggaggttgcg gtgagccaag attgcgccac tgcactccag
cctgggcaaa cagagggaga 1920 ctccatcgcc ccccccaaca aaaaaaaaag
tttcccatac actggcctgc cccaaaaccc 1980 actaacaatt ttagcaaaac
agtccaggcc aaagaggaag catttcatgt tcaataagaa 2040 acccagccat
tccgcatggc tggttcctga gtggctctgg tgatactctc cagccacctg 2100
ctgacattca gaatctcaga cctcgggact gctgttgcgg taccgtgtgt ctgacacctg
2160 ccagcagccc tttgctatct gcgcgcagga tgggggtgac tgcccagaca
ttcccgctag 2220 ataggctctg atttccgggg cagcctttca gatgcggcag
acatacaaca cctgtacttt 2280 agagttttaa gggaaaaaaa atcagaagtg
ctggttagat agtaaaaact taggataact 2340 tagaaaggct agttttagct
tcctttgtgg ctccctggtg caaaacaatt agcagttatg 2400 caatggacct
gattctagtt tattctaatt aagaagtgag gccgagtttg acttcgttcc 2460
tgaatacaat cttgagtaac tgggaaagtc tgagtgaaag gatggcctca ttctctttct
2520 aatcttgctg gtttcaagat tagaaaatgg cattatttga tctgaaatgt
ttgagaagac 2580 acgaataaag ttacttgggc agaaaaaaaa aaaaaaaaa 2619 10
384 PRT Homo sapiens 10 Met Asp Pro Ala Gly Gly Pro Arg Gly Val Leu
Pro Arg Pro Cys Arg 1 5 10 15 Val Leu Val Leu Leu Asn Pro Arg Gly
Gly Lys Gly Lys Ala Leu Gln 20 25 30 Leu Phe Arg Ser His Val Gln
Pro Leu Leu Ala Glu Ala Glu Ile Ser 35 40 45 Phe Thr Leu Met Leu
Thr Glu Arg Arg Asn His Ala Arg Glu Leu Val 50 55 60 Arg Ser Glu
Glu Leu Gly Arg Trp Asp Ala Leu Val Val Met Ser Gly 65 70 75 80 Asp
Gly Leu Met His Glu Val Val Asn Gly Leu Met Glu Arg Pro Asp 85 90
95 Trp Glu Thr Ala Ile Gln Lys Pro Leu Cys Ser Leu Pro Ala Gly Ser
100 105 110 Gly Asn Ala Leu Ala Ala Ser Leu Asn His Tyr Ala Gly Tyr
Glu Gln 115 120 125 Val Thr Asn Glu Asp Leu Leu Thr Asn Cys Thr Leu
Leu Leu Cys Arg 130 135 140 Arg Leu Leu Ser Pro Met Asn Leu Leu Ser
Leu His Thr Ala Ser Gly 145 150 155 160 Leu Arg Leu Phe Ser Val Leu
Ser Leu Ala Trp Gly Phe Ile Ala Asp 165 170 175 Val Asp Leu Glu Ser
Glu Lys Tyr Arg Arg Leu Gly Glu Met Arg Phe 180 185 190 Thr Leu Gly
Thr Phe Leu Arg Leu Ala Ala Leu Arg Thr Tyr Arg Gly 195 200 205 Arg
Leu Ala Tyr Leu Pro Val Gly Arg Val Gly Ser Lys Thr Pro Ala 210 215
220 Ser Pro Val Val Val Gln Gln Gly Pro Val Asp Ala His Leu Val Pro
225 230 235 240 Leu Glu Glu Pro Val Pro Ser His Trp Thr Met Val Pro
Asp Glu Asp 245 250 255 Phe Val Leu Ile Leu Ala Leu Leu His Ser His
Leu Gly Ser Glu Met 260 265 270 Phe Ala Ala Pro Met Gly Arg Cys Ala
Ala Gly Val Met His Leu Phe 275 280 285 Tyr Val Arg Ala Gly Val Ser
Arg Ala Met Leu Leu Arg Phe Phe Leu 290 295 300 Ala Met Glu Lys Gly
Arg His Met Glu Tyr Glu Cys Pro Tyr Leu Val 305 310 315 320 Tyr Val
Pro Val Val Ala Phe Arg Leu Glu Pro Lys Asp Gly Lys Gly 325 330 335
Val Phe Ala Val Asp Gly Glu Leu Met Val Ser Glu Ala Val Gln Gly 340
345 350 Gln Val His Pro Asn Tyr Phe Trp Met Val Ser Gly Cys Val Glu
