U.S. patent application number 11/782458 was filed with the patent office on 2008-04-10 for sphingosine-1-phosphate lyase polypeptides, polynucleotides and modulating agents and methods of use therefor.
This patent application is currently assigned to CHILDREN'S HOSPITAL & RESEARCH CENTER AT OAKLAND. Invention is credited to Henrik Fyrst, Julie D. Saba.
Application Number | 20080085995 11/782458 |
Document ID | / |
Family ID | 25472928 |
Filed Date | 2008-04-10 |
United States Patent
Application |
20080085995 |
Kind Code |
A1 |
Saba; Julie D. ; et
al. |
April 10, 2008 |
SPHINGOSINE-1-PHOSPHATE LYASE POLYPEPTIDES, POLYNUCLEOTIDES AND
MODULATING AGENTS AND METHODS OF USE THEREFOR
Abstract
Compositions, methods and kits for diagnosing and treating
cancer are provided. Therapeutic compositions may comprise agents
that modulate the expression or activity of a
sphingosine-1-phosphate lyase (SPL). Such compositions may be
administered to a mammal afflicted with cancer. Diagnostic methods
and kits may employ an agent suitable for detecting alterations in
endogenous SPL. Such methods and kits may be used to detect the
presence of a cancer or to evaluate the prognosis of a known
disease. SPL polypeptides, polynucleotides and antibodies are also
provided.
Inventors: |
Saba; Julie D.; (Oakland,
CA) ; Fyrst; Henrik; (Alameda, CA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 5400
SEATTLE
WA
98104
US
|
Assignee: |
CHILDREN'S HOSPITAL & RESEARCH
CENTER AT OAKLAND
5700 Martin Luther King Jr. Way
Oakland
CA
94609-1673
|
Family ID: |
25472928 |
Appl. No.: |
11/782458 |
Filed: |
July 24, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10979085 |
Nov 1, 2004 |
7262044 |
|
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11782458 |
Jul 24, 2007 |
|
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|
10053510 |
Jan 17, 2002 |
6830881 |
|
|
10979085 |
Nov 1, 2004 |
|
|
|
09356643 |
Jul 19, 1999 |
6569666 |
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10053510 |
Jan 17, 2002 |
|
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08939309 |
Sep 29, 1997 |
6423527 |
|
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09356643 |
Jul 19, 1999 |
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Current U.S.
Class: |
536/23.1 ;
435/320.1 |
Current CPC
Class: |
Y10S 435/975 20130101;
A61P 35/00 20180101; A61P 43/00 20180101; C12N 9/88 20130101; A01K
2217/075 20130101; C12Q 2600/136 20130101; C12Q 1/6886 20130101;
Y10S 435/81 20130101; A01K 2217/05 20130101 |
Class at
Publication: |
536/023.1 ;
435/320.1 |
International
Class: |
C07H 21/02 20060101
C07H021/02; C12N 15/00 20060101 C12N015/00 |
Claims
1.-30. (canceled)
31. An isolated polynucleotide comprising the polynucleotide
sequence of SEQ ID NO:9, or the complement thereof.
32. An isolated polynucleotide encoding the polypeptide of SEQ ID
NO:10, or a portion of such a polypeptide that has
sphingosine-1-phosphate lyase activity.
33. An isolated polynucleotide comprising at least 100 contiguous
nucleotides of SEQ ID NO:9, or the complement thereof.
34. A recombinant expression vector comprising a polynucleotide
according to any one of claims 31-33.
35. An isolated host cell transformed or transfected with an
expression vector according to claim 34.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/979,085, filed Nov. 1, 2004, now pending;
which is a continuation of U.S. patent application Ser. No.
10/053,510, filed Jan. 17, 2002, now U.S. Pat. No. 6,830,881; which
is a continuation-in-part of U.S. patent application Ser. No.
09/356,643, filed Jul. 19, 1999, now U.S. Pat. No. 6,569,666; which
is a continuation-in-part of U.S. patent application Ser. No.
08/939,309, filed Sep. 29, 1997, now U.S. Pat. No. 6,423,527, which
applications are incorporated herein by reference in their
entirety.
STATEMENT REGARDING SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is
200116.sub.--402C5_SEQUENCE_LISTING.txt. The text file is 95 KB,
was created on Jul. 23, 2007, and is being submitted electronically
via EFS-Web, concurrent with the filing of the specification.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to cancer detection
and therapy. The invention is more particularly related to
sphingosine-1-phosphate lyase polynucleotides and polypeptides, and
to agents that modulate the expression and/or activity of such
polypeptides. Such agents may be used, for example, to diagnose
and/or treat cancers such as breast and colon cancer.
[0005] 2. Description of the Related Art
[0006] Breast cancer is a significant health problem for women in
the United States and throughout the world. Although advances have
been made in detection and treatment of the disease, breast cancer
remains the most common form of cancer, and the second leading
cause of cancer death, in American women. Among African-American
women and women between 15 and 54 years of age, breast cancer is
the leading cause of cancer death. One out of every eight women in
the United States will develop breast cancer, a risk which has
increased 52% during 1950-1990. In 1994, it is estimated that
182,000 new cases of female breast cancer were diagnosed, and
46,000 women died from the disease.
[0007] No vaccine or other universally successful method for the
prevention or treatment of breast cancer is currently available.
Management of the disease currently relies on a combination of
early diagnosis (through routine breast screening procedures) and
aggressive treatment, which may include one or more of a variety of
treatments such as surgery, radiotherapy, chemotherapy and hormone
therapy. The course of treatment for a particular breast cancer is
often selected based on a variety of prognostic parameters,
including an analysis of specific tumor markers. However, the use
of established markers often leads to a result that is difficult to
interpret.
[0008] With current therapies, tumor invasiveness and metastasis is
a critical determinant in the outcome for breast cancer patients.
Although the five year survival for women diagnosed with localized
breast cancer is about 90%, the five year survival drops to 18% for
women whose disease has metastasized. Present therapies are
inadequate for inhibiting tumor invasiveness for the large
population of women with this severe disease.
[0009] Colon cancer is the second most frequently diagnosed
malignancy in the United States as well as the second most common
cause of cancer death. The five-year survival rate for patients
with colorectal cancer detected in an early localized stage is 92%;
unfortunately, only 37% of colorectal cancer is diagnosed at this
stage. The survival rate drops to 64% if the cancer is allowed to
spread to adjacent organs or lymph nodes, and to 7% in patients
with distant metastases.
[0010] The prognosis of colon cancer is directly related to the
degree of penetration of the tumor through the bowel wall and the
presence or absence of nodal involvement, consequently, early
detection and treatment are especially important. Currently,
diagnosis is aided by the use of screening assays for fecal occult
blood, sigmoidoscopy, colonoscopy and double contrast barium
enemas. Treatment regimens are determined by the type and stage of
the cancer, and include surgery, radiation therapy and/or
chemotherapy. Recurrence following surgery (the most common form of
therapy) is a major problem and is often the ultimate cause of
death. In spite of considerable research into therapies for the
disease, colon cancer remains difficult to diagnose and treat. In
spite of considerable research into therapies for these and other
cancers, colon cancer remains difficult to diagnose and treat
effectively. Accordingly, improvements are needed in the treatment,
diagnosis and prevention of breast and colon cancer. The present
invention fulfills this need and further provides other related
advantages.
BRIEF SUMMARY OF THE INVENTION
[0011] Briefly stated, the present invention provides compositions
and methods for the diagnosis and therapy of cancer. Within one
aspect, the present invention provides isolated polynucleotides
comprising a sequence selected from the group consisting of: (a) a
sequence shown in SEQ ID NO:15; (b) nucleotide sequences that
hybridize to a polynucleotide complementary to a sequence shown in
SEQ ID NO:15 under moderately stringent conditions, wherein the
nucleotide sequences encode polypeptides having
sphingosine-1-phosphate lyase activity; and (c) nucleotide
sequences that encode a polypeptide encoded by a sequence shown in
SEQ ID NO:15.
[0012] Within a related aspect, an isolated polynucleotide is
provided that encodes a polypeptide shown in SEQ ID NO:16, or a
variant of such a polypeptide that has sphingosine-1-phosphate
lyase activity. Recombinant expression vectors comprising any of
the foregoing polynucleotides, and host cells transformed or
transfected with such expression vectors, are also provided.
[0013] Within further aspects, SPL polypeptides are provided. Such
polypeptides may be encoded by any of the foregoing
polynucleotides. Alternatively, a polypeptide may comprise an amino
acid sequence shown in SEQ ID NO:16, or a variant thereof, wherein
the polypeptide has sphingosine-1-phosphate lyase activity.
[0014] Within a further aspect, the present invention provides
isolated polynucleotides comprising at least 100 nucleotides
complementary to a sequence shown in SEQ ID NO:15.
[0015] Within other aspects, methods are provided for preparing a
sphingosine-1-phosphate lyase, comprising culturing a host cell
transformed or transfected with a polynucleotide as described above
under conditions promoting expression of the polynucleotide and
recovering a sphingosine-1-phosphate lyase.
[0016] In further aspects, the present invention provides methods
for identifying an agent that modulates sphingosine-1-phosphate
lyase activity. In one such aspect, the method comprises: (a)
contacting a candidate agent with a polypeptide comprising a
sequence shown in SEQ ID NO:16, or a variant of such a sequence
having sphingosine-1-phosphate lyase activity, wherein the step of
contacting is carried out under conditions and for a time
sufficient to allow the candidate agent to interact with the
polypeptide; and (b) subsequently measuring the ability of the
polypeptide to degrade sphingosine-1-phosphate or a derivative
thereof, relative to an ability in the absence of candidate agent.
The step of contacting may be performed by incubating a cell
expressing the polypeptide with the candidate modulator, and the
step of measuring the ability to degrade sphingosine-1-phosphate
may be performed using an in vitro assay and a cellular
extract.
[0017] The present invention further provides pharmaceutical
compositions comprising an agent that modulates
sphingosine-1-phosphate lyase activity in combination with a
pharmaceutically acceptable carrier. Such agents preferably
increase sphingosine-1-phosphate lyase activity. Such inhibition
may be achieved by increasing expression of an endogenous SPL gene,
or by increasing the ability of an endogenous SPL to degrade
sphingosine-1-phosphate. Within certain preferred embodiments, a
modulating agent comprises a polynucleotide or an antibody or an
antigen-binding fragment thereof.
[0018] Within still further aspects, the present invention provides
methods for modulating sphingosine-1-phosphate activity, comprising
contacting a sphingosine-1-phosphate lyase with an effective amount
of an agent that modulates sphingosine-1-phosphate lyase activity,
wherein the step of contacting is performed under conditions and
for a time sufficient to allow the agent and the
sphingosine-1-phosphate lyase to interact. To modulate
sphingosine-1-phosphate lyase activity in a cell, a cell expressing
sphingosine-1-phosphate may be contacted with such an agent.
[0019] Within related aspects, the present invention provides
methods for inhibiting the growth of a cancer cell, comprising
contacting a cancer cell with an agent that increases
sphingosine-1-phosphate lyase activity. In a preferred embodiment,
the cancer cell is a breast cancer cell.
[0020] The present invention also provides methods for inhibiting
the development and/or metastasis of a cancer in a mammal,
comprising administering to a mammal an agent that increases
sphingosine-1-phosphate lyase activity. Within certain embodiments,
an agent may comprise, or be linked to, a targeting component, such
as an anti-tumor antibody or a component that binds to an estrogen
receptor.
[0021] Within other aspects, methods for diagnosing cancer in a
mammal are provided, comprising detecting an alteration in an
endogenous sphingosine-1-phosphate lyase gene in a sample obtained
from a mammal, and therefrom diagnosing a cancer in the mammal. In
certain embodiments the cancer is breast or colon cancer and the
sample is a breast tumor biopsy.
[0022] In related aspects, the present invention provides methods
for evaluating a cancer prognosis, comprising determining the
presence or absence of an alteration in an endogenous
sphingosine-1-phosphate lyase gene in a sample obtained from a
mammal afflicted with cancer, and therefrom determining a
prognosis.
[0023] The present invention further provides isolated antibodies
that bind to a polypeptide having a sequence shown in SEQ ID NO:16.
Such antibodies may be polyclonal or monoclonal, and may increase
the ability of a polypeptide having a sequence shown in SEQ ID
NO:16 degrade sphingosine-1-phosphate.
[0024] In still further aspects, the present invention provides
methods for detecting sphingosine-1-phosphate lyase in a sample,
comprising: (a) contacting a sample with an antibody as described
above under conditions and for a time sufficient to allow the
antibody to bind to sphingosine-1-phosphate lyase; and (b)
detecting in the sample the presence of sphingosine-1-phosphate
lyase bound to the antibody.
[0025] Kits for use in the above methods are also provided. A kit
for detecting sphingosine-1-phosphate lyase in a sample comprises
an antibody as described above and a buffer or detection reagent. A
kit for detecting an alteration in a sphingosine-1-phosphate gene
in a sample comprises a polynucleotide and a detection reagent.
[0026] The present invention further provides for a homozygous null
mutant Drosophila melanogaster fly line the genome of which
comprises a P-element transposon insertion in the coding region of
the sphingosine phosphate lyase (SPL) gene wherein said gene
encodes the sequence set forth in SEQ ID NO:16, and wherein said
fly line has a flightless phenotype. In a related embodiment, the
homozygous mutant flies demonstrate abnormal developmental
patterning of thoracic muscles of the T2 segment.
[0027] The present invention also provides methods for testing an
agent capable of inhibiting the development and/or metastasis of a
cancer in a mammal, comprising contacting SPL mutant Drosophila
progeny with growth medium comprising a test agent suspected of
inhibiting mammalian sphingosine kinase, and detecting the
restoration of flight ability in the progeny. In a related
embodiment, the homozygous mutant flies used in this method
demonstrate abnormal developmental patterning of thoracic muscles
of the T2 segment.
[0028] The present invention further provides for methods for
determining the presence of a cancer in a patient, comprising the
steps of: (a) obtaining a biological sample from the patient; (b)
contacting the biological sample with at least one oligonucleotide
that is at least partially complementary to the sequence set forth
in SEQ ID NO:7; (c) detecting in the sample an amount of said
oligonucleotide that hybridizes to the polynucleotide; and
comparing the amount of oligonucleotide that hybridizes to the
polynucleotide to a predetermined cut-off value, and therefrom
determining the presence of the cancer in the patient.
BRIEF DESCRIPTION OF THE SEQUENCE IDENTIFIERS
[0029] SEQ ID NO:1 is the determined cDNA sequence of S. cerevisiae
SPL
[0030] SEQ ID NO:2 is the amino acid sequence of S. cerevisiae SPL
encoded by the polynucleotide sequence set forth in SEQ ID NO:1
[0031] SEQ ID NO:3 is the determined cDNA sequence of C. elegans
SPL
[0032] SEQ ID NO:4 is the amino acid sequence of C. elegans SPL
encoded by the polynucleotide sequence set forth in SEQ ID NO:3
[0033] SEQ ID NO:5 is the determined cDNA sequence of the mouse
SPL
[0034] SEQ ID NO:6 is the amino acid sequence of mouse SPL encoded
by the polynucleotide sequence set forth in SEQ ID NO:5
[0035] SEQ ID NO:7 is the determined cDNA sequence of the
full-length human SPL
[0036] SEQ ID NO:8 is the amino acid sequence of human SPL encoded
by the polynucleotide sequence set forth in SEQ ID NO:7
[0037] SEQ ID NO:9 is the determined cDNA sequence of a human SPL
with a deletion
[0038] SEQ ID NO:10 is the amino acid sequence of a human SPL with
a deletion, encoded by the polynucleotide sequence set forth in SEQ
ID NO:9
[0039] SEQ ID NO:11 is the amino acid sequence of C. elegans SPL
encoded by the polynucleotide sequence set forth in SEQ ID
NO:12
[0040] SEQ ID NO:12 is the determined cDNA sequence of a C. elegans
SPL
[0041] SEQ ID NO:13 is a PCR primer
[0042] SEQ ID NO:14 is a PCR primer
[0043] SEQ ID NO:15 is the determined cDNA sequence encoding the
Drosophila melanogaster SPL
[0044] SEQ ID NO:16 is the amino acid sequence of the Drosophila
melanogaster SPL, encoded by the cDNA sequence set forth in SEQ ID
NO:15
[0045] SEQ ID NO:17 is the determined cDNA sequence of a human SPL
as set forth in Genbank Accession No: AF144638.
[0046] SEQ ID NO:18 is the amino acid sequence of a human SPL
encoded by the polynucleotide sequence provided in SEQ ID
NO:17.
[0047] SEQ ID NO:19 is the amino acid sequence of a first
Drosophila melanogaster SK protein.
[0048] SEQ ID NO:20 is the amino acid sequence of a second
Drosophila melanogaster SK protein.
[0049] SEQ ID NO:21 is the amino acid sequence of a human SK
protein.
DETAILED DESCRIPTION OF THE INVENTION
[0050] As noted above, the present invention is generally directed
to compositions and methods for the diagnosis and therapy of
cancers such as breast cancer. The invention is more particularly
related to sphingosine-1-phosphate lyase (SPL) polypeptides, which
have the ability to cleave sphingosine-1-phosphate into inactive
metabolites, and to polynucleotides encoding such polypeptides.
Sphingosine-1-phosphate (S-1-P) is an endogenous sphingolipid
metabolite present in most mammalian cells and in serum. Like other
sphingolipid metabolites such as ceramide and sphingosine, S-1-P
participates in specific signal transduction pathways. The results
of S-1-P signaling are diverse and dependent upon the cell type
being examined. However, many of the effects of S-1-P signaling,
which include promotion of cellular proliferation, enhancement of
migration, inhibition of apoptosis and stimulation of angiogenesis,
influence the transformation, growth, drug resistance, vascularity
and metastatic capacity of cancer cells. The gene encoding the
enzyme responsible for S-1-P synthesis is sphingosine kinase, SK,
and S-1-P degradation is sphingosine phosphate lyase, SPL and S-1-P
phosphatase, S-1-PP. Several observations support the notion that
SPL may be a cancer related gene. First, altered expression of SPL
in human tumors compared to corresponding normal tissue from the
same patient has been shown. Second, human SPL maps to 10q21, a
chromosomal region frequently deleted in a variety of human
cancers. Taken together, these observations raise the possibility
that SPL may be potentially effective targets for pharmacological
intervention in the treatment of cancer.
[0051] Agents that decrease the expression or activity of
endogenous SPL polypeptides are encompassed by the present
invention. Such modulating agents may be identified using methods
described herein and used, for example, in cancer therapy. It has
also been found, within the context of the present invention, that
the detection of alterations in an endogenous SPL sequence can be
used to diagnose cancer, and to assess the prognosis for recovery.
The present invention further provides such diagnostic methods and
kits.
[0052] As used herein, the term "polypeptide" encompasses amino
acid chains of any length, including full length endogenous (i.e.,
native) SPL proteins and variants of endogenous sequences.
"Variants" are polypeptides that differ in sequence from a native
SPL only in substitutions, deletions and/or other modifications,
such that the variant retains SPL activity, which may be determined
using a representative method described herein SPL polypeptide
variants generally encompassed by the present invention will
typically exhibit at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity along its
length, to an SPL polypeptide sequence set forth herein. Within an
SPL polypeptide variant, amino acid substitutions are preferably
made at no more than 50% of the amino acid residues in the native
polypeptide, and more preferably at no more than 25% of the amino
acid residues. Such substitutions are preferably conservative. A
conservative substitution is one in which an amino acid is
substituted for another amino acid that has similar properties,
such that one skilled in the art of peptide chemistry would expect
the secondary structure and hydropathic nature of the polypeptide
to be substantially unchanged. In general, the following amino
acids represent conservative changes: (1) ala, pro, gly, glu, asp,
gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met,
ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
Substitutions, deletions and/or amino acid additions may be made at
any location(s) in the polypeptide, provided that the modification
does not diminish the SPL activity of the variant. Thus, a variant
may comprise only a portion of a native SPL sequence. In addition,
or alternatively, variants may contain additional amino acid
sequences (such as, for example, linkers, tags and/or ligands),
preferably at the amino and/or carboxy termini. Such sequences may
be used, for example, to facilitate purification, detection or
cellular uptake of the polypeptide.
[0053] When comparing polypeptide sequences, two sequences are said
to be "identical" if the sequence of amino acids in the two
sequences is the same when aligned for maximum correspondence, as
described below. Comparisons between two sequences are typically
performed by comparing the sequences over a comparison window to
identify and compare local regions of sequence similarity. A
"comparison window" as used herein, refers to a segment of at least
about 20 contiguous positions, usually 30 to about 75, 40 to about
50, in which a sequence may be compared to a reference sequence of
the same number of contiguous positions after the two sequences are
optimally aligned.
[0054] Optimal alignment of sequences for comparison may be
conducted using the Megalign program in the Lasergene suite of
bioinformatics software (DNASTAR, Inc., Madison, Wis.), using
default parameters. This program embodies several alignment schemes
described in the following references: Dayhoff, M. O. (1978) A
model of evolutionary change in proteins--Matrices for detecting
distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein
Sequence and Structure, National Biomedical Research Foundation,
Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990)
Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in
Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;
Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E.
W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971)
Comb. Theor 11:105; Saitou, N. Nei, M. (1987) Mol. Biol. Evol.
4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical
Taxonomy--the Principles and Practice of Numerical Taxonomy,
Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D.
J. (1983) Proc. Natl. Acad., Sci. USA 80:726-730.
[0055] Alternatively, optimal alignment of sequences for comparison
may be conducted by the local identity algorithm of Smith and
Waterman (1981) Add. APL. Math 2:482, by the identity alignment
algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by
the search for similarity methods of Pearson and Lipman (1988)
Proc. Natl. Acad. Sci. USA 85: 2444, by computerized
implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA,
and TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by
inspection.
[0056] One preferred example of algorithms that are suitable for
determining percent sequence identity and sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al.
(1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0
can be used, for example with the parameters described herein, to
determine percent sequence identity for the polynucleotides and
polypeptides of the invention. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information. For amino acid sequences, a scoring
matrix can be used to calculate the cumulative score. Extension of
the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T and X determine the sensitivity and speed
of the alignment.
[0057] The SPL activity of an SPL polypeptide or variant thereof
may generally be assessed using an in vitro assay that detects the
degradation of labeled substrate (i.e., sphingosine-1-phosphate, or
a derivative thereof). Within such assays, pyridoxal 5'-phosphate
is a requirement for SPL activity. In addition, the reaction
generally proceeds optimally at pH 7.4-7.6 and requires chelators
due to sensitivity toward heavy metal ions. The substrate should be
a D-erythro isomer, but in derivatives of sphingosine-1-phosphate
the type and chain length of sphingoid base may vary. In general,
an assay as described by Van Veldhoven and Mannaerts, J. Biol.
Chem. 266:12502-07, 1991 may be employed. Briefly, a solution
(e.g., a cellular extract) containing the polypeptide may be
incubated with 40 .mu.M substrate at 37.degree. C. for 1 hour in
the presence of, for example, 50 mM sucrose, 100 mM K-phosphate
buffer pH 7.4, 25 mM NaF, 0.1% (w/v) Triton X-100, 0.5 mM EDTA, 2
mM DTT, 0.25 mM pyridoxal phosphate. Reactions may then be
terminated and analyzed by thin-layer chromatography to detect the
formation of labeled fatty aldehydes and further metabolites. In
general, a polypeptide has SPL activity if, within such an assay:
(1) the presence of 2-50 .mu.g polypeptide (or 0.1-10 mg/mL)
results in a statistically significant increase in the level of
substrate degradation, preferably a two-fold increase, relative to
the level observed in the absence of polypeptide; and (2) the
increase in the level of substrate degradation is pyridoxal
5'-phosphate dependent.
[0058] Within certain embodiments, an in vitro assay for SPL
activity may be performed using cellular extracts prepared from
cells that express the polypeptide of interest. Preferably, in the
absence of a gene encoding an SPL polypeptide, such cells do not
produce a significant amount of endogenous SPL (i.e., a cellular
extract should not contain a detectable increase in the level of
SPL, as compared to buffer alone without extract). It has been
found, within the context of the present invention, that yeast
cells containing deletion of the SPL gene (BST1) are suitable for
use in evaluating the SPL activity of a polypeptide. bst1.DELTA.
cells can be generated from S. cerevisiae using standard
techniques, such as PCR, as described herein. A polypeptide to be
tested for SPL activity may then be expressed in bst1.DELTA. cells,
and the level of SPL activity in an extract containing the
polypeptide may be compared to that of an extract prepared from
cells that do not express the polypeptide. For such a test, a
polypeptide is preferably expressed on a high-copy yeast vector
(such as pYES2, which is available from Invitrogen) yielding more
than 20 copies of the gene per cell. In general, a polypeptide has
SPL activity if, when expressed using such a vector in a
bst1.DELTA. cell, a cellular extract results in a two-fold increase
in substrate degradation over the level observed for an extract
prepared from cells not expressing the polypeptide.
[0059] A further test for SPL activity may be based upon functional
complementation in the bst1.DELTA. strain. It has been found,
within the context of the present invention, that bst1.DELTA. cells
are highly sensitive to D-erythro-sphingosine. In particular,
concentrations as low as 10 .mu.M sphingosine completely inhibit
the growth of bst1.DELTA. cells. Such a level of sphingosine has no
effect on the growth of wildtype cells. A polypeptide having SPL
activity as provided above significantly diminishes (i.e., by at
least two fold) the sphingosine sensitivity when expressed on a
high-copy yeast vector yielding more than 20 copies of the gene per
cell.
[0060] In general, SPL polypeptides, and polynucleotides encoding
such polypeptides, may be prepared using any of a variety of
techniques that are well known in the art. For example, a DNA
sequence encoding native SPL may be prepared by amplification from
a suitable cDNA or genomic library using, for example, polymerase
chain reaction (PCR) or hybridization techniques. Libraries may
generally be prepared and screened using methods well known to
those of ordinary skill in the art, such as those described in
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989. cDNA
libraries may be prepared from any of a variety of sources known to
contain enzymes having SPL activity. SPL activity is ubiquitous
with regard to species and mammalian tissues, with the exception of
platelets, in which SPL activity is notably absent. In rat tissues,
the highest levels of activity have been demonstrated in intestinal
mucosa, liver and Harderian gland, with low activity in skeletal
muscle and heart. Activity has also been demonstrated in a number
of human (hepatoma cell line HB 8065, cervical carcinoma HeLa),
mouse (hepatoma line BW1, mouse embryo 3T3-L1, Swiss 3T3 cells) and
other cell lines, as well as in human cultured fibroblasts.
Preferred cDNA libraries may prepared from human liver, intestine
or brain tissues or cells. Other libraries that may be employed
will be apparent to those of ordinary skill in the art. Primers for
use in amplification may be readily designed based on the sequence
of a native SPL polypeptide or polynucleotide, as provided
herein.
[0061] Alternatively, an endogenous SPL gene may be identified
using a screen for cDNAs that complement the BST1 deletion in
yeast. A cDNA expression library may be generated using a
regulatable yeast expression vector (e.g., pYES, which is available
from Invitrogen, Inc.) and standard techniques. A yeast bst1.DELTA.
strain may then be transformed with the cDNA library, and
endogenous cDNAs having the ability to functionally complement the
yeast lyase defect (i.e., restore the ability to grow in the
presence of D-erythro-sphingosine) may be isolated.
[0062] An endogenous SPL gene may also be identified based on
cross-reactivity of the protein product with anti-SPL antibodies,
which may be prepared as described herein. Such screens may
generally be performed using standard techniques (see Huynh et al.,
"Construction and Screening cDNA Libraries in .lamda.gt11," in D.
M. Glover, ed., DNA Cloning: A Practical Approach, 1:49-78, 1984
(IRL Press, Oxford)).
[0063] Polynucleotides encompassed by the present invention include
DNA and RNA molecules that comprise an endogenous SPL gene
sequence. Such polynucleotides include those that comprise a
sequence recited in any one of SEQ ID NOs:1-16. Also encompassed
are other polynucleotides that encode an SPL amino acid sequence
encoded by such polynucleotides, as well as polynucleotides that
encode variants of a native SPL sequence that retain SPL activity.
Polynucleotides that are substantially homologous to a sequence
complementary to an endogenous SPL gene are also within the scope
of the present invention. "Substantial homology," as used herein
refers to polynucleotides that are capable of hybridizing under
moderately stringent conditions to a polynucleotide complementary
to an SPL polynucleotide sequence provided herein, provided that
the encoded SPL polypeptide variant retains SPL activity. Suitable
moderately stringent conditions include prewashing in a solution of
5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at
50-65.degree. C., 5.times.SSC, overnight; followed by washing twice
at 65.degree. C. for 20 minutes with each of 2.times., 0.5.times.
and 0.2.times.SSC containing 0.1% SDS. Nucleotide sequences that,
because of code degeneracy, encode a polypeptide encoded by any of
the above sequences are also encompassed by the present
invention.
[0064] Polypeptides of the present invention may be prepared by
expression of recombinant DNA encoding the polypeptide in cultured
host cells. Preferably, the host cells are bacteria, yeast, insect
or mammalian cells, and more preferably the host cells are S.
cerevisiae bst1.DELTA. cells. The recombinant DNA may be cloned
into any expression vector suitable for use within the host cell
and transfected into the host cell using techniques well known to
those of ordinary skill in the art. A suitable expression vector
contains a promoter sequence that is active in the host cell. A
tissue-specific or conditionally active promoter may also be used.
Preferred promoters express the polypeptide at high levels.
[0065] Optionally, the construct may contain an enhancer, a
transcription terminator, a poly(A) signal sequence, a bacterial or
mammalian origin of replication and/or a selectable marker, all of
which are well known in the art. Enhancer sequences may be included
as part of the promoter region or separately. Transcription
terminators are sequences that stop RNA polymerase-mediated
transcription. The poly(A) signal may be contained within the
termination sequence or incorporated separately. A selectable
marker includes any gene that confers a phenotype on the host cell
that allows transformed cells to be identified. Such markers may
confer a growth advantage under specified conditions. Suitable
selectable markers for bacteria are well known and include
resistance genes for ampicillin, kanamycin and tetracycline.
Suitable selectable markers for mammalian cells include hygromycin,
neomycin, genes that complement a deficiency in the host (e.g.,
thymidine kinase and TK-cells) and others well known in the art.
For yeast cells, one suitable selectable marker is URA3, which
confers the ability to grow on medium without uracil.
[0066] DNA sequences expressed in this manner may encode a native
SPL polypeptide (e.g., human), or may encode portions or other
variants of native SPL polypeptide. DNA molecules encoding variants
of a native SPL may generally be prepared using standard
mutagenesis techniques, such as oligonucleotide-directed
site-specific mutagenesis, and sections of the DNA sequence may be
removed to permit preparation of truncated polypeptides.
[0067] To generate cells that express a polynucleotide encoding an
SPL polypeptide, cells may be transfected, transformed or
transduced using any of a variety of techniques known in the art.
Any number of transfection, transformation, and transduction
protocols known to those in the art may be used, for example those
outlined in Current Protocols in Molecular Biology, John Wiley
& Sons, New York. N.Y., or in numerous kits available
commercially (e.g., Invitrogen Life Technologies, Carlsbad,
Calif.). Such techniques may result in stable transformants or may
be transient. One suitable transfection technique is
electroporation, which may be performed on a variety of cell types,
including mammalian cells, yeast cells and bacteria, using
commercially available equipment. Optimal conditions for
electroporation (including voltage, resistance and pulse length)
are experimentally determined for the particular host cell type,
and general guidelines for optimizing electroporation may be
obtained from manufacturers. Other suitable methods for
transfection will depend upon the type of cell used (e.g., the
lithium acetate method for yeast), and will be apparent to those of
ordinary skill in the art. Following transfection, cells may be
maintained in conditions that promote expression of the
polynucleotide within the cell. Appropriate conditions depend upon
the expression system and cell type, and will be apparent to those
skilled in the art.
[0068] SPL polypeptides may be expressed in transfected cells by
culturing the cell under conditions promoting expression of the
transfected polynucleotide. Appropriate conditions will depend on
the specific host cell and expression vector employed, and will be
readily apparent to those of ordinary skill in the art. For
commercially available expression vectors, the polypeptide may
generally be expressed according to the manufacturer's
instructions. For certain purposes, expressed polypeptides of this
invention may be isolated in substantially pure form. Preferably,
the polypeptides are isolated to a purity of at least 80% by
weight, more preferably to a purity of at least 95% by weight, and
most preferably to a purity of at least 99% by weight. In general,
such purification may be achieved using, for example, the standard
techniques of ammonium sulfate fractionation, SDS-PAGE
electrophoresis, and/or affinity chromatography.
[0069] The present invention further provides antibodies that bind
to an SPL polypeptide. Antibodies may function as modulating agents
(as discussed further below) to inhibit or block SPL activity in
vivo. Alternatively, or in addition, antibodies may be used within
screens for endogenous SPL polypeptides or modulating agents, for
purification of SPL polypeptides, for assaying the level of SPL
within a sample and/or for studies of SPL expression. Such
antibodies may be polyclonal or monoclonal, and are generally
specific for one or more SPL polypeptides and/or one or more
variants thereof. Within certain preferred embodiments, antibodies
are polyclonal.
[0070] Antibodies may be prepared by any of a variety of techniques
known to those of ordinary skill in the art (see, e.g., Harlow and
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, 1988). In one such technique, an immunogen comprising
an SPL polypeptide or antigenic portion thereof is initially
injected into a suitable animal (e.g., mice, rats, rabbits, sheep
and goats), preferably according to a predetermined schedule
incorporating one or more booster immunizations. The use of rabbits
is preferred. To increase immunogenicity, an immunogen may be
linked to, for example, glutaraldehyde or keyhole limpet hemocyanin
(KLH). Following injection, the animals are bled periodically to
obtain post-immune serum containing polyclonal anti-SPL antibodies.
Polyclonal antibodies may then be purified from such antisera by,
for example, affinity chromatography using an SPL polypeptide or
antigenic portion thereof coupled to a suitable solid support. Such
polyclonal antibodies may be used directly for screening purposes
and for Western blots.
[0071] More specifically, an adult rabbit (e.g., NZW) may be
immunized with 10 .mu.g purified (e.g., using a nickel-column) SPL
polypeptide emulsified in complete Freund's adjuvant (1:1 v/v) in a
volume of 1 mL. Immunization may be achieved via injection in at
least six different subcutaneous sites. For subsequent
immunizations, 5 .mu.g of an SPL polypeptide may be emulsified in
complete Freund's adjuvant and injected in the same manner.
Immunizations may continue until a suitable serum antibody titer is
achieved (typically a total of about three immunizations). The
rabbit may be bled immediately before immunization to obtain
pre-immune serum, and then 7-10 days following each
immunization.