Pro 355 360 365 Pro Pro Ser Trp Lys Pro Gln Gln Met Pro Pro Pro Glu
Glu Pro Leu 370 375 380 11 618 PRT Homo sapiens 11 Met Ala Pro Pro
Pro Pro Pro Leu Ala Ala Ser Thr Pro Leu Leu His 1 5 10 15 Gly Glu
Phe Gly Ser Tyr Pro Ala Arg Gly Pro Arg Phe Ala Leu Thr 20 25 30
Leu Thr Ser Gln Ala Leu His Ile Gln Arg Leu Arg Pro Lys Pro Glu 35
40 45 Ala Arg Pro Arg Gly Gly Leu Val Pro Leu Ala Glu Val Ser Gly
Cys 50 55 60 Cys Thr Leu Arg Ser Arg Ser Pro Ser Asp Ser Ala Ala
Tyr Phe Cys 65 70 75 80 Ile Tyr Thr Tyr Pro Arg Gly Arg Arg Gly Ala
Arg Arg Arg Ala Thr 85 90 95 Arg Thr Phe Arg Ala Asp Gly Ala Ala
Thr Tyr Glu Glu Asn Arg Ala 100 105 110 Glu Ala Gln Arg Trp Ala Thr
Ala Leu Thr Cys Leu Leu Arg Gly Leu 115 120 125 Pro Leu Pro Gly Asp
Gly Glu Ile Thr Pro Asp Leu Leu Pro Arg Pro 130 135 140 Pro Arg Leu
Leu Leu Leu Val Asn Pro Phe Gly Gly Arg Gly Leu Ala 145 150 155 160
Trp Gln Trp Cys Lys Asn His Val Leu Pro Met Ile Ser Glu Ala Gly 165
170 175 Leu Ser Phe Asn Leu Ile Gln Thr Glu Arg Gln Asn His Ala Arg
Glu 180 185 190 Leu Val Gln Gly Leu Ser Leu Ser Glu Trp Asp Gly Ile
Val Thr Val 195 200 205 Ser Gly Asp Gly Leu Leu His Glu Val Leu Asn
Gly Leu Leu Asp Arg 210 215 220 Pro Asp Trp Glu Glu Ala Val Lys Met
Pro Val Gly Ile Leu Pro Cys 225 230 235 240 Gly Ser Gly Asn Ala Leu
Ala Gly Ala Val Asn Gln His Gly Gly Phe 245 250 255 Glu Pro Ala Leu
Gly Leu Asp Leu Leu Leu Asn Cys Ser Leu Leu Leu 260 265 270 Cys Arg
Gly Gly Gly His Pro Leu Asp Leu Leu Ser Val Thr Leu Ala 275 280 285
Ser Gly Ser Arg Cys Phe Ser Phe Leu Ser Val Ala Trp Gly Phe Val 290
295 300 Ser Asp Val Asp Ile Gln Ser Glu Arg Phe Arg Ala Leu Gly Ser
Ala 305 310 315 320 Arg Phe Thr Leu Gly Thr Val Leu Gly Leu Ala Thr
Leu His Thr Tyr 325 330 335 Arg Gly Arg Leu Ser Tyr Leu Pro Ala Thr
Val Glu Pro Ala Ser Pro 340 345 350 Thr Pro Ala His Ser Leu Pro Arg
Ala Lys Ser Glu Leu Thr Leu Thr 355 360 365 Pro Asp Pro Ala Pro Pro
Met Ala His Ser Pro Leu His Arg Ser Val 370 375 380 Ser Asp Leu Pro
Leu Pro Leu Pro Gln Pro Ala Leu Ala Ser Pro Gly 385 390 395 400 Ser
Pro Glu Pro Leu Pro Ile Leu Ser Leu Asn Gly Gly Gly Pro Glu 405 410
415 Leu Ala Gly Asp Trp Gly Gly Ala Gly Asp Ala Pro Leu Ser Pro Asp
420 425 430 Pro Leu Leu Ser Ser Pro Pro Gly Ser Pro Lys Ala Ala Leu
His Ser 435 440 445 Pro Val Ser Glu Gly Ala Pro Val Ile Pro Pro Ser
Ser Gly Leu Pro 450 455 460 Leu Pro Thr Pro Asp Ala Arg Val Gly Ala
Ser Thr Cys Gly Pro Pro 465 470 475 480 Asp His Leu Leu Pro Pro Leu
Gly Thr Pro Leu Pro Pro Asp Trp Val 485 490 495 Thr Leu Glu Gly Asp
Phe Val Leu Met Leu Ala Ile Ser Pro Ser His 500 505 510 Leu Gly Ala
Asp Leu Val Ala Ala Pro His Ala Arg Phe Asp Asp Gly 515 520 525 Leu
Val His Leu Cys Trp Val Arg Ser Gly Ile Ser Arg Ala Ala Leu 530 535