[0072] For certain embodiments, monoclonal antibodies may be
desired. Monoclonal antibodies may be prepared, for example, using
the technique of Kohler and Milstein, Eur. J. Immunol. 6:511-519,
1976, and improvements thereto. Briefly, these methods involve the
preparation of immortal cell lines capable of producing antibodies
having the desired specificity (i.e., reactivity with the
polypeptide of interest). Such cell lines may be produced, for
example, from spleen cells obtained from an animal immunized as
described above. The spleen cells are then immortalized by, for
example, fusion with a myeloma cell fusion partner, preferably one
that is syngeneic with the immunized animal. For example, the
spleen cells and myeloma cells may be combined with a nonionic
detergent for a few minutes and then plated at low density on a
selective medium that supports the growth of hybrid cells, but not
myeloma cells. A preferred selection technique uses HAT
(hypoxanthine, aminopterin, thymidine) selection. After a
sufficient time, usually about 1 to 2 weeks, colonies of hybrids
are observed. Single colonies are selected and tested for binding
activity against the polypeptide. Hybridomas having high reactivity
and specificity are preferred.
[0073] Monoclonal antibodies may be isolated from the supernatants
of growing hybridoma colonies. In addition, various techniques may
be employed to enhance the yield, such as injection of the
hybridoma cell line into the peritoneal cavity of a suitable
vertebrate host, such as a mouse. Monoclonal antibodies may then be
harvested from the ascites fluid or the blood. Contaminants may be
removed from the antibodies by conventional techniques, such as
chromatography, gel filtration, precipitation, and extraction.
[0074] As noted above, the present invention provides agents that
modulate, preferably inhibit, the expression (transcription or
translation), stability and/or activity of an SPL polypeptide. To
identify such a modulating agent, any of a variety of screens may
be performed. Candidate modulating agents may be obtained using
well known techniques from a variety of sources, such as plants,
fungi or libraries of chemicals, small molecules or random
peptides. Antibodies that bind to an SPL polypeptide, and
anti-sense polynucleotides that hybridize to a polynucleotides that
encodes an SPL, may be candidate modulating agents. Preferably, a
modulating agent has a minimum of side effects and is non-toxic.
For some applications, agents that can penetrate cells are
preferred.
[0075] Screens for modulating agents that decrease SPL expression
or stability may be readily performed using well known techniques
that detect the level of SPL protein or mRNA. Suitable assays
include RNAse protection assays, in situ hybridization, ELISAs,
Northern blots and Western blots. Such assays may generally be
performed using standard methods (see Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold
Spring Harbor, N.Y., 1989). For example, to detect mRNA encoding
SPL, a nucleic acid probe complementary to all or a portion of the
SPL gene sequence may be employed in a Northern blot analysis of
mRNA prepared from suitable cells. Alternatively, real-time PCR can
also be used to detect levels of mRNA encoding SPL (see Gibson et
al., Genome Research 6:995-1001, 1996; Heid et al., Genome Research
6:986-994, 1996). The first-strand cDNA to be used in the
quantitative real-time PCR is synthesized from 20 .mu.g of total
RNA that is first treated with DNase I (e.g., Amplification Grade,
Gibco BRL Life Technology, Gaitherburg, Md.), using Superscript
Reverse Transcriptase (RT) (e.g., Gibco BRL Life Technology,
Gaitherburg, Md.). Real-time PCR is performed, for example, with a
GeneAmp.TM. 5700 sequence detection system (PE Biosystems, Foster
City, Calif.). The 5700 system uses SYBR.TM. green, a fluorescent
dye that only intercalates into double stranded DNA, and a set of
gene-specific forward and reverse primers. The increase in
fluorescence is monitored during the whole amplification process.
The optimal concentration of primers is determined using a
checkerboard. The PCR reaction is performed in 25 .mu.l volumes
that include 2.5 .mu.l of SYBR green buffer, 2 .mu.l of cDNA
template and 2.5 .mu.l each of the forward and reverse primers for
the SPL gene, or other gene of interest. The cDNAs used for RT
reactions are diluted approximately 1:10 for each gene of interest
and 1:100 for the .beta.-actin control. In order to quantitate the
amount of specific cDNA (and hence initial mRNA) in the sample, a
standard curve is generated for each run using the plasmid DNA
containing the gene of interest. Standard curves are generated
using the Ct values determined in the real-time PCR which are
related to the initial cDNA concentration used in the assay.
Standard dilution ranging from 20-2.times.10.sup.6 copies of the
SPL gene or other gene of interest are used for this purpose. In
addition, a standard curve is generated for .beta.-actin ranging
from 200 fg-2000 fg. This enables standardization of the initial
RNA content of a sample to the amount of .beta.-actin for
comparison purposes. The mean copy number for each sample tested is
normalized to a constant amount of .beta.-actin, allowing the
evaluation of the observed expression levels of SPL or other gene
of interest.
[0076] To detect SPL protein, a reagent that binds to the protein
(typically an antibody, as described herein) may be employed within
an ELISA or Western assay. Following binding, a reporter group
suitable for direct or indirect detection of the reagent is
employed (i.e., the reporter group may be covalently bound to the
reagent or may be bound to a second molecule, such as Protein A,
Protein G, immunoglobulin or lectin, which is itself capable of
binding to the reagent). Suitable reporter groups include, but are
not limited to, enzymes (e.g., horseradish peroxidase), substrates,
cofactors, inhibitors, dyes, radionuclides, luminescent groups,
fluorescent groups and biotin. Such reporter groups may be used to
directly or indirectly detect binding of the reagent to a sample
component using standard methods known to those of ordinary skill
in the art.
[0077] To use such assays for identifying a modulating agent, the
level of SPL protein or mRNA may be evaluated in cells treated with
one or more candidate modulating agents. An increase or decrease in
SPL levels may be measured by evaluating the level of SPL mRNA
and/or protein in the presence and absence of candidate modulating
agent. For example, an antisense modulating agent may be evaluated
by assaying the effect on SPL levels. Suitable cells for use in
such assays include the breast cancer cell lines MCF-7 (ATCC
Accession Number HTB-22) and MDA-MB-231 (ATCC Accession Number
HTB-26). A candidate modulator may be tested by transfecting the
cells with a polynucleotide encoding the candidate and evaluating
the effect of expression of the polynucleotide on SPL levels.
Alternatively, the cells may be contacted with a candidate
modulator, typically in an amount ranging from about 10 nM to about
10 mM. A candidate that results in a statistically significant
change in the level of SPL mRNA and/or protein is a modulating
agent.
[0078] Alternatively, or in addition, a candidate modulating agent
may be tested for the ability to inhibit or increase SPL activity,
using an in vitro assay as described herein (see Van Veldhoven and
Mannaerts, J. Biol. Chem. 266:12502-07, 1991) that detects the
degradation of labeled substrate (i.e., sphingosine-1-phosphate, or
a derivative thereof). Briefly, a solution (e.g., a cellular
extract) containing an SPL polypeptide (e.g., 10 nM to about 10 mM)
may be incubated with a candidate modulating agent (typically 1 nM
to 10 mM, preferably 10 nM to 1 mM) and a substrate (e.g., 40
.mu.M) at 37.degree. C. for 1 hour in the presence of, for example,
50 mM sucrose, 100 mM K-phosphate buffer pH 7.4, 25 mM NaF, 0.1%
(w/v) Triton X-100, 0.5 mM EDTA, 2 mM DTT, 0.25 mM pyridoxal
phosphate. Reactions may then be terminated and analyzed by
thin-layer chromatography to detect the formation of labeled fatty
aldehydes and further metabolites. A modulating agent (e.g., an
antibody) that increases SPL activity results in a statistically
significant increase in the degradation of sphingosine-1-phosphate,
relative to the level of degradation in the absence of modulating
agent. Such modulating agents may be used to increase SPL activity
in a cell culture or a mammal, as described below.
[0079] A modulating agent may additionally comprise, or may be
associated with, a targeting component that serves to direct the
agent to a desired tissue or cell type. As used herein, a
"targeting component" may be any substance (such as a compound or
cell) that, when linked to a compound enhances the transport of the
compound to a target tissue, thereby increasing the local
concentration of the compound. Targeting components include
antibodies or fragments thereof, receptors, ligands and other
molecules that bind to cells of, or in the vicinity of, the target
tissue. Known targeting components include hormones, antibodies
against cell surface antigens, lectins, adhesion molecules, tumor
cell surface binding ligands, steroids, cholesterol, lymphokines,
fibrinolytic enzymes and other drugs and proteins that bind to a
desired target site. In particular, anti-tumor antibodies and
compounds that bind to an estrogen receptor may serve as targeting
components. An antibody employed in the present invention may be an
intact (whole) molecule, a fragment thereof, or a functional
equivalent thereof. Examples of antibody fragments are F(ab')2,
-Fab', Fab and F[v] fragments, which may be produced by
conventional methods or by genetic or protein engineering. Linkage
may be via any suitable covalent bond using standard techniques
that are well known in the art. Such linkage is generally covalent
and may be achieved by, for example, direct condensation or other
reactions, or by way of bi- or multi-functional linkers.
[0080] For in vivo use, a modulating agent as described herein is
generally incorporated into a pharmaceutical composition prior to
administration. A pharmaceutical composition comprises one or more
modulating agents in combination with a physiologically acceptable
carrier. To prepare a pharmaceutical composition, an effective
amount of one or more modulating agents is mixed with any
pharmaceutical carrier(s) known to those skilled in the art to be
suitable for the particular mode of administration. A
pharmaceutical carrier may be liquid, semi-liquid or solid.
Solutions or suspensions used for parenteral, intradermal,
subcutaneous or topical application may include, for example, a
sterile diluent (such as water), saline solution, fixed oil,
polyethylene glycol, glycerine, propylene glycol or other synthetic
solvent; antimicrobial agents (such as benzyl alcohol and methyl
parabens); antioxidants (such as ascorbic acid and sodium
bisulfite) and chelating agents (such as ethylenediaminetetraacetic
acid (EDTA)); buffers (such as acetates, citrates and phosphates).
If administered intravenously, suitable carriers include
physiological saline or phosphate buffered saline (PBS), and
solutions containing thickening and solubilizing agents, such as
glucose, polyethylene glycol, polypropylene glycol and mixtures
thereof. In addition, other pharmaceutically active ingredients
(including other anti-cancer agents) and/or suitable excipients
such as salts, buffers and stabilizers may, but need not, be
present within the composition.
[0081] A modulating agent may be prepared with carriers that
protect it against rapid elimination from the body, such as time
release formulations or coatings. Such carriers include controlled
release formulations, such as, but not limited to, implants and
microencapsulated delivery systems, and biodegradable,
biocompatible polymers, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid
and others known to those of ordinary skill in the art.
[0082] Administration may be achieved by a variety of different
routes, including oral, parenteral, nasal, intravenous,
intradermal, subcutaneous or topical. Preferred modes of
administration depend upon the nature of the condition to be
treated or prevented. An amount that, following administration,
inhibits, prevents or delays the progression and/or metastasis of a
cancer is considered effective. Preferably, the amount administered
is sufficient to result in regression, as indicated by 50% mass or
by scan dimensions. The precise dosage and duration of treatment is
a function of the disease being treated and may be determined
empirically using known testing protocols or by testing the
compositions in model systems known in the art and extrapolating
therefrom. Controlled clinical trials may also be performed.
Dosages may also vary with the severity of the condition to be
alleviated. A pharmaceutical composition is generally formulated
and administered to exert a therapeutically useful effect while
minimizing undesirable side effects. The composition may be
administered one time, or may be divided into a number of smaller
doses to be administered at intervals of time. For any particular
subject, specific dosage regimens may be adjusted over time
according to the individual need.
[0083] As an alternative to direct administration of a modulating
agent, a polynucleotide encoding a modulating agent may be
administered. Such a polynucleotide may be present in a
pharmaceutical composition within any of a variety of delivery
systems known to those of ordinary skill in the art, including
nucleic acid, bacterial and viral expression systems, and colloidal
dispersion systems such as liposomes. Appropriate nucleic acid
expression systems contain the necessary DNA sequences for
expression in the patient (such as a suitable promoter and
terminating signal, as described above). The DNA may also be
"naked," as described, for example, in Ulmer et al., Science
259:1745-49, 1993.
[0084] Various viral vectors that can be used to introduce a
nucleic acid sequence into the targeted patient's cells include,
but are not limited to, vaccinia or other pox virus, herpes virus,
retrovirus, or adenovirus. Techniques for incorporating DNA into
such vectors are well known to those of ordinary skill in the art.
Another delivery system for polynucleotides is a colloidal
dispersion system. Colloidal dispersion systems include
macromolecule complexes, nanocapsules, microspheres, beads, and
lipid-based systems including oil-in-water emulsions, micelles,
mixed micelles, and liposomes. The preparation and use of liposomes
is well known to those of ordinary skill in the art.
[0085] Within certain aspects of the present invention, one or more
modulating agents may be used to modulate SPL expression and/or
activity in vitro, in a cell or in a mammal. In vitro, an SPL
polypeptide may be contacted with a modulating agent that increases
or decreases SPL activity (e.g., certain antibodies). For use
within a cell or a mammal, such modulation may be achieved by
contacting a target cell with an effective amount of a modulating
agent, as described herein. Administration to a mammal may
generally be achieved as described above.
[0086] As noted above, increase of SPL expression and/or activity
provides a method for inhibiting the growth (i.e., proliferation)
of a cancer cell, either in culture or in a mammal afflicted with
cancer. In vivo, such increase may also be used to inhibit cancer
development, progression and/or metastasis. Accordingly, one or
more modulating agents as provided herein may be administered as
described above to a mammal in need of anti-cancer therapy.
Patients that may benefit from administration of a modulating agent
are those afflicted with cancer. Such patients may be identified
based on standard criteria that are well known in the art. Within
preferred embodiments, a patient is afflicted with breast cancer,
as identified based on tissue biopsy and microscopic evaluation,
using techniques well known in the art. In particular, patients
whose tumor cells contain a tissue-specific deletion and/or
alteration within an endogenous SPL gene may benefit from
administration of a modulating agent, as provided herein.
[0087] Within other aspects, the present invention provides methods
and kits for diagnosing cancer and/or identifying individuals with
a risk for metastasis that is higher or lower than average. It has
been found, within the context of the present invention, that
certain human tumor cells contain an altered SPL gene. In
particular, certain brain tumor cells contain a deletion of amino
acid residues 354 to 433 of the human SPL sequence set forth in SEQ
ID NO:8 (cDNA and amino acid sequence of the SPL containing the
deletion are set forth in SEQ ID NOs:9 and 10, respectively).
Specific alterations present in other tumor cells, such as breast
tumor cells, may be readily identified using standard techniques,
such as PCR. Alterations that may be associated with a particular
tumor include amino acid deletions, insertions, substitutions and
combinations thereof. Methods in which the presence or absence of
such an alteration is determined may generally be used to detect
cancer and to evaluate the prognosis for a patient known to be
afflicted with cancer.
[0088] To detect an altered SPL gene, any of a variety of
well-known techniques may be used including, but not limited to,
PCR and hybridization techniques. Any sample that may contain
cancerous cells may be assayed. In general, suitable samples are
tumor biopsies. Within a preferred embodiment, a sample is a breast
tumor biopsy.
[0089] Kits for diagnosing or evaluating the prognosis of a cancer
generally comprise reagents for use in the particular assay to be
employed. In general, a kit of the present invention comprises one
or more containers enclosing elements, such as primers, probes,
reagents or buffers, to be used in an assay. For example, a kit may
contain one or more polynucleotide primers or probes comprising at
least 15 nucleotides complementary to a polynucleotide encoding
SPL. In certain embodiments, the primers or probes comprise at
least 10, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,
95 or 100 nucleotides, and preferably at least 150 or 200
nucleotides, complementary to an SPL mRNA or to a polynucleotide
encoding SPL. Such probe(s) may be used to detect an altered SPL
gene by hybridization. For example, a kit may contain one probe
that hybridizes to a region of an SPL gene that is not generally
altered in tumors (a control) and a second probe that hybridizes to
a region commonly deleted in breast cancer. A sample that contains
mRNA that hybridizes to the first probe, and not to the second
(using standard techniques) contains an altered SPL gene. Suitable
control probes include probes that hybridize to a portion of the
SPL gene outside of the commonly deleted region encoding amino acid
resides 354 to 433; suitable probes for an altered region include
probes that hybridize to a portion of the SPL gene that encodes
amino acid residues 354 to 433. Alternatively, a kit may comprise
one or more primers for PCR analyses, which may be readily designed
based upon the sequences provided herein by those of ordinary skill
in the art. Optionally, a kit may further comprise one or more
solutions, compounds or detection reagents for use within an assay
as described above.
[0090] In a related aspect of the present invention, kits for
detecting SPL are provided. Such kits may be designed for detecting
the level of SPL or nucleic acid encoding SPL within a sample, or
may detect the level of SPL activity as described herein. A kit for
detecting the level of SPL, or nucleic acid encoding SPL, typically
contains a reagent that binds to the SPL protein, DNA or RNA. To
detect nucleic acid encoding SPL, the reagent may be a nucleic acid
probe or a PCR primer. To detect SPL protein, the reagent is
typically an antibody. The kit may also contain a reporter group
suitable for direct or indirect detection of the reagent as
described above.
[0091] Within further aspects, the present invention provides
transgenic mammals in which SPL activity is reduced, compared to a
wild-type animal. Such animals may contain an alteration, insertion
or deletion in an endogenous SPL gene, or may contain DNA encoding
a modulating agent that modulates expression or activity of an SPL
gene. In certain aspects, such animals may contain DNA encoding a
modulating agent that increases expression or activity of an SPL
gene. Transgenic animals may be generated using techniques that are
known to those of ordinary skill in the art. For example, a
transgenic animal containing an insertion or deletion in the coding
region for the SPL gene may be generated from embryonic stem cells,
using standard techniques. Such stem cells may be generated by
first identifying the full genomic sequence of the gene encoding
the SPL, and then creating an insertion or deletion in the coding
region in embryonic stem cells. Alternatively, appropriate
genetically altered embryonic stem cells may be identified from a
bank. Using the altered stem cells, hybrid animals may be generated
with one normal SPL gene and one marked, abnormal gene. These
hybrids may be mated, and homozygous progeny identified.
[0092] Transgenic animals may be used for a variety of purposes,
which will be apparent to those of ordinary skill in the art. For
example, such animals may be used to prepare cell lines from
different tissues, using well known techniques. Such cell lines may
be used, for example, to evaluate the effect of the alteration, and
to test various candidate modulators.
[0093] The invention further provides Drosophila melanogaster
animal models that exhibit a flightless phenotype, where the
phenotype results from the disruption of an endogenous SPL gene as
described in greater detail below. By flightless phenotype is meant
that the subject non-mammalian animal models spontaneously develop
a reduced number of muscle fibers comprising the dorsal
longitudinal muscles (DLM) and have compensatory hypertrophy in the
remaining fibers. In certain aspects, the non-mammalian animal
model of the present invention may also demonstrate abnormal
developmental patterning of thoracic muscles of the T2 segment. In
a preferred embodiment, the above phenotypes result in an inability
to fly. The subject non-mammalian animal models, within a preferred
embodiment, demonstrate altered activity of the endogenous SPL. In
a particularly illustrative embodiment, said D. melanogaster animal
models have decreased activity of endogenous SPL.
[0094] Within further aspects, the present invention provides
mutant strains of Drosophila melanogaster. In a preferred
embodiment, the strain contains a mutation in the SPL gene. In a
further embodiment of the present invention the D. melanogaster
strain are heterozygous for a P-element transposon which sits in
the coding region of the gene encoding the SPL protein set forth in
SEQ ID NO:16. In a preferred embodiment, the flies are homozygous
insertional mutants in the coding region of the gene encoding the
SPL protein set forth in SEQ ID NO:16. In yet a further embodiment
of the present invention, the homozygous mutant strain of fly has a
flightless phenotype. In certain embodiments, the mutant flies have
a reduced number of muscle fibers comprising the dorsal
longitudinal muscles and have compensatory hypertrophy in the
remaining fibers. In certain aspects, the mutant flies of the
present invention may also demonstrate abnormal developmental
patterning of thoracic muscles of the T2 segment.
[0095] Flies heterozygous for a P-element transposon which sits in
the coding region of the SPL gene or genes and disrupts production
of SPL proteins may be obtained from the Drosophila Genome Project.
Homozygous insertional mutants can be made, using techniques known
in the art, by genetically crossing and evaluating progeny for the
presence of homozygous insertional mutants (based on presence of
rosy eye color, encoded by a recessive marker carried on the
P-element). Expression of the SPL gene can be evaluated using any
number of assays known to the skilled artisan, for example, by
Northern blot analysis. To determine the SPL function of each
genotype, +/+, +/- and -/- flies may be homogenized using standard
techniques and whole extracts can be assayed for SPL activity using
assays as described herein. The transposon can be mobilized by
crossing SPL mutant flies with flies carrying an actively
transcribed transposase gene, which should cause the P-element to
be excised in the chromosomes of both somatic cells and in the
germline. Germline transposon loss is heritable and can be
identified in progeny by virtue of eye color. Progeny which lost
both the transposase gene and the P-element can then be isolated
and the restored SPL allele can be homozygosed.
[0096] Mutations in Drosophila melanogaster as described herein
which permanently block expression of a functional protein can be
created in several ways, such as with P-element transposon
insertions or chemical or radiation induced mutagenesis. Exemplary
strains of mutant flies are available through the Drosophila Genome
Project, at the University of California at Berkeley (Adams, M. et
al 2000. The genome sequence of Drosophila melanogaster. Science.
287:2185-2195.). Alternatively, insertional mutant of interest may
be obtained by using local hop strategies essentially as described
in Tower, J. et al (Tower, J., et al. 1993. Preferential
transposition of Drosophila P elements to nearby chromosomal sites.
Genetics. 133:347-359.), hereby incorporated by reference in its
entirety. Transposons can be mobilized by crossing in a transposase
gene, followed by crossing the transposase back out (reintroducing
genetic stability). Mutant flies can be identified using techniques
know to those of skill in the art. For example, mutant flies can be
identified by probing Southern blots prepared from extracts from
flies generated in the screen using the target gene as probe.
Subsequently, crosses can be performed to introduce a mutant allele
of interest, (e.g. SPL) and generate homozygosity at both mutant
alleles (e.g. SPL and new transposon integration sites). Mutants
can be screened for a phenotype of interest, for example the
ability to restore flight to an SPL mutant when the mutated allele
is homozygous (predicting a recessive phenotype).
[0097] In one aspect of the present invention, fly genetic
manipulation may entail mating or "crossing" of flies and selection
for or against progeny expressing various phenotypic markers.
Exemplary techniques for fly genetic manipulation of the present
invention are know in the art and are described, for example in,
Ashburner, M., and J. Roote. 2000. Laboratory culture of
Drosophila. In Drosophila Protocols. W. Sullivan, M. Ashburner, and
R. Hawley, editors. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y. 585-600. Phenotypic markers may be used to
identify the inheritance of chromosomes, engineered transposable
elements, or transposase genes used to facilitate their
mobilization. Marker mutations affecting eye color, bristle shape,
wing morphology and cuticle pigmentation, for example, may be
employed in the crosses for the mutant flies of the present
invention. Within one aspect of the present invention, it may be
desirable to select the individuals which contain a collection of
markers indicating the desired genotype. In another aspect of the
present invention, balancer chromosomes may be used to create the
ability to identify recessive mutations present in the heterozygous
state. Balancer chromosomes may be employed to prevent homologous
recombination during meiotic prophase in females. The presence of
both dominant and recessive lethal markers allows one to determine
the presence or absence of the balancer chromosomes and
simultaneously to follow the homologous chromosomes, which may
themselves not contain a dominant marker. One particularly
illustrative cross of the present invention is to eliminate the
P-element insertion in the Drosophila SPL gene and establish
phenotypic reversion, as described herein in the Examples.
[0098] Selective markers to allow for selection of mutant flies is
provided for in the present invention. Exemplary selective markers
of the present invention may comprise a wild type rosy (ry.sup.+)
allele carried on the transposon to allow for selection for or
against the stable transposon. Introduction of an active
transposase is selected for by presence of the dominant marker,
Stubble (short bristle phenotype) in the first cross, and is
selected against to identify progeny which have lost the
transposase, restoring genetic stability in the second cross. Other
illustrative markers include Curly 0 (CyO) which is lethal when
present in two copies, allowing selection for heterozygotes
containing the CyO balancer and another allele of interest
originally containing the transposon (e.g., SPL). By selecting
against rosy eye color, progeny in which the transposon has been
excised from the locus of interest, e.g., SPL, can be identified.
Expansion of this "reverted" allele in the population can be
achieved in the third cross, and the desired allele can be
homozygosed in the final cross, to determine whether restoration of
the intact allele of interest, for example SPL, is associated with
a desired phenotype of interest, such as restoration of flight.
[0099] In another aspect of the present invention, transgenic flies
can be created using P-elements to overexpress or misexpress
proteins of interest, such as SPL. In one embodiment of the
invention, GAL4-mediated ectopic gene expression is employed,
essentially as described (van Roessel, P., and A. Brand. 2000.
GAL4-mediated ectopic gene expression in Drosophila. In Drosophila
Protocols. W. Sullivan, M. Ashburner, and R. Hawley, editors. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 439-448.).
The GAL4 protein is a yeast transcription factor capable of
activating transcription of Drosophila genes which have been
engineered to contain upstream sequences recognized by the GAL4
protein. Various mutants can be created with a gene of interest
expressed in specific tissue distributions, a construct containing
the gene of interest (reporter) under regulation of a GAL4
containing promoter is introduced into embryos, and a genetic
marker allows identification of progeny containing this construct.
Illustrative GAL4 containing promoters include, but are not limited
to, pUAS. The use of embryos of a strain containing an active
P-transposase increases the efficiency of transgene integration,
although many of the embryos die. These progeny can then be crossed
to various available lines containing GAL4 transgenes (driver)
expressed under control of tissue-specific promoters. In one aspect
of the present invention, GAL4 driver constructs which allow
expression during embryogenesis are used.
[0100] The following Examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
Example 1
Isolation and Characterization of SPL cDNA from Yeast
[0101] This Example illustrates the preparation of an S. cerevisiae
cDNA molecule encoding an endogenous SPL polypeptide.
[0102] Wild-type yeast cells (SGP3 (Garrett and Broach, Genes and
Dev. 3:1336-1348, 1989); leu2-3,112 trp1 ura3-52 his3 ade8
ras1::HIS3) were transformed with a yeast genomic library carried
on the pRS202 high-copy shuttle vector (Sikorski and Heiter,
Genetics 122:19-27, 1989) containing a selectable nutritional
marker (URA3). pRS202 is a modified version of the pRS306 vector,
into which a 2 micron plasmid piece was inserted. Inserts from this
library are approximately 6-8 kb in length. Wild type yeast were
transformed with the high copy library as described by Ito et al.,
J. Bact. 153:163-68, 1983, selected for uracil prototrophy (i.e.,
the ability to grow on medium lacking uracil), and transformants
were pooled and replated at a concentration of 10.sup.6 cells per
plate onto 1 mM D-erythro-sphingosine plates.
[0103] Six transformants which grew large colonies on 1 mM
D-erythro-sphingosine plates were grown in selective medium, and
control SGP3 colonies were grown in minimal medium, at 30.degree.
C. until saturated. Absorbance at 660 nm was used to correct for
small variations in cell concentration between cultures. Serial
dilutions were performed, and cells were template-inoculated onto 1
mM D-erythro-sphingosine plates and incubated at 30.degree. C. for
48 hours.
[0104] The most highly represented insert, 13-1, was subcloned and
sequenced, and named BST1 (bestower of sphingosine tolerance;
GenBank accession number U51031; Saccharomyces cerevisiae genome
database accession number YDR294C). The BST1 nucleotide sequence
encodes a previously unknown predicted protein of 65,523
kilodaltons and 589 amino acids in length. This sequence is 23%
identical to gadA and gadB, two nearly identical E. coli genes
encoding glutamate decarboxylase (GAD), a
pyridoxal-5'-phosphate-dependent enzyme which catalyzes synthesis
of the neurotransmitter .gamma.-amino butyric acid. BST1 has been
localized to S. cerevisiae chromosome 4. The DNA sequence of BST1
is provided in SEQ ID NO:1, which encodes the amino acid sequence
set forth in SEQ ID NO:2.
[0105] To explore the function of BST1, a deletion strain was
created through homologous recombination using a NEO selectable
marker (Wach et al., Yeast 10:1793-1808, 1994). Genomic BST1 was
replaced with kanMX (Wach et al., Yeast 10:1793-1808, 1994), which
confers resistance to G418. Disruption was confirmed using PCR
amplification of genomic DNA from G418 resistant clones, using
primers to genomic sequence just 5' and 3' to the region replaced
by the disruption. Deletion of BST1 and all subsequent biological
studies were performed in both SGP3 and in JK93d (Hietman et al.,
Proc. Natl. Acad. Sci. USA 88:1948-52, 1991); ura3-52 leu2-3,112
his4 trp1 rme1). Heterozygous diploids were sporulated, and spores
segregated 2:2 for G418 resistance. Both G418 resistant and
sensitive progeny were viable, indicating that BST1 is not an
essential gene.
[0106] Analysis of GAD activity in cytosolic extracts from wild
type, BST1 overexpression and bst1.DELTA. strains indicated that
BST1 does not encode the S. cervisiae homologue of GAD. However,
deletion of BST1 was associated with severe sensitivity to
D-erythro-sphingosine. Concentrations as low as 10 .mu.M
sphingosine completely inhibited growth of bst1.DELTA. strains but
had no effect on the viability of wild type cells. In comparison to
the control strain, the bst1.DELTA. strain also demonstrated
greater sensitivity to 100 .mu.M phytosphingosine, the long chain
base endogenous to S. cerevisia. No difference between the growth
of wild type and BST1 overexpression strains on phytosphingosine,
which is only minimally toxic to wild type cells at this
concentration, was observed.
[0107] To determine whether differences in sphingosine uptake or
metabolism were responsible for these sensitivity differences, BST1
wild type, overexpression and bst1.DELTA. strains were exposed to
[C3-.sup.3H]labeled sphingosine (American Radiolabeled Chemical,
Inc., St. Louis, Mo.), washed in sterile water and subjected to
Bligh-Dyer extractions (Bligh and Dyer, Can. J. Buichem. Physiol.
37:911-17, 1959). There were no major differences in sphingosine
recovery among the three strains. However, the aqueous phase from
the bst1.DELTA. strain contained a ten-fold increase in
radioactivity over that of control and BST1 overexpression strains.
Thin layer chromatography (TLC) analysis of the lipid fractions in
butanol:acetic acid:water (3:1:1) revealed a sphingosine band which
appeared equivalent in each strain.
[0108] Radioactive sphingosine-1-phosphate (S-1-P) was also
observed in the extracts from the bst1.DELTA. strain, but not in
the wild type or BST1 overexpression strains. This compound
accumulated rapidly, reaching a plateau by 60 minutes. Three
separate TLC conditions were used to confirm the presence of S-1-P.
These conditions, along with the resulting RF values, are shown
below: [0109] butanol:water:acetic acid (3:1:1).47 [0110]
chloroform:methanol:water (60:35:8).22 [0111]
chloroform:methanol:water:acetic acid (30:30:2:5).33
[0112] Hyperaccumulation of S-1-P and hypersensitivity to
D-erythro-sphingosine suggeset a failure to metabolize S-1-P,
indicating that BST1 is a yeast SPL. To confirm this
identification, lyase activity in BST1 wild type, overexpression
and deletion strains were evaluated as described by Veldhoven and
Mannaerts, J. Biol. Chem. 266:12502-07, 1991, using unlabeled
D-erythro-dihydrosphingosine-1-phosphate (Biomol, Plymouth Meeting,
Pa.) and D-erythro-dihydrosphingosine [4,5-.sup.3H]1-phosphate
(American Radiolabeled Chemicals, Inc., St. Louis, Mo.). Specific
activity was 100 mCi/mmol. SPL activity was found to correlate with
BST1 expression, confirming BST1 to be the yeast homologue of
sphingosine-1-phosphate lyase.
[0113] These results indication that BST1 is a yeast SPL, and that
SPL catalyzes a rate-limiting step in sphingolipid catabolism.
Regulation of SPL activity may therefore result in regulation of
intracellular S-1-P levels.
Example 2
Isolation and Characterization of SPL cDNA from C. elegans and
Mouse
[0114] This Example illustrates the identification of endogenous
SPL cDNAs from C. elegans and Mus musculus.
[0115] Comparison of the yeast BST1 sequence to sequences within
the GenBank database identified a full length gene from C. elegans
that was identified during the systematic sequencing of the C.
elegans genome. This cDNA sequence is set forth in SEQ ID NO:3 and
was found to encode SPL, the sequence set forth in SEQ ID NO:4.
This and other DNA homology searches described hereinwere performed
via the National Center for Biotechnology Information website using
BLAST search program.
[0116] Using both S. cerevisiae and C. elegans SPL sequences to
search the EST database, an expressed sequence tag from early
embryonic cells of the mouse (day 8 embryo, strain C57BL/6J) was
identified. The cDNA clone containing this putative mouse SPL was
purchased from Genome Systems, Inc (St. Louis, Mo.). Completion of
the full length cDNA sequence revealed an 1707 bp open reading
frame. This mouse sequence showed significant homology to BST1 and
to other pyridoxal phosphate-binding enzymes such as glutamate
decarboxylase, with greatest conservation surrounding the predicted
pyridoxal phosphate-binding lysine. Since the two genes encoding
mouse glutamate decarboxylase have been identified previously, and
the identified sequence was unique and had no known function, it
was a likely candidate mouse SPL gene.
[0117] To confirm the SPL activity of the mouse gene, a two step
process was undertaken. First, the sequence was cloned into the
high-copy yeast expression vector, pYES2 (Invitrogen, Inc.,
Carlsbad, Calif.), in which the gene of interest is placed under
control of the yeast GAL promoter and is, therefore,
transcriptionally activated by galactose and repressed by glucose.
pYES2 also contains the URA3 gene (which provides transformants the
ability to grow in media without uracil) and an ampicillin
resistance marker and origin of replication functional in E.
coli.
[0118] The expression vector containing the full-length mouse SPL
gene was then introduced into the yeast bst1.DELTA. strain which as
noted above, is extremely sensitive to D-erythro-sphingosine, as a
result of metabolism of sphingosine to S-1-P. S-1-P cannot be
further degraded in the absence of SPL activity and
overaccumulates, causing growth inhibition. Transformation was
performed using the lithium acetate method (Ito et al., J. Bact.
153:163-68, 1983). Transformants were grown on medium containing 20
g/L galactose and selected for uracil prototrophy.
[0119] Transformants were then evaluated for sphingosine
resistance. Strains of interest were grown to saturation in liquid
culture for 2-3 days. They were then resuspended in minimal medium,
placed in the first row of a 96-well plate and diluted serially
from 1:2 to 1:4000 across the plate. The cultures were then
template inoculated onto a control plate (YPD) and a plate
containing minimal synthetic media supplemented with 50 .mu.M
D-erythro-sphingosine (Sigma Chemical Co., St. Louis, Mo.) and
0.0015% NP40 (Sigma Chemical Co.). At this concentration of NP40,
no effects on cell viability were observed. Plates were incubated
at 30.degree. C. for two days and assessed visually for differences
in growth. Transformants containing the mouse SPL gene were
resistant to sphingosine present in galactose-containing plates. A
strain transformed with vector alone remained sensitive to
sphingosine. Therefore, the mouse SPL gene was capable of reversing
the sphingosine-sensitive phenotype of a yeast bst1.DELTA.
strain.