540 Leu Arg Leu Phe Leu Ala Met Glu Arg Gly Ser His Phe Ser Leu Gly
545 550 555 560 Cys Pro Gln Leu Gly Tyr Ala Ala Ala Arg Ala Phe Arg
Leu Glu Pro 565 570 575 Leu Thr Pro Arg Gly Val Leu Thr Val Asp Gly
Glu Gln Val Glu Tyr 580 585 590 Gly Pro Leu Gln Ala Gln Met His Pro
Gly Ile Gly Thr Leu Leu Thr
595 600 605 Gly Pro Pro Gly Cys Pro Gly Arg Glu Pro 610 615 12 481
PRT Homo sapiens 12 Ile Ile Ala Val Glu Glu Thr Asp Val His Gly Lys
His Gln Gly Ser 1 5 10 15 Gly Lys Trp Gln Lys Met Glu Lys Pro Tyr
Ala Phe Thr Val His Cys 20 25 30 Val Lys Arg Ala Arg Arg His Arg
Trp Lys Trp Ala Gln Val Thr Phe 35 40 45 Trp Cys Pro Glu Glu Gln
Leu Cys His Leu Trp Leu Gln Thr Leu Arg 50 55 60 Glu Met Leu Glu
Lys Leu Thr Ser Arg Pro Lys His Leu Leu Val Phe 65 70 75 80 Ile Asn
Pro Phe Gly Gly Lys Gly Gln Gly Lys Arg Ile Tyr Glu Arg 85 90 95
Lys Val Ala Pro Leu Phe Thr Leu Ala Ser Ile Thr Thr Asp Ile Ile 100
105 110 Val Thr Glu His Ala Asn Gln Ala Lys Glu Thr Leu Tyr Glu Ile
Asn 115 120 125 Ile Asp Lys Tyr Asp Gly Ile Val Cys Val Gly Gly Asp
Gly Met Phe 130 135 140 Ser Glu Val Leu His Gly Leu Ile Gly Arg Thr
Gln Arg Ser Ala Gly 145 150 155 160 Val Asp Gln Asn His Pro Arg Ala
Val Leu Val Pro Ser Ser Leu Arg 165 170 175 Ile Gly Ile Ile Pro Ala
Gly Ser Thr Asp Cys Val Cys Tyr Ser Thr 180 185 190 Val Gly Thr Ser
Asp Ala Glu Thr Ser Ala Leu His Ile Val Val Gly 195 200 205 Asp Ser
Leu Ala Met Asp Val Ser Ser Val His His Asn Ser Thr Leu 210 215 220
Leu Arg Tyr Ser Val Ser Leu Leu Gly Tyr Gly Phe Tyr Gly Asp Ile 225
230 235 240 Ile Lys Asp Ser Glu Lys Lys Arg Trp Leu Gly Leu Ala Arg
Tyr Asp 245 250 255 Phe Ser Gly Leu Lys Thr Phe Leu Ser His His Cys
Tyr Glu Gly Thr 260 265 270 Val Ser Phe Leu Pro Ala Gln His Thr Val
Gly Ser Pro Arg Asp Arg 275 280 285 Lys Pro Cys Arg Ala Gly Cys Phe
Val Cys Arg Gln Ser Lys Gln Gln 290 295 300 Leu Glu Glu Glu Gln Lys
Lys Ala Leu Tyr Gly Leu Glu Ala Ala Glu 305 310 315 320 Asp Val Glu
Glu Trp Gln Val Val Cys Gly Lys Phe Leu Ala Ile Asn 325 330 335 Ala
Thr Asn Met Ser Cys Ala Cys Arg Arg Ser Pro Arg Gly Leu Ser 340 345
350 Pro Ala Ala His Leu Gly Asp Gly Ser Ser Asp Leu Ile Leu Ile Arg
355 360 365 Lys Cys Ser Arg Phe Asn Phe Leu Arg Phe Leu Ile Arg His
Thr Asn 370 375 380 Gln Gln Asp Gln Phe Asp Phe Thr Phe Val Glu Val
Tyr Arg Val Lys 385 390 395 400 Lys Phe Gln Phe Thr Ser Lys His Met
Glu Asp Glu Asp Ser Asp Leu 405 410 415 Lys Glu Gly Gly Lys Lys Arg
Phe Gly His Ile Cys Ser Ser His Pro 420 425 430 Ser Cys Cys Cys Thr
Val Ser Asn Ser Ser Trp Asn Cys Asp Gly Glu 435 440 445 Val Leu His
Ser Pro Ala Ile Glu Val Arg Val His Cys Gln Leu Val 450 455 460 Arg
Leu Phe Ala Arg Gly Ile Glu Glu Asn Pro Lys Pro Asp Ser His 465 470