[0120] In order to determine whether the mouse SPL gene was able to
restore biochemical SPL activity to the bst1.DELTA. strain, the
untransformed bst1.DELTA. strain, and the bst1.DELTA. strain
transformed with pYES2 containing either BST1 or the putative mouse
SPL gene were grown to exponential phase (A.sub.600=1.0) in either
minimal (JS16) or uracil medium containing galactose as a carbon
source. Whole cell extracts were prepared from each strain as
described above, adjusted for protein concentration, and evaluated
for sphingosine phosphate lyase activity as described above, using
.sup.3H-dihydrosphingosine-1-phosphate (American Radiolabeled
Chemicals, Inc., St. Louis, Mo.). Qualitative analysis of product
was performed by autoradiography. Quantitative measurement was
performed by scraping TLC plates and determining radioactivity
present using a standard scintillation counter.
[0121] The results of the sphingosine phosphate lyase assays
showned that expression of both the yeast and mouse sequences
restored SPL activity to the bst1.DELTA. strain, whereas vector
alone had no effect, confirming the identity of the mouse sequence
as SPL.
[0122] To determine whether the expression of the mouse SPL
transcript coincided with previously reported tissue-specific SPL
activity in the mouse, total RNA was obtained from a variety of
mouse tissues and probed with the complete mouse SPL cDNA sequence.
Northern analysis was performed as described by Thomas, Proc. Natl.
Acad. Sci. USA 77:5201, 1980, using a full length mouse SPL cDNA
probe labeled by random labeling technique (Cobianchi and Wilson,
Meth. Enzymol. 152:94-110, 1987). This analysis revealed a pattern
of expression consistent with the known SPL activity in various
mouse tissues, providing further confirmation that this sequence
encodes mouse SPL.
Example 3
Isolation and Characterization of Human SPL cDNA
[0123] This Example illustrates the identification of an endogenous
human cDNA.
[0124] An EST database was searched using the mouse SPL sequence
described herein. Two distinct EST sequences having strong homology
to the mouse sequence were identified from human sources. One of
these sequences corresponded to the C-terminus, and the other
corresponded to the N-terminus. Primers were designed based on
these sequences, and a DNA fragment was amplified by PCR from a
human expression library made from human glioblastoma multiforme
tissue RNA. The fragment was sequenced and was shown to contain a
deletion, so the primers were used to amplify the gene from human
fibroblast RNA. This gene has the sequence provided in SEQ ID NO:7
and encodes the polypeptide sequence provided in SEQ ID NO:8. The
cDNA and amino acid sequences of the SPL containing the deletion
are set forth in SEQ ID NOs:9 and 10, respectively.
Example 4
Isolation and Characterization of C. Elegans SPL cDNA
[0125] This Example illustrates the identification of a cDNA
molecule encoding a primary C. elegans sphingosine phosphate
lyase.
[0126] The human SPL cDNA sequence was used to screen the ACEdb C.
elegans genome database. A potential C. elegans open reading frame
of unknown function present on YAC Y66H1B showed substantial (40%)
homology to yeast, human and mouse SPL cDNA sequences. To clone
this sequence, a coupled reverse transcriptase/polymerase chain
reaction was performed using the Access RT-PCR system (see below).
Template was C. elegans total RNA, and primers were:
[0127] 5'-GAGGAATTCATGGATTCGGTTAAGCACACAACCG-3'
[0128] 5'-AGCCTCGAGTTAATTAGAAGTTGAAGGTGGAGC-3'
[0129] This resulted in a DNA fragment cSPL2, which was ligated
into the yeast expression vector pYES2, obtained from Invitrogen.
Inc. (Carlsbad, Calif.). Genes expressed using this system are
regulated under the control of the GAL promoter, which allows
expression in the presence of galactose and not in the presence of
glucose. The nucleotide sequence of cSPL2 is set forth in SEQ ID
NO:12, with the encoded amino acid sequence set forth in SEQ ID
NO:11
[0130] cSPL2 was further analyzed for its ability to complement the
sphingosine sensitive phenotype of a yeast dpl1 mutant, the
previously described yeast strain JS16 which contains a large
deletion in DPL1, the S. cerevisiae sphingosine phosphate lyase
gene (Zhou and Saba, Biochem Biophys Res Commun 242:502-507, 1998).
Transformation of JS16 with pYES2 or the C. elegans SPL-pYES2
construct was performed by the lithium acetate method (Ito et al.,
J. Bact. 153:163-168, 1983). Transformants were selected for uracil
prototrophy and evaluated for sphingosine resistance using the
dilutional assay described by Zhou and Saba, Biochem Biophys Res
Commun 242:502-507, 1998. Cells were grown in minimal or
uracil.sup.- media containing either 20 g glucose or galactose per
liter, as indicated. D-erythro-sphingosine and NP40 were obtained
from Sigma Chemical Company (St. Louis, Mo.).
[0131] The results demonstrate that cSPL2 convincingly complemented
the yeast mutant, restoring enzyme activity. In each plate, yeast
were grown to saturation in overnight liquid cultures, spun down,
resuspended in 200 microliters of water and dispensed into the
first (left-most) well of each horizontal row. Yeast were then
further diluted into sterile water, so the second well was 1:2,
third well was 1:4, fourth well was 1:40, fifth was 1:400 and sixth
was 1:4000 dilution from the original on the left. The toxicity of
sphingosine is cell number dependent, because it disperses itself
in cell membranes. Therefore, the concentration of sphingosine in
the plate is not the only thing affecting toxicity, and these
dilutional assays show differences in tolerance/sensitivity. So, a
strain which can grow in the sixth row is about 4,000 times more
resistant to sphingosine than one which can grow only in the first
row.
[0132] The mutant yeast strain containing cSPL2 also demonstrated
substantial SPL activity. The sphingosine phosphate lyase assay
used whole cell extracts of yeast containing either pYES2 vector
alone or (cSPL2) C. elegans SPL-pYES2. Extracts were prepared as
described by Saba et al., J Biol Chem 272:26087, 1997. SPL activity
was determined essentially as described, using
.sup.3H-dihydrosphingosine-1-phosphate substrate (see Zhou and
Saba, Biochem Biophys Res Commun 242:502-507, 1998). Substrate for
SPL assay (.sup.3H-dihydrosphingosine-1-phosphate) was obtained
from American Radiolabeled Chemicals, Inc. (St. Louis, Mo.). Access
RT-PCR system was obtained from Promega Corp. (Madison, Wis.).
[0133] Enzyme activity in (cSPL2) C. elegans SPL-pYES2 was
appreciably greater than that of the vector control. These results
indicate that cSPL2 encodes the primary C. elegans SPL.
Example 5
Developmental Defects Induced by RNA Interference in C. elegans
[0134] In order to determine the effect of blocking cSPL2
expression on the development of C. elegans, RNA interference
studies were undertaken. The cSPL2 cDNA was cloned into pBluescript
such that the insert was flanked by the T3 and T7 promoter regions.
RNA complementary to each strand was synthesized from these
promoters using an in vitro transcription kit (Promega, Madison,
Wis.). The two strands were annealed to make double stranded RNA
(dsRNA) and injected into the distal gonads of 12 wild-type (N2
Bristol) young adult C. elegans hermaphrodites. As controls,
uninjected hermaphrodites as well as hermaphrodites injected with a
dsRNA that does not produce a visible phenotype were handled in
parallel. Eight hours after injection, each hermaphrodite was
transferred to a fresh culture plate and 12 hour cohorts of F1
progeny were established. Progeny were observed daily with a
dissecting microscope until most animals reached adulthood and the
culture plates became too crowded with F2 progeny. Compared to
control F1s, animals inheriting cSPL-2 dsRNA developed slowly,
moved sluggishly, were thin and pale, and did not pump food
actively. These animals reach adulthood approximately 24 hours
later than controls. Adult hermaphrodites that inherited cSPL-2
dsRNA were markedly different from controls especially in the gonad
and uterus. Control animals had abundant nuclei in the distal gonad
and a row of developing oocytes in the proximal gonad. Affected
hermaphrodites had poorly developed distal gonads with fewer
nuclei. Control adults had embryos of progressive stages of
development in the uterus, whereas the number of developing oocytes
in the proximal gonad of affected hermaphrodites was reduced. The
embryos in the uterus of affected progeny were also abnormal. Those
near the vulva were at late developmental stages indicating a
defect in egg laying. There was not a uniform progression of
developmental stages in adjacent embryos suggesting a defect in
ovulation or development, and some of the embryos showed abnormal
patterns of cell division. In summary, inhibition of C. elegans SPL
expression through the use of RNA interference leads to poor
feeding, developmental abnormalities and impaired fertility in the
progeny. These results suggest that SPL is an essential gene in C.
elegans.
Example 6
Isolation and Characterization of SPL cDNA from Drosophila
melanogaster
[0135] In order to seek out the Drosophila melanogaster SPL cDNA
and genomic sequence, the D. melanogaster genomic database was
searched for sequences which demonstrated significant homology to
human SPL cDNA. This led to identification of two full-length cDNA
clones (LP04413 and GH3783) which were confirmed by sequence and
restriction analysis. The two clones are predicted based on
alternative 5' exon usage and may be expressed in different
subcellular locations. The predicted Drosophila melanogaster SPL is
located on the right arm of chromosome II, position 53F8-12. The
cDNA sequence for Drosophila melanogaster SPL is set forth in SEQ
ID NO:15 and encodes the SPL protein set forth in SEQ ID NO:16. The
Drosophila SPL predicted protein sequence set forth in SEQ ID NO:16
is 49%, 49% and 43% identical to human, mouse and yeast SPL protein
sequences, respectively.
[0136] In order to evaluate whether these clones contained a
functional SPL gene, they were recloned into the yeast expression
vector, pYES2, and this construct was transformed into a
dpl1.DELTA. strain. Expression of clones containing the potential
Drosophila melanogaster SPL fully complement the dpl1.DELTA.
strain's sensitivity to 50 .mu.M D-erythro-sphingosine. Further,
whole cell extracts of dpl1 strains containing either pYES2-LP04413
or pYES2-GH3783 demonstrate restoration of SPL enzyme activity to
wild type levels or greater, although not as high as a DPL1
overexpressing strain (DPL OE).
Example 7
Generation and Characterization of SPL Transposon Mutant D.
melanogaster
[0137] Flies heterozygous for a P-element transposon which sits in
the coding region of both of the above transcripts described in
Example 6 and presumably disrupts both SPL proteins were obtained
from the Drosophila Genome Project. These flies were genetically
crossed using techniques well known to ordinarily skilled artisans,
and progeny were evaluated for the presence of homozygous
insertional mutants (based on presence of rosy eye color, encoded
by a recessive marker carried on the P-element). Northern blot
analysis from wild type and SPL insertional mutant flies indicated
that no SPL gene expression occurred in the latter.
[0138] To determine the SPL function of each genotype, +/+, +/-and
-/- flies were homogenized and whole extracts assayed for SPL
activity. It was observed that SPL genotype corresponded with SPL
activity with +/+>+/->-/-. Initial evaluation of homozygous
mutants indicated that adult SPL mutants were flightless,
suggesting a potential defect in either muscle development or
energetics of the adult fly. Flight analysis was carried out
essentially as described (Vigoreaux, J., J. Saide, K.
Valgeirdottir, and M. Pardue. 1993. Flightin, a novel myofibrillar
protein of Drosophila stretch-activated muscles. J. Cell Biol.
121:587-598) by determining the percentage of flies that were
flightless or exhibited downward, upward, or lateral flight
capabilities in control Canton-S flies as compared to mutant
flies.
[0139] The transposon was mobilized by crossing SPL mutant flies
with flies carrying an actively transcribed transposase gene, which
caused the P-element to be excised in the chromosomes of both
somatic cells and in the germline. Germline transposon loss is
heritable and was identified in progeny by virtue of eye color.
Progeny which lost both the transposase gene and the P-element were
then isolated and the restored SPL allele was homozygosed. Progeny
which had lost the P-element at the SPL locus demonstrated
restoration of flight, indicating that the phenotype correlated
with the P-element insertional mutation. To determine the etiology
of the Sightlessness of -/- flies, flies were sectioned through the
thoracic region and indirect flight muscles were evaluated by both
light and electron microscopy. These studies revealed a reduced
number of muscle fibers comprising the dorsal longitudinal muscles
with evidence of what appears to be compensatory hypertrophy in the
fibers which remained. Electron microscopy revealed no
ultrastructural defects in the myocytes which remained.
[0140] In order to determine whether the loss of SPL expression was
due to excess accumulation of S-1-P in the developing adult fly, we
salvaged the developing flight muscles of homozygous SPL mutant
progeny by adding D,L-threo-dihydrosphingosine, an inhibitor of
mammalian sphingosine kinase, to the growth media. A significant
proportion of homozygous SPL mutant progeny demonstrated
restoration of flight when grown on media supplemented with
D,L-threo-dihydrosphingosine.
[0141] Northern analysis was performed to investigate SPL
expression throughout development. These studies indicated that SPL
expression begins at 8-12 hours of embryonic development and
remains detectible throughout larval stages and pupation.
[0142] Therefore, the Drosophila melanogaster model described
herein can be used to identify pharmacologic suppressors of SPL
mutant flies' inability to fly. Drugs which alter SPL activity or
expression may be effective treatment for at least some kinds of
cancer. Therefore, the fact that a fruitfly SPL null mutant
containing a P-element insertion within the SPL coding region is
flightless provides an excellent model in which to screen and
identify compounds that modulate SPL activity. Thus, other
chemicals created through rational drug design approaches can be
screened using this method. The Drosophila melanogaster model
described herein can thus be used to screen an array of rationally
designed chemicals with homology to sphingolipids for their ability
to restore flight to SPL mutant progeny. Candidate drugs identified
using this method can then be further evaluated in an in vitro
yeast screen.
Example 8
Further Characterization of Developmental Expression Patterns of
SPL in SPL Transposon Mutant D. melanogaster
[0143] Northern analysis is carried out and extended to include
adult samples, and blots are reprobed with SPL specific probes
using the following approaches. Once genes are confirmed to encode
the predicted enzyme, DNA probes or riboprobes for SPL and S-1-P
phosphatase are labeled either radioactively or with digoxygenin.
For Northern analysis, full-length probes are labeled by random
priming with [.alpha.-.sup.32P]dATP. Hybridization is carried out
under standard conditions against an RNA blot prepared from total
RNA of flies harvested at different stages of development (embryos
at hours 0-4, 4-8, 8-12, 12-24, larval instars 1.sup.st, 2.sup.nd,
3.sup.rd, early and late pupal stages, and adults). For in situ
hybridization purposes, .sup.3H labeling is the most sensitive
approach, and the very low energy of the beta particle emitted
causes it to travel only short distances through the radiographic
emulsion, allowing precise localization for the probe. However,
digoxygenin labeling provides the advantage of being able to
visualize hybridization with much higher spatial resolution because
of the ability to directly visualize the tissue. Random primer
labeling of DNA are performed with either tritium or digoxygenin
labeled nucleotides. In situ hybridization is performed as
described in Blair, S. (Blair S., 2000. Imaginal discs. In
Drosophila Protocols. W. Sullivan, M. Ashburner, and R. Hawley,
editors. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. 159-175), hereby incorporated by reference in its
entirety.
Example 9
Characterization of Sphingolipid Species in the Drosophila
melanogaster
[0144] Without being bound by theory, it is hypothesized that the
phenotype of the SPL mutant Drosophila is caused by an abnormal
level of S-1-P during development. Further, without being bound by
theory, it is the inventors hypothesis that phosphorylated
sphingoid base species are responsible for regulating cell
proliferation, migration and other events required for both tumor
formation and normal developmental processes in this model
organism. Therefore, characterization of sphingolipid species in
Drosophila was determined.
[0145] Method: Wild type (Canton S) whole fly extracts were
prepared by mechanical disruption. Lipids were isolated by
two-phase extraction and derivatized with the fluorescent molecule
o-pthalaldehyde essentially as described in Caligan, et al. hereby
incorporated by reference in its entirety (Caligan, T. B., K.
Peters, J. Ou, E. Wang, J. Saba, and A. H. Merrill, Jr. 2000. A
high-performance liquid chromatographic method to measure
sphingosine 1-phosphate and related compounds from sphingosine
kinase assays and other biological samples. Analytical
Biochemistry. 281:36-44). Derivatized lipid extracts were separated
by HPLC using a C.sub.18 ODS column (LUNA 4.6.times.250 mm) and
mobile phase MeOH/H.sub.20/1M TBAP 82:17:0.9, pH 4.8. Standards
included commercially available C.sub.10, C.sub.12, C.sub.14,
C.sub.16, C.sub.18 and C.sub.20 sphingosines, as well as the
phosphorylated forms of these standards, prepared by incubation of
sphingosine standards with extract from a yeast strain which
overexpresses the major yeast sphingosine kinase, LCB4.
[0146] Results: Drosophila extracts contained only sphingolipid
species which comigrated with C.sub.14 sphingosine and C.sub.14
sphingosine-1-phosphate (S-1-P) standards under the stated
conditions. To verify the identity of the peaks in fly extracts
which comigrated with C.sub.14 sphingosine and C.sub.14S-1-P
standards, extracts and standards were compared in four different
mobile phase buffers. The peak identified as C.sub.14 sphingosine
comigrated with the C.sub.14 sphingosine standard under all four
conditions (Table 1). However, the peak identified as C.sub.14S-1-P
demonstrated a slight difference from the C.sub.14S-1-P standard
under conditions which exploit differences in charge (MeOH/10 mM
KP/1 M TBAP, pH 7.2, 81:18:1). TABLE-US-00001 TABLE 1 Sphingolipid
Identification C.sub.14S C.sub.14S-1-P Mobile Phase C.sub.14S std
in extract C.sub.14S-1-P std in extract MeOH/H.sub.20/1M 19.1 min
19.0 min 14.8 min 14.8 min TBAP pH 4.8 82.1:17:0.9 MeOH/H.sub.20/1M
27.3 min 27.1 min 22.5 min 22.1 min TBAP pH 4.8 79.1:20:0.9 MeOH/10
mM KP/ 21.9 min 22.0 min 18.3 min 17.2 min 1M TBAP pH 5.5 81:18.1
MeOH/10 mM KP/ 21.4 min 21.8 min 15.0 min 17.1 min 1M TBAP pH 7.2
81:18.1
[0147] This finding is likely to be due to a chemical modification
of the phosphate group, since a phosphatase capable of
dephosphorylating the C.sub.14S-1-P standard does not recognize
this substrate. Mass spectroscopy is utilized to identify the
phosphate group modification of this S-1-P species. Herein, this
sphingolipid is referred to as "modified C.sub.14S-1-P."
Example 10
Characterization of Sphingolipid Species in the Drosophila SPL
Mutant
[0148] Differences in the quantity or type of sphingolipid species
present in mutant versus wild type adult flies and during various
stages of development was determined as described below.
[0149] Methods were as described in Example 9.
[0150] Results: The modified C.sub.14S-1-P peak was ten-fold higher
in the Drosophila SPL mutant than in the wild type (using an
internal standard to normalize for extraction variation),
supporting the notion that the phenotype of the SPL mutant may be
due to abnormal accumulation of phosphorylated sphingoid bases and
resulting abnormalities in signaling. C.sub.14 sphingosine was also
increased in the mutant, but to a lesser extent (Table 2). No other
peaks in the mutant demonstrated a significant difference in
comparison to wild type controls. TABLE-US-00002 TABLE 2
Sphingolipid Quantification (nmol/200 mg flies) Line (n = 3)
modified C.sub.14S-1-P C.sub.14S Canton S (wild type) 0.49 .+-.
0.07 2.61 .+-. 0.27 SPL mutant 4.49 .+-. 0.53 5.27 .+-. 0.73
Example 11
Characterization of the SPL Activity Encoded by ESTs LP04413/GH3783
and Which is Absent in Insertional Mutant 11393
[0151] Drosophila ESTs LP04413 and GH3783 encode a protein with
strong homology to other sphingosine phosphate lyases (SPL). Mutant
11393 which demonstrates the flight defect and dorsal longitudinal
muscle (DLM) abnormalities described above in Example 7, contains a
p-element insertion within this locus. Initial results using a
standard SPL assay and a radiolabelled C.sub.18DHS-1-P substrate
indicated that Drosophila ESTs LP04413 and GH3783 encode an SPL,
since expression restored SPL activity to a yeast SPL mutant.
However, the activity conferred by the EST expression in yeast was
not pronounced. Since Drosophila extracts contain C.sub.14
sphingosine and a modified species of C.sub.14S-1-P, it was
hypothesized that the C.sub.18DHS-1-P was not a favorable substrate
for the major Drosophila lyase. Further, residual lyase activity
observed in the mutant indicated the presence of more than one SPL
activity in Drosophila. Therefore, the optimal substrate of the SPL
encoded by ESTs LP04413 and GH3783 was determined and this activity
was differentiated from other SPL activities in Drosophila.
[0152] Methods: Wild type (Canton S) and mutant whole fly extracts
were prepared by mechanical disruption. Standard SPL assays using
C.sub.18 DHS-1-P substrate were performed as previously described
(Van Veldhoven, P. P., and G. P. Mannaerts. 1991. Subcellular
localization and membrane topology of sphingosine-1-phosphate lyase
in rat liver. J Biol. Chem. 266:12502-12507). An HPLC-based SPL
assay was established, to allow for various non-radioactive
substrates to be evaluated. For this assay, C.sub.14S-1-P,
C.sub.18DHS-1-P and modified C.sub.14S-1-P were prepared by drying
down the lipid extract from 15 mg of 11939 flies, plus 200 .mu.mol
C.sub.14S-1-P standard and 200 .mu.mol C.sub.18DHS-1-P standard.
Lipids were resuspended in 25 .mu.l of 1% Triton X-100 in potassium
phosphate buffer, pH 7.4. 175 .mu.l of reaction buffer (KP buffer,
NaF, DTT, EDTA, sucrose) were added, and mixture was tip sonicated
for 20 seconds, followed by addition of 50 .mu.g of protein from
whole cell extract of flies (CS or 11939) or Adpl1:lcb4 yeast
overexpressing the fly lyase. Incubation proceeded for 1 hr at
37.degree. C. Reaction was stopped by adding 175 .mu.l of MeOH
containing 0.2% acetic acid. The reaction was applied to STRATA C18
column in 40% MeOH containing 0.1% acetic acid. The column was
washed with 600 .mu.l of 40% MeOH containing 0.1% acetic acid.
Lipids were eluted with 1 ml of 90% MeOH/10% 10 mM K-Phosphate, pH
7.2. Samples were dried and resuspended in MeOH, treated with
o-pthalaldehyde and injected on the HPLC. The degradation of S-1-P
standards and modified C.sub.14S-1-P were compared to standards
incubated in the absence of protein extracts.
[0153] Results: An activity which metabolizes modified
C.sub.14S-1-P is present in wild type fly extracts but is absent in
the mutant fly extracts. Residual SPL activity does exist in the
mutant fly. This activity is distinct from that encoded by
LP04413/GH3783, in that it metabolizes C.sub.14S-1-P and
C.sub.18DHS-1-P with an efficacy similar to or better than wild
type. The pH curve of the residual SPL activity in mutant flies is
identical to that seen in wild type flies (against a
C.sub.18DHS-1-P substrate), indicating that this activity is not
disrupted in the mutant.
Example 12
Further Characterization of the Drosophila melanogaster SPL Mutant
Phenotype
[0154] Adult SPL mutant flies demonstrated inability to fly and
abnormal patterning of indirect flight muscles. The adult SPL
mutant flies consistently demonstrated abnormal patterning of DLMs,
although the number of remaining DLMs varied in each mutant. In
this Example, it was determined whether the abnormal muscle
development was limited to the adult fly, or whether the defect was
also present at earlier developmental stages.
[0155] Methods: Larval locomotor assay. Third instar larvae were
placed on a clear agar substrate that overlays a grid. A light
source at one end provided a photactic stimulus. Distance traveled
was scored during three minute trials. Larval muscle microscopy.
Larvae were filleted during the third instar and pinned with the
dorsal cuticle down. The viscera were removed to allow an
unobstructed view of the body wall muscles using polarized light.
Muscles were refractile due to the presence of filamentous arrays
in each muscle fiber.
[0156] Results: 11393 mutant larvae demonstrated significant
defects in locomotion in comparison to wild type larvae, although
phototactic response is intact. In all mutant larvae examined, the
T2-dorsal oblique muscles exhibited alterations in number and/or
size. Fused, hypertrophied residual dorsal obliques were observed
in the mutants.
[0157] Since the four pairs of dorsal obliques in thoracic segment
two create scaffolds which give rise during pupation to the DLM
structures of the adult, it is likely that the developmental defect
seen in the adult is the result of a process which begins much
earlier, during larval development or embryogenesis.
Example 13
Human SPL Expression Patterns in Cancer
[0158] To determine if SPL expression is altered in human tumors,
we utilized a cancer profiling array which contains more than 240
cDNA pairs representing tumor tissue and corresponding normal
tissue from the same patient. By utilizing tissue pairs from one
patient, differences between gene expression in tumor and normal
tissue which might be due to person to person variability should
not confound the interpretation of results. Additionally, each blot
was normalized for loading using four separate housekeeping genes.
Traditional hybridization techniques were utilized to probe this
cDNA blot with a 300 nucleotide 3' fragment of human SPL cDNA (SEQ
ID NO:7), which was obtained from the previously described cloning
experiments. Analysis of the array indicated that, whereas human
SPL expression is matched closely in most tissue pairs, it is
significantly reduced in colon cancer specimens, with a 50%
reduction in expression in colloid cancer of the colon and 61%
reduction in adenocarcinoma of the colon. Reduced SPL expression
was also seen in adenocarcinaom of the uterus. None of the tumors
in which SPL expression is diminished demonstrate SK
overexpression. Thus, altered SPL expression is observed in primary
human tumors. Therefore, modulating the activity of SPL protein
either by altering gene expression or through direct action on the
protein may provide a useful treatment for individuals afflicted
with an SPL-related cancer. Furthermore, SPL expression serves as a
useful diagnostic marker of cancer in humans.
[0159] All of the above U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign
patent applications and non-patent publications referred to in this
specification and/or listed in the Application Data Sheet, are
incorporated herein by reference, in their entirety.
[0160] From the foregoing it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
21 1 1770 DNA S. cerevisiae CDS (1)...(1770) 1 atg agt gga gta tca
aat aaa aca gta tca att aat ggt tgg tat ggc 48 Met Ser Gly Val Ser
Asn Lys Thr Val Ser Ile Asn Gly Trp Tyr Gly 1 5 10 15 atg cca att
cat tta cta agg gaa gaa ggc gac ttt gcc cag ttt atg 96 Met Pro Ile
His Leu Leu Arg Glu Glu Gly Asp Phe Ala Gln Phe Met 20 25 30 att
cta acc atc aac gaa tta aaa ata gcc ata cat ggt tac ctc aga 144 Ile
Leu Thr Ile Asn Glu Leu Lys Ile Ala Ile His Gly Tyr Leu Arg 35 40
45 aat acc cca tgg tac aac atg ttg aag gat tat ttg ttt gtg atc ttt
192 Asn Thr Pro Trp Tyr Asn Met Leu Lys Asp Tyr Leu Phe Val Ile Phe
50 55 60 tgt tac aag cta ata agt aat ttt ttt tat ctg ttg aaa gtt
tat ggg 240 Cys Tyr Lys Leu Ile Ser Asn Phe Phe Tyr Leu Leu Lys Val
Tyr Gly 65 70 75 80 ccg gtg agg tta gca gtg aga aca tac gag cat agt
tcc aga aga ttg 288 Pro Val Arg Leu Ala Val Arg Thr Tyr Glu His Ser
Ser Arg Arg Leu 85 90 95 ttt cgt tgg tta ttg gac tca cca ttt ttg
agg ggt acc gta gaa aag 336 Phe Arg Trp Leu Leu Asp Ser Pro Phe Leu
Arg Gly Thr Val Glu Lys 100 105 110 gaa gtc aca aag gtc aaa caa tcg
atc gaa gac gaa cta att aga tcg 384 Glu Val Thr Lys Val Lys Gln Ser
Ile Glu Asp Glu Leu Ile Arg Ser 115 120 125 gac tct cag tta atg aat
ttc cca cag ttg cca tcc aat ggg ata cct 432 Asp Ser Gln Leu Met Asn
Phe Pro Gln Leu Pro Ser Asn Gly Ile Pro 130 135 140 cag gat gat gtt
att gaa gag cta aat aaa ttg aac gac ttg ata cca 480 Gln Asp Asp Val
Ile Glu Glu Leu Asn Lys Leu Asn Asp Leu Ile Pro 145 150 155 160 cat
acc caa tgg aag gaa gga aag gtc tct ggt gcc gtt tac cac ggt 528 His
Thr Gln Trp Lys Glu Gly Lys Val Ser Gly Ala Val Tyr His Gly 165 170
175 ggt gat gat ttg atc cac tta caa aca atc gca tac gaa aaa tat tgc
576 Gly Asp Asp Leu Ile His Leu Gln Thr Ile Ala Tyr Glu Lys Tyr Cys
180 185 190 gtt gcc aat caa tta cat ccc gat gtc ttt cct gcc gta cgt
aaa atg 624 Val Ala Asn Gln Leu His Pro Asp Val Phe Pro Ala Val Arg
Lys Met 195 200 205 gaa tcc gaa gtg gtt tct atg gtt tta aga atg ttt
aat gcc cct tct 672 Glu Ser Glu Val Val Ser Met Val Leu Arg Met Phe
Asn Ala Pro Ser 210 215 220 gat aca ggt tgt ggt acc aca act tca ggt
ggt aca gaa tcc ttg ctt 720 Asp Thr Gly Cys Gly Thr Thr Thr Ser Gly
Gly Thr Glu Ser Leu Leu 225 230 235 240 tta gca tgt ctg agc gct aaa
atg tat gcc ctt cat cat cgt gga atc 768 Leu Ala Cys Leu Ser Ala Lys
Met Tyr Ala Leu His His Arg Gly Ile 245 250 255 acc gaa cca gaa ata
att gct ccc gta act gca cat gct ggg ttt gac 816 Thr Glu Pro Glu Ile