475 480 Ser 13 537 PRT Homo sapiens 13 Met Gly Ala Thr Gly Ala Ala
Glu Pro Leu Gln Ser Val Leu Trp Val 1 5 10 15 Lys Gln Gln Arg Cys
Ala Val Ser Leu Glu Pro Ala Arg Ala Leu Leu 20 25 30 Arg Trp Trp
Arg Ser Pro Gly Pro Gly Ala Gly Ala Pro Gly Ala Asp 35 40 45 Ala
Cys Ser Val Pro Val Ser Glu Ile Ile Ala Val Glu Glu Thr Asp 50 55
60 Val His Gly Lys His Gln Gly Ser Gly Lys Trp Gln Lys Met Glu Lys
65 70 75 80 Pro Tyr Ala Phe Thr Val His Cys Val Lys Arg Ala Arg Arg
His Arg 85 90 95 Trp Lys Trp Ala Gln Val Thr Phe Trp Cys Pro Glu
Glu Gln Leu Cys 100 105 110 His Leu Trp Leu Gln Thr Leu Arg Glu Met
Leu Glu Lys Leu Thr Ser 115 120 125 Arg Pro Lys His Leu Leu Val Phe
Ile Asn Pro Phe Gly Gly Lys Gly 130 135 140 Gln Gly Lys Arg Ile Tyr
Glu Arg Lys Val Ala Pro Leu Phe Thr Leu 145 150 155 160 Ala Ser Ile
Thr Thr Asp Ile Ile Val Thr Glu His Ala Asn Gln Ala 165 170 175 Lys
Glu Thr Leu Tyr Glu Ile Asn Ile Asp Lys Tyr Asp Gly Ile Val 180 185
190 Cys Val Gly Gly Asp Gly Met Phe Ser Glu Val Leu His Gly Leu Ile
195 200 205 Gly Arg Thr Gln Arg Ser Ala Gly Val Asp Gln Asn His Pro
Arg Ala 210 215 220 Val Leu Val Pro Ser Ser Leu Arg Ile Gly Ile Ile
Pro Ala Gly Ser 225 230 235 240 Thr Asp Cys Val Cys Tyr Ser Thr Val
Gly Thr Ser Asp Ala Glu Thr 245 250 255 Ser Ala Leu His Ile Val Val
Gly Asp Ser Leu Ala Met Asp Val Ser 260 265 270 Ser Val His His Asn
Ser Thr Leu Leu Arg Tyr Ser Val Ser Leu Leu 275 280 285 Gly Tyr Gly
Phe Tyr Gly Asp Ile Ile Lys Asp Ser Glu Lys Lys Arg 290 295 300 Trp
Leu Gly Leu Ala Arg Tyr Asp Phe Ser Gly Leu Lys Thr Phe Leu 305 310
315 320 Ser His His Cys Tyr Glu Gly Thr Val Ser Phe Leu Pro Ala Gln
His 325 330 335 Thr Val Gly Ser Pro Arg Asp Arg Lys Pro Cys Arg Ala
Gly Cys Phe 340 345 350 Val Cys Arg Gln Ser Lys Gln Gln Leu Glu Glu
Glu Gln Lys Lys Ala 355 360 365 Leu Tyr Gly Leu Glu Ala Ala Glu Asp
Val Glu Glu Trp Gln Val Val 370 375 380 Cys Gly Lys Phe Leu Ala Ile
Asn Ala Thr Asn Met Ser Cys Ala Cys 385 390 395 400 Arg Arg Ser Pro
Arg Gly Leu Ser Pro Ala Ala His Leu Gly Asp Gly 405 410 415 Ser Ser
Asp Leu Ile Leu Ile Arg Lys Cys Ser Arg Phe Asn Phe Leu 420 425 430
Arg Phe Leu Ile Arg His Thr Asn Gln Gln Asp Gln Phe Asp Phe Thr 435
440 445 Phe Val Glu Val Tyr Arg Val Lys Lys Phe Gln Phe Thr Ser Lys
His 450 455 460 Met Glu Asp Glu Asp Ser Asp Leu Lys Glu Gly Gly Lys
Lys Arg Phe 465 470 475 480 Gly His Ile Cys Ser Ser His Pro Ser Cys
Cys Cys Thr Val Ser Asn 485 490 495 Ser Ser Trp Asn Cys Asp Gly Glu
Val Leu His Ser Pro Ala Ile Glu 500 505 510 Val Arg Val His Cys Gln
Leu Val Arg Leu Phe Ala Arg Gly Ile Glu 515 520 525 Glu Asn Pro Lys
Pro Asp Ser His Ser 530 535
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