Ile Ala Pro Val Thr Ala His Ala Gly Phe Asp 260 265 270 aaa gct gct
tat tac ttt ggc atg aag cta cgc cac gtg gag cta gat 864 Lys Ala Ala
Tyr Tyr Phe Gly Met Lys Leu Arg His Val Glu Leu Asp 275 280 285 cca
acg aca tat caa gtg gac ctg gga aaa gtg aaa aaa ttc atc aat 912 Pro
Thr Thr Tyr Gln Val Asp Leu Gly Lys Val Lys Lys Phe Ile Asn 290 295
300 aag aac aca att tta ctg gtc ggt tcc gct cca aac ttt cct cat ggt
960 Lys Asn Thr Ile Leu Leu Val Gly Ser Ala Pro Asn Phe Pro His Gly
305 310 315 320 att gcc gat gat att gaa gga ttg ggt aaa ata gca caa
aaa tat aaa 1008 Ile Ala Asp Asp Ile Glu Gly Leu Gly Lys Ile Ala
Gln Lys Tyr Lys 325 330 335 ctt cct tta cac gtc gac agt tgt cta ggt
tcc ttt att gtt tca ttt 1056 Leu Pro Leu His Val Asp Ser Cys Leu
Gly Ser Phe Ile Val Ser Phe 340 345 350 atg gaa aag gct ggt tac aaa
aat ctg cca tta ctt gac ttt aga gtc 1104 Met Glu Lys Ala Gly Tyr
Lys Asn Leu Pro Leu Leu Asp Phe Arg Val 355 360 365 ccg gga gtc acc
tca ata tca tgt gac act cat aaa tat gga ttt gca 1152 Pro Gly Val
Thr Ser Ile Ser Cys Asp Thr His Lys Tyr Gly Phe Ala 370 375 380 cca
aaa ggc tcg tca gtt ata atg tat aga aac agc gac tta cga atg 1200
Pro Lys Gly Ser Ser Val Ile Met Tyr Arg Asn Ser Asp Leu Arg Met 385
390 395 400 cat cag tat tac gta aat cct gct tgg act ggc ggg tta tat
ggc tct 1248 His Gln Tyr Tyr Val Asn Pro Ala Trp Thr Gly Gly Leu
Tyr Gly Ser 405 410 415 cct aca tta gca ggg tcc agg cct ggt gct att
gtc gta ggt tgt tgg 1296 Pro Thr Leu Ala Gly Ser Arg Pro Gly Ala
Ile Val Val Gly Cys Trp 420 425 430 gcc act atg gtc aac atg ggt gaa
aat ggg tac att gag tcg tgc caa 1344 Ala Thr Met Val Asn Met Gly
Glu Asn Gly Tyr Ile Glu Ser Cys Gln 435 440 445 gaa ata gtc ggt gca
gca atg aag ttt aaa aaa tac atc cag gaa aac 1392 Glu Ile Val Gly
Ala Ala Met Lys Phe Lys Lys Tyr Ile Gln Glu Asn 450 455 460 att cca
gac ctg aat ata atg ggc aac cct aga tat tca gtc att tca 1440 Ile
Pro Asp Leu Asn Ile Met Gly Asn Pro Arg Tyr Ser Val Ile Ser 465 470
475 480 ttt tct tca aag acc ttg aac ata cac gaa cta tct gac agg ttg
tcc 1488 Phe Ser Ser Lys Thr Leu Asn Ile His Glu Leu Ser Asp Arg
Leu Ser 485 490 495 aag aaa ggc tgg cat ttc aat gcc cta caa aag ccg
gtt gca cta cac 1536 Lys Lys Gly Trp His Phe Asn Ala Leu Gln Lys
Pro Val Ala Leu His 500 505 510 atg gcc ttc acg aga ttg agc gct cat
gtt gtg gat gag atc tgc gac 1584 Met Ala Phe Thr Arg Leu Ser Ala
His Val Val Asp Glu Ile Cys Asp 515 520 525 att tta cgt act acc gtg
caa gag ttg aag agc gaa tca aat tct aaa 1632 Ile Leu Arg Thr Thr
Val Gln Glu Leu Lys Ser Glu Ser Asn Ser Lys 530 535 540 cca tcc cca
gac gga act agc gct cta tat ggt gtc gcc ggg agc gtt 1680 Pro Ser
Pro Asp Gly Thr Ser Ala Leu Tyr Gly Val Ala Gly Ser Val 545 550 555
560 aaa act gct ggc gtt gca gac aaa ttg att gtg gga ttc cta gac gca
1728 Lys Thr Ala Gly Val Ala Asp Lys Leu Ile Val Gly Phe Leu Asp
Ala 565 570 575 tta tac aag ttg ggt cca gga gag gat acc gcc acc aag
tag 1770 Leu Tyr Lys Leu Gly Pro Gly Glu Asp Thr Ala Thr Lys * 580
585 2 589 PRT S. cerevisiae 2 Met Ser Gly Val Ser Asn Lys Thr Val
Ser Ile Asn Gly Trp Tyr Gly 1 5 10 15 Met Pro Ile His Leu Leu Arg
Glu Glu Gly Asp Phe Ala Gln Phe Met 20 25 30 Ile Leu Thr Ile Asn
Glu Leu Lys Ile Ala Ile His Gly Tyr Leu Arg 35 40 45 Asn Thr Pro
Trp Tyr Asn Met Leu Lys Asp Tyr Leu Phe Val Ile Phe 50 55 60 Cys
Tyr Lys Leu Ile Ser Asn Phe Phe Tyr Leu Leu Lys Val Tyr Gly 65 70
75 80 Pro Val Arg Leu Ala Val Arg Thr Tyr Glu His Ser Ser Arg Arg
Leu 85 90 95 Phe Arg Trp Leu Leu Asp Ser Pro Phe Leu Arg Gly Thr
Val Glu Lys 100 105 110 Glu Val Thr Lys Val Lys Gln Ser Ile Glu Asp
Glu Leu Ile Arg Ser 115 120 125 Asp Ser Gln Leu Met Asn Phe Pro Gln
Leu Pro Ser Asn Gly Ile Pro 130 135 140 Gln Asp Asp Val Ile Glu Glu
Leu Asn Lys Leu Asn Asp Leu Ile Pro 145 150 155 160 His Thr Gln Trp
Lys Glu Gly Lys Val Ser Gly Ala Val Tyr His Gly 165 170 175 Gly Asp
Asp Leu Ile His Leu Gln Thr Ile Ala Tyr Glu Lys Tyr Cys 180 185 190
Val Ala Asn Gln Leu His Pro Asp Val Phe Pro Ala Val Arg Lys Met 195
200 205 Glu Ser Glu Val Val Ser Met Val Leu Arg Met Phe Asn Ala Pro
Ser 210 215 220 Asp Thr Gly Cys Gly Thr Thr Thr Ser Gly Gly Thr Glu
Ser Leu Leu 225 230 235 240 Leu Ala Cys Leu Ser Ala Lys Met Tyr Ala
Leu His His Arg Gly Ile 245 250 255 Thr Glu Pro Glu Ile Ile Ala Pro
Val Thr Ala His Ala Gly Phe Asp 260 265 270 Lys Ala Ala Tyr Tyr Phe
Gly Met Lys Leu Arg His Val Glu Leu Asp 275 280 285 Pro Thr Thr Tyr
Gln Val Asp Leu Gly Lys Val Lys Lys Phe Ile Asn 290 295 300 Lys Asn
Thr Ile Leu Leu Val Gly Ser Ala Pro Asn Phe Pro His Gly 305 310 315
320 Ile Ala Asp Asp Ile Glu Gly Leu Gly Lys Ile Ala Gln Lys Tyr Lys
325 330 335 Leu Pro Leu His Val Asp Ser Cys Leu Gly Ser Phe Ile Val
Ser Phe 340 345 350 Met Glu Lys Ala Gly Tyr Lys Asn Leu Pro Leu Leu
Asp Phe Arg Val 355 360 365 Pro Gly Val Thr Ser Ile Ser Cys Asp Thr
His Lys Tyr Gly Phe Ala 370 375 380 Pro Lys Gly Ser Ser Val Ile Met
Tyr Arg Asn Ser Asp Leu Arg Met 385 390 395 400 His Gln Tyr Tyr Val
Asn Pro Ala Trp Thr Gly Gly Leu Tyr Gly Ser 405 410 415 Pro Thr Leu
Ala Gly Ser Arg Pro Gly Ala Ile Val Val Gly Cys Trp 420 425 430 Ala
Thr Met Val Asn Met Gly Glu Asn Gly Tyr Ile Glu Ser Cys Gln 435 440
445 Glu Ile Val Gly Ala Ala Met Lys Phe Lys Lys Tyr Ile Gln Glu Asn
450 455 460 Ile Pro Asp Leu Asn Ile Met Gly Asn Pro Arg Tyr Ser Val
Ile Ser 465 470 475 480 Phe Ser Ser Lys Thr Leu Asn Ile His Glu Leu
Ser Asp Arg Leu Ser 485 490 495 Lys Lys Gly Trp His Phe Asn Ala Leu
Gln Lys Pro Val Ala Leu His 500 505 510 Met Ala Phe Thr Arg Leu Ser
Ala His Val Val Asp Glu Ile Cys Asp 515 520 525 Ile Leu Arg Thr Thr
Val Gln Glu Leu Lys Ser Glu Ser Asn Ser Lys 530 535 540 Pro Ser Pro
Asp Gly Thr Ser Ala Leu Tyr Gly Val Ala Gly Ser Val 545 550 555 560
Lys Thr Ala Gly Val Ala Asp Lys Leu Ile Val Gly Phe Leu Asp Ala 565
570 575 Leu Tyr Lys Leu Gly Pro Gly Glu Asp Thr Ala Thr Lys 580 585
3 1629 DNA C. elegans CDS (1)...(1629) 3 atg gat ttt gca ctg gag
caa tat cat agt gca aag gat ttg tta ata 48 Met Asp Phe Ala Leu Glu
Gln Tyr His Ser Ala Lys Asp Leu Leu Ile 1 5 10 15 ttt gag ctt cga
aag ttc aat cca att gtt ctg gtt tct agt act att 96 Phe Glu Leu Arg
Lys Phe Asn Pro Ile Val Leu Val Ser Ser Thr Ile 20 25 30 gtt gca
aca tac gta ctc acc aat ctg aga cat atg cat tta gat gaa 144 Val Ala
Thr Tyr Val Leu Thr Asn Leu Arg His Met His Leu Asp Glu 35 40 45
atg ggc atc cgg aaa cgt ttg agc act tgg ttt ttc acc act gta aag 192
Met Gly Ile Arg Lys Arg Leu Ser Thr Trp Phe Phe Thr Thr Val Lys 50
55 60 cgt gtg cct ttc atc agg aaa atg att gac aaa caa cta aac gaa
gta 240 Arg Val Pro Phe Ile Arg Lys Met Ile Asp Lys Gln Leu Asn Glu
Val 65 70 75 80 aag gac gag ctt gag aaa agt ctg aga att gtg gat cga
agc acc gaa 288 Lys Asp Glu Leu Glu Lys Ser Leu Arg Ile Val Asp Arg
Ser Thr Glu 85 90 95 tac ttc act aca atc cca agc cat tca gtt gga
aga act gaa gta ctt 336 Tyr Phe Thr Thr Ile Pro Ser His Ser Val Gly
Arg Thr Glu Val Leu 100 105 110 cgc ctt gct gcc atc tat gat gat ttg
gaa gga cca gct ttt ttg gaa 384 Arg Leu Ala Ala Ile Tyr Asp Asp Leu
Glu Gly Pro Ala Phe Leu Glu 115 120 125 gga aga gta tct gga gca gtc
ttc aat aga gaa gac gac aag gac gaa 432 Gly Arg Val Ser Gly Ala Val
Phe Asn Arg Glu Asp Asp Lys Asp Glu 130 135 140 cgg gag atg tat gag
gag gtg ttc gga aaa ttt gcc tgg acc aac cca 480 Arg Glu Met Tyr Glu
Glu Val Phe Gly Lys Phe Ala Trp Thr Asn Pro 145 150 155 160 ctt tgg
cca aaa ttg ttc cct gga gtg aga atc atg gag gct gaa gtt 528 Leu Trp
Pro Lys Leu Phe Pro Gly Val Arg Ile Met Glu Ala Glu Val 165 170 175
gtt cgc atg tgt tgt aat atg atg aat gga gat tcg gag aca tgt gga 576
Val Arg Met Cys Cys Asn Met Met Asn Gly Asp Ser Glu Thr Cys Gly 180
185 190 act atg tca act ggt gga tcc att tca att ctt ttg gcg tgc ctg
gct 624 Thr Met Ser Thr Gly Gly Ser Ile Ser Ile Leu Leu Ala Cys Leu
Ala 195 200 205 cat cgt aat cgt ctt ttg aaa aga gga gaa aag tac aca
gag atg att 672 His Arg Asn Arg Leu Leu Lys Arg Gly Glu Lys Tyr Thr
Glu Met Ile 210 215 220 gtc cca tca tcc gtc cat gca gcg ttc ttc aaa
gct gcc gaa tgt ttc 720 Val Pro Ser Ser Val His Ala Ala Phe Phe Lys
Ala Ala Glu Cys Phe 225 230 235 240 cgt atc aaa gtt cgc aag att cca
gtt gat cct gtt act ttc aaa gta 768 Arg Ile Lys Val Arg Lys Ile Pro
Val Asp Pro Val Thr Phe Lys Val 245 250 255 gac ctt gtc aaa atg aaa
gcc gca att aac aag aga aca tgt atg tta 816 Asp Leu Val Lys Met Lys
Ala Ala Ile Asn Lys Arg Thr Cys Met Leu 260 265 270 gtt gga tct gct
cca aac ttt cca ttt gga act gtt gat gac att gaa 864 Val Gly Ser Ala
Pro Asn Phe Pro Phe Gly Thr Val Asp Asp Ile Glu 275 280 285 gct att
gga cag cta gga ctt gaa tat gac atc cca gtt cat gtt gat 912 Ala Ile
Gly Gln Leu Gly Leu Glu Tyr Asp Ile Pro Val His Val Asp 290 295 300
gct tgt ctt ggt ggt ttc ctt ctt cca ttc ctt gaa gaa gac gag att 960
Ala Cys Leu Gly Gly Phe Leu Leu Pro Phe Leu Glu Glu Asp Glu Ile 305
310 315 320 cgc tat gac ttc cgt gtt cct ggt gta tct tcg att tct gca
gat agt 1008 Arg Tyr Asp Phe Arg Val Pro Gly Val Ser Ser Ile Ser
Ala Asp Ser 325 330 335 cac aaa tac gga ctc gct cca aag ggg tca tca
gtt gtt ctt tat cgc 1056 His Lys Tyr Gly Leu Ala Pro Lys Gly Ser
Ser Val Val Leu Tyr Arg 340 345 350 aat aag gaa ctt ctt cat aat cag
tac ttc tgt gat gct gat tgg caa 1104 Asn Lys Glu Leu Leu His Asn
Gln Tyr Phe Cys Asp Ala Asp Trp Gln 355 360 365 gga ggt atc tat gca
tcg gct act atg gaa gga tca cgc gct ggg cac 1152 Gly Gly Ile Tyr
Ala Ser Ala Thr Met Glu Gly Ser Arg Ala Gly His 370 375 380 aac att
gca ctt tgc tgg gcc gca atg ctt tat cac gct cag gaa gga 1200 Asn
Ile Ala Leu Cys Trp Ala Ala Met Leu Tyr His Ala Gln Glu Gly 385 390
395 400 tac aag gcc aat gct aga aag att gtt gac act aca aga aag att
aga 1248 Tyr Lys Ala Asn Ala Arg Lys Ile Val Asp Thr Thr Arg Lys
Ile Arg 405 410 415 aat gga ctt tca aac att aag gga atc aaa tta caa
ggg cca agt gat 1296 Asn Gly Leu Ser Asn Ile Lys Gly Ile Lys Leu
Gln Gly Pro Ser Asp 420 425 430 gtt tgt att gtt agc tgg aca acc aat
gat gga gtt gaa ctc tac aga 1344 Val Cys Ile Val Ser Trp Thr Thr
Asn Asp Gly Val Glu Leu Tyr Arg 435 440 445 ttc cat aac ttc atg aag
gaa aaa cat tgg caa ctg aat gga ctt caa 1392 Phe His Asn Phe Met
Lys Glu Lys His Trp Gln Leu Asn Gly Leu Gln 450 455 460 ttc cca gct
gga gtt cat atc atg gtc act atg aat cat act cat cct 1440 Phe Pro
Ala Gly Val His Ile Met Val Thr Met Asn His Thr His Pro 465 470 475
480 gga ctc gct gaa gct ttc gtc gcc gat tgc aga gct gca gtt gag ttt
1488 Gly Leu Ala Glu Ala Phe Val Ala Asp Cys Arg Ala Ala Val Glu
Phe 485 490 495 gtc aaa agc cac aaa cca tcg gaa tcc gac aag aca agt
gaa gca gcc 1536 Val Lys Ser His Lys Pro Ser Glu Ser Asp Lys Thr
Ser Glu Ala Ala 500 505 510 atc tac gga ctt gct caa agt att cca gac
cga tcg ctt gtt cac gag 1584 Ile Tyr Gly Leu Ala Gln Ser Ile Pro
Asp Arg Ser Leu Val His Glu 515 520 525 ttt gct cac agc tat atc gat
gct gtt tat gct tta aca gag tga 1629 Phe Ala His Ser Tyr Ile Asp
Ala Val Tyr Ala Leu Thr Glu * 530 535 540 4 542 PRT C. elegans 4
Met Asp Phe Ala Leu Glu Gln Tyr His Ser Ala Lys Asp Leu Leu Ile 1 5
10
15 Phe Glu Leu Arg Lys Phe Asn Pro Ile Val Leu Val Ser Ser Thr Ile
20 25 30 Val Ala Thr Tyr Val Leu Thr Asn Leu Arg His Met His Leu
Asp Glu 35 40 45 Met Gly Ile Arg Lys Arg Leu Ser Thr Trp Phe Phe
Thr Thr Val Lys 50 55 60 Arg Val Pro Phe Ile Arg Lys Met Ile Asp
Lys Gln Leu Asn Glu Val 65 70 75 80 Lys Asp Glu Leu Glu Lys Ser Leu
Arg Ile Val Asp Arg Ser Thr Glu 85 90 95 Tyr Phe Thr Thr Ile Pro
Ser His Ser Val Gly Arg Thr Glu Val Leu 100 105 110 Arg Leu Ala Ala
Ile Tyr Asp Asp Leu Glu Gly Pro Ala Phe Leu Glu 115 120 125 Gly Arg
Val Ser Gly Ala Val Phe Asn Arg Glu Asp Asp Lys Asp Glu 130 135 140
Arg Glu Met Tyr Glu Glu Val Phe Gly Lys Phe Ala Trp Thr Asn Pro 145
150 155 160 Leu Trp Pro Lys Leu Phe Pro Gly Val Arg Ile Met Glu Ala
Glu Val 165 170 175 Val Arg Met Cys Cys Asn Met Met Asn Gly Asp Ser
Glu Thr Cys Gly 180 185 190 Thr Met Ser Thr Gly Gly Ser Ile Ser Ile
Leu Leu Ala Cys Leu Ala 195 200 205 His Arg Asn Arg Leu Leu Lys Arg
Gly Glu Lys Tyr Thr Glu Met Ile 210 215 220 Val Pro Ser Ser Val His
Ala Ala Phe Phe Lys Ala Ala Glu Cys Phe 225 230 235 240 Arg Ile Lys
Val Arg Lys Ile Pro Val Asp Pro Val Thr Phe Lys Val 245 250 255 Asp
Leu Val Lys Met Lys Ala Ala Ile Asn Lys Arg Thr Cys Met Leu 260 265
270 Val Gly Ser Ala Pro Asn Phe Pro Phe Gly Thr Val Asp Asp Ile Glu
275 280 285 Ala Ile Gly Gln Leu Gly Leu Glu Tyr Asp Ile Pro Val His
Val Asp 290 295 300 Ala Cys Leu Gly Gly Phe Leu Leu Pro Phe Leu Glu
Glu Asp Glu Ile 305 310 315 320 Arg Tyr Asp Phe Arg Val Pro Gly Val
Ser Ser Ile Ser Ala Asp Ser 325 330 335 His Lys Tyr Gly Leu Ala Pro
Lys Gly Ser Ser Val Val Leu Tyr Arg 340 345 350 Asn Lys Glu Leu Leu
His Asn Gln Tyr Phe Cys Asp Ala Asp Trp Gln 355 360 365 Gly Gly Ile
Tyr Ala Ser Ala Thr Met Glu Gly Ser Arg Ala Gly His 370 375 380 Asn
Ile Ala Leu Cys Trp Ala Ala Met Leu Tyr His Ala Gln Glu Gly 385 390
395 400 Tyr Lys Ala Asn Ala Arg Lys Ile Val Asp Thr Thr Arg Lys Ile
Arg 405 410 415 Asn Gly Leu Ser Asn Ile Lys Gly Ile Lys Leu Gln Gly
Pro Ser Asp 420 425 430 Val Cys Ile Val Ser Trp Thr Thr Asn Asp Gly
Val Glu Leu Tyr Arg 435 440 445 Phe His Asn Phe Met Lys Glu Lys His
Trp Gln Leu Asn Gly Leu Gln 450 455 460 Phe Pro Ala Gly Val His Ile
Met Val Thr Met Asn His Thr His Pro 465 470 475 480 Gly Leu Ala Glu
Ala Phe Val Ala Asp Cys Arg Ala Ala Val Glu Phe 485 490 495 Val Lys
Ser His Lys Pro Ser Glu Ser Asp Lys Thr Ser Glu Ala Ala 500 505 510
Ile Tyr Gly Leu Ala Gln Ser Ile Pro Asp Arg Ser Leu Val His Glu 515
520 525 Phe Ala His Ser Tyr Ile Asp Ala Val Tyr Ala Leu Thr Glu 530
535 540 5 1707 DNA Mus musculus CDS (1)...(1707) 5 atg ccc gga acc
gac ctc ctc aag ctg aag gac ttc gag cct tat ttg 48 Met Pro Gly Thr
Asp Leu Leu Lys Leu Lys Asp Phe Glu Pro Tyr Leu 1 5 10 15 gag att
ttg gaa tct tat tcc aca aaa gcc aag aat tat gtg aat gga 96 Glu Ile
Leu Glu Ser Tyr Ser Thr Lys Ala Lys Asn Tyr Val Asn Gly 20 25 30
tat tgc acc aaa tat gag ccc tgg cag ctc att gcg tgg agt gtc ctg 144
Tyr Cys Thr Lys Tyr Glu Pro Trp Gln Leu Ile Ala Trp Ser Val Leu 35
40 45 tgt act ctg ctg ata gtc tgg gtg tat gag ctt atc ttc cag cca
gag 192 Cys Thr Leu Leu Ile Val Trp Val Tyr Glu Leu Ile Phe Gln Pro
Glu 50 55 60 agt tta tgg tct cgg ttt aaa aaa aaa tta ttt aag ctt
atc agg aag 240 Ser Leu Trp Ser Arg Phe Lys Lys Lys Leu Phe Lys Leu
Ile Arg Lys 65 70 75 80 atg cca ttt att gga cgt aag atc gaa caa cag
gtg agc aaa gcc aag 288 Met Pro Phe Ile Gly Arg Lys Ile Glu Gln Gln
Val Ser Lys Ala Lys 85 90 95 aag gat ctt gtc aag aac atg cca ttc
cta aag gtg gac aag gat tat 336 Lys Asp Leu Val Lys Asn Met Pro Phe
Leu Lys Val Asp Lys Asp Tyr 100 105 110 gtg aaa act ctg cct gct cag
ggt atg ggc aca gct gag gtt ctg gag 384 Val Lys Thr Leu Pro Ala Gln
Gly Met Gly Thr Ala Glu Val Leu Glu 115 120 125 aga ctc aag gag tac
agc tcc atg gat ggt tcc tgg caa gaa ggg aaa 432 Arg Leu Lys Glu Tyr
Ser Ser Met Asp Gly Ser Trp Gln Glu Gly Lys 130 135 140 gcc tca gga
gct gtg tac aat ggg gaa ccg aag ctc acg gag ctg ctg 480 Ala Ser Gly
Ala Val Tyr Asn Gly Glu Pro Lys Leu Thr Glu Leu Leu 145 150 155 160
gtg cag gct tat gga gaa ttc acg tgg agc aat cca ctg cat cca gat 528
Val Gln Ala Tyr Gly Glu Phe Thr Trp Ser Asn Pro Leu His Pro Asp 165
170 175 atc ttc cct gga ttg cgg aag tta gag gca gaa atc gtt agg atg
act 576 Ile Phe Pro Gly Leu Arg Lys Leu Glu Ala Glu Ile Val Arg Met
Thr 180 185 190 tgt tcc ctc ttc aat ggg gga cca gat tcc tgt gga tgt
gtg act tct 624 Cys Ser Leu Phe Asn Gly Gly Pro Asp Ser Cys Gly Cys
Val Thr Ser 195 200 205 ggg gga acg gaa agc atc ctg atg gcc tgc aaa
gct tac cgg gac ttg 672 Gly Gly Thr Glu Ser Ile Leu Met Ala Cys Lys
Ala Tyr Arg Asp Leu 210 215 220 gcg tta gag aag ggg atc aaa act cca
gaa att gtg gct ccc gag agt 720 Ala Leu Glu Lys Gly Ile Lys Thr Pro
Glu Ile Val Ala Pro Glu Ser 225 230 235 240 gcc cat gct gca ttc gac
aaa gca gct cat tat ttt ggg atg aag att 768 Ala His Ala Ala Phe Asp
Lys Ala Ala His Tyr Phe Gly Met Lys Ile 245 250 255 gtc cga gtt gca
ctg aaa aag aac atg gag gtg gat gtg cag gca atg 816 Val Arg Val Ala
Leu Lys Lys Asn Met Glu Val Asp Val Gln Ala Met 260 265 270 aag aga
gcc atc tcc agg aac aca gct atg ctg gtc tgt tct acc cca 864 Lys Arg
Ala Ile Ser Arg Asn Thr Ala Met Leu Val Cys Ser Thr Pro 275 280 285
cag ttt cct cat ggt gtg atg gat cct gtc ccc gaa gtg gcc aag tta 912
Gln Phe Pro His Gly Val Met Asp Pro Val Pro Glu Val Ala Lys Leu 290
295 300 act gtc aga tat aaa atc cca ctc cat gtg gat gct tgt ctg ggg
ggc 960 Thr Val Arg Tyr Lys Ile Pro Leu His Val Asp Ala Cys Leu Gly
Gly 305 310 315 320 ttc ctc att gtc ttc atg gag aaa gca ggg tac cca
ctg gag aaa cca 1008 Phe Leu Ile Val Phe Met Glu Lys Ala Gly Tyr
Pro Leu Glu Lys Pro 325 330 335 ttt gat ttc cgg gtg aaa ggt gtg acc
agc att tca gca gat act cat 1056 Phe Asp Phe Arg Val Lys Gly Val
Thr Ser Ile Ser Ala Asp Thr His 340 345 350 aag tat ggc tat gct cct
aaa ggt tca tca gtg gtg atg tac tct aac 1104 Lys Tyr Gly Tyr Ala
Pro Lys Gly Ser Ser Val Val Met Tyr Ser Asn 355 360 365 gag aag tac
agg acg tac cag ttc ttt gtt ggt gca gac tgg caa ggt 1152 Glu Lys
Tyr Arg Thr Tyr Gln Phe Phe Val Gly Ala Asp Trp Gln Gly 370 375 380
ggt gtc tac gca tct cca agc ata gct ggc tca cgg cct ggt ggc atc
1200 Gly Val Tyr Ala Ser Pro Ser Ile Ala Gly Ser Arg Pro Gly Gly
Ile 385 390 395 400 att gca gcc tgt tgg gcg gcc ttg atg cac ttc ggt
gag aac ggc tat 1248 Ile Ala Ala Cys Trp Ala Ala Leu Met His Phe
Gly Glu Asn Gly Tyr 405 410 415 gtt gaa gct acc aaa cag atc atc aaa
act gct cgc ttc ctg aag tca 1296 Val Glu Ala Thr Lys Gln Ile Ile
Lys Thr Ala Arg Phe Leu Lys Ser 420 425 430 gaa ctg gaa aac atc aaa
aac atc ttc att ttc ggt gat cct caa ttg 1344 Glu Leu Glu Asn Ile
Lys Asn Ile Phe Ile Phe Gly Asp Pro Gln Leu 435 440 445 tca gtt att
gct ctg gga tcc aac gat ttt gac att tac cga cta tct 1392 Ser Val
Ile Ala Leu Gly Ser Asn Asp Phe Asp Ile Tyr Arg Leu Ser 450 455 460
aat atg atg tct gct aag ggg tgg aat ttt aac tac ctg cag ttc cca
1440 Asn Met Met Ser Ala Lys Gly Trp Asn Phe Asn Tyr Leu Gln Phe
Pro 465 470 475 480 aga agc att cat ttc tgc att acg tta gta cat act
cgg aag cga gtg 1488 Arg Ser Ile His Phe Cys Ile Thr Leu Val His
Thr Arg Lys Arg Val 485 490 495 gcg atc cag ttc cta aag gat atc cgg
gaa tca gtc aca caa atc atg 1536 Ala Ile Gln Phe Leu Lys Asp Ile
Arg Glu Ser Val Thr Gln Ile Met 500 505 510 aag aat cct aaa gct aag
acc aca gga atg ggt gcc atc tat ggc atg 1584 Lys Asn Pro Lys Ala
Lys Thr Thr Gly Met Gly Ala Ile Tyr Gly Met 515 520 525 gcc cag gca
acc att gac agg aag ctg gtt gca gaa ata tcc tcc gtc 1632 Ala Gln
Ala Thr Ile Asp Arg Lys Leu Val Ala Glu Ile Ser Ser Val 530 535 540
ttc ttg gac tgc ctt tat act acg gac ccc gtg act cag ggc aac cag
1680 Phe Leu Asp Cys Leu Tyr Thr Thr Asp Pro Val Thr Gln Gly Asn
Gln 545 550 555 560 atg aac ggt tct cca aag ccc cgc tga 1707 Met
Asn Gly Ser Pro Lys Pro Arg * 565 6 568 PRT Mus musculus 6 Met Pro
Gly Thr Asp Leu Leu Lys Leu Lys Asp Phe Glu Pro Tyr Leu 1 5 10 15
Glu Ile Leu Glu Ser Tyr Ser Thr Lys Ala Lys Asn Tyr Val Asn Gly 20
25 30 Tyr Cys Thr Lys Tyr Glu Pro Trp Gln Leu Ile Ala Trp Ser Val
Leu 35 40 45 Cys Thr Leu Leu Ile Val Trp Val Tyr Glu Leu Ile Phe
Gln Pro Glu 50 55 60 Ser Leu Trp Ser Arg Phe Lys Lys Lys Leu Phe
Lys Leu Ile Arg Lys 65 70 75 80 Met Pro Phe Ile Gly Arg Lys Ile Glu
Gln Gln Val Ser Lys Ala Lys 85 90 95 Lys Asp Leu Val Lys Asn Met
Pro Phe Leu Lys Val Asp Lys Asp Tyr 100 105 110 Val Lys Thr Leu Pro
Ala Gln Gly Met Gly Thr Ala Glu Val Leu Glu 115 120 125 Arg Leu Lys
Glu Tyr Ser Ser Met Asp Gly Ser Trp Gln Glu Gly Lys 130 135 140 Ala
Ser Gly Ala Val Tyr Asn Gly Glu Pro Lys Leu Thr Glu Leu Leu 145 150
155 160 Val Gln Ala Tyr Gly Glu Phe Thr Trp Ser Asn Pro Leu His Pro
Asp 165 170 175 Ile Phe Pro Gly Leu Arg Lys Leu Glu Ala Glu Ile Val
Arg Met Thr 180 185 190 Cys Ser Leu Phe Asn Gly Gly Pro Asp Ser Cys
Gly Cys Val Thr Ser 195 200 205 Gly Gly Thr Glu Ser Ile Leu Met Ala
Cys Lys Ala Tyr Arg Asp Leu 210 215 220 Ala Leu Glu Lys Gly Ile Lys
Thr Pro Glu Ile Val Ala Pro Glu Ser 225 230 235 240 Ala His Ala Ala
Phe Asp Lys Ala Ala His Tyr Phe Gly Met Lys Ile 245 250 255 Val Arg
Val Ala Leu Lys Lys Asn Met Glu Val Asp Val Gln Ala Met 260 265 270
Lys Arg Ala Ile Ser Arg Asn Thr Ala Met Leu Val Cys Ser Thr Pro 275
280 285 Gln Phe Pro His Gly Val Met Asp Pro Val Pro Glu Val Ala Lys
Leu 290 295 300 Thr Val Arg Tyr Lys Ile Pro Leu His Val Asp Ala Cys
Leu Gly Gly 305 310 315 320 Phe Leu Ile Val Phe Met Glu Lys Ala Gly
Tyr Pro Leu Glu Lys Pro 325 330 335 Phe Asp Phe Arg Val Lys Gly Val
Thr Ser Ile Ser Ala Asp Thr His 340 345 350 Lys Tyr Gly Tyr Ala Pro
Lys Gly Ser Ser Val Val Met Tyr Ser Asn 355 360 365 Glu Lys Tyr Arg
Thr Tyr Gln Phe Phe Val Gly Ala Asp Trp Gln Gly 370 375 380 Gly Val
Tyr Ala Ser Pro Ser Ile Ala Gly Ser Arg Pro Gly Gly Ile 385 390 395
400 Ile Ala Ala Cys Trp Ala Ala Leu Met His Phe Gly Glu Asn Gly Tyr
405 410 415 Val Glu Ala Thr Lys Gln Ile Ile Lys Thr Ala Arg Phe Leu
Lys Ser 420 425 430 Glu Leu Glu Asn Ile Lys Asn Ile Phe Ile Phe Gly
Asp Pro Gln Leu 435 440 445 Ser Val Ile Ala Leu Gly Ser Asn Asp Phe
Asp Ile Tyr Arg Leu Ser 450 455 460 Asn Met Met Ser Ala Lys Gly Trp
Asn Phe Asn Tyr Leu Gln Phe Pro 465 470 475 480 Arg Ser Ile His Phe
Cys Ile Thr Leu Val His Thr Arg Lys Arg Val 485 490 495 Ala Ile Gln
Phe Leu Lys Asp Ile Arg Glu Ser Val Thr Gln Ile Met 500 505 510 Lys
Asn Pro Lys Ala Lys Thr Thr Gly Met Gly Ala Ile Tyr Gly Met 515 520
525 Ala Gln Ala Thr Ile Asp Arg Lys Leu Val Ala Glu Ile Ser Ser Val
530 535 540 Phe Leu Asp Cys Leu Tyr Thr Thr Asp Pro Val Thr Gln Gly
Asn Gln 545 550 555 560 Met Asn Gly Ser Pro Lys Pro Arg 565 7 1707
DNA Homo sapiens CDS (1)...(1707) 7 atg cct agc aca gac ctt ctg atg
ttg aag gcc ttt gag ccc tac tta 48 Met Pro Ser Thr Asp Leu Leu Met
Leu Lys Ala Phe Glu Pro Tyr Leu 1 5 10 15 gag att ttg gaa gta tac
tcc aca aaa gcc aag aat tat gta aat gga 96 Glu Ile Leu Glu Val Tyr
Ser Thr Lys Ala Lys Asn Tyr Val Asn Gly 20 25 30 cat tgc acc aag
tat gag ccc tgg cag cta att gca tgg agt gtc gtg 144 His Cys Thr Lys
Tyr Glu Pro Trp Gln Leu Ile Ala Trp Ser Val Val 35 40 45 tgg acc
ctg ctg ata gtc tgg gga tat gag ttt gtc ttc cag cca gag 192 Trp Thr
Leu Leu Ile Val Trp Gly Tyr Glu Phe Val Phe Gln Pro Glu 50 55 60
agt tta tgg tca agg ttt aaa aag aaa tgt ttt aag ctc acc agg aag 240
Ser Leu Trp Ser Arg Phe Lys Lys Lys Cys Phe Lys Leu Thr Arg Lys 65
70 75 80 atg ccc att att ggt cgt aag att caa gac aag ttg aac aag
acc aag 288 Met Pro Ile Ile Gly Arg Lys Ile Gln Asp Lys Leu Asn Lys
Thr Lys 85 90 95 gat gat att agc aag aac atg tca ttc ctg aaa gtg
gac aaa gag tat 336 Asp Asp Ile Ser Lys Asn Met Ser Phe Leu Lys Val
Asp Lys Glu Tyr 100 105 110 gtg aaa gct tta ccc tcc cag ggt ctg agc
tca tct gct gtt ttg gag 384 Val Lys Ala Leu Pro Ser Gln Gly Leu Ser
Ser Ser Ala Val Leu Glu 115 120 125 aaa ctt aag gag tac agc tct atg
gac gcc ttc tgg caa gag ggg aga 432 Lys Leu Lys Glu Tyr Ser Ser Met
Asp Ala Phe Trp Gln Glu Gly Arg 130 135 140 gcc tct gga aca gtg tac
agt ggg gag gag aag ctc act gag ctc ctt 480 Ala Ser Gly Thr Val Tyr
Ser Gly Glu Glu Lys Leu Thr Glu Leu Leu 145 150 155 160 gtg aag gct
tat gga gat ttt gca tgg agt aac ccc ctg cat cca gat 528 Val Lys Ala
Tyr Gly Asp Phe Ala Trp Ser Asn Pro Leu His Pro Asp 165 170 175 atc
ttc cca gga cta cgc aag ata gag gca gaa att gtg agg ata gct 576 Ile
Phe Pro Gly Leu Arg Lys Ile Glu Ala Glu Ile Val Arg Ile Ala 180 185
190 tgt tcc ctg ttc aat ggg gga cca gat tcg tgt gga tgt gtg act tct
624 Cys Ser Leu Phe Asn Gly Gly Pro Asp Ser Cys Gly Cys Val Thr Ser
195 200 205 ggg gga aca gaa agc ata ctc atg gcc tgc aaa gca tgt cgg
gat ctg 672 Gly Gly Thr Glu Ser Ile Leu Met Ala Cys Lys Ala Cys Arg
Asp Leu 210 215 220 gcc ttt gag aag ggg atc aaa act cca gaa att gtg
gct ccc caa agt 720 Ala Phe Glu Lys Gly Ile Lys Thr Pro Glu Ile Val
Ala Pro Gln Ser 225 230 235 240 gcc cat gct gca ttt aac aaa gca gcc
agt tac ttt ggg atg aag att 768 Ala His Ala Ala Phe Asn Lys Ala Ala
Ser Tyr Phe Gly Met Lys Ile 245 250 255 gtg cgg gtc cca ttg acg aag
atg atg gag gtg gat gtg agg gca atg 816 Val Arg Val Pro
Leu Thr Lys Met Met Glu Val Asp Val Arg Ala Met 260 265 270 aga aga
gct atc tcc agg aac act gcc atg ctc gtc tgt tct acc cca 864 Arg Arg
Ala Ile Ser Arg Asn Thr Ala Met Leu Val Cys Ser Thr Pro 275 280 285
cag ttt cct cat ggt gta ata gat cct gtc cct gaa gtg gcc aag ctg 912
Gln Phe Pro His Gly Val Ile Asp Pro Val Pro Glu Val Ala Lys Leu 290
295 300 gct gtc aaa tac aaa ata ccc ctt cat gtc gac gct tgt ctg gga
ggc 960 Ala Val Lys Tyr Lys Ile Pro Leu His Val Asp Ala Cys Leu Gly
Gly 305 310 315 320 ttc ctc atc gtc ttt atg gag aaa gca gga tac cca
ctg gag cac cca 1008 Phe Leu Ile Val Phe Met Glu Lys Ala Gly Tyr
Pro Leu Glu His Pro 325 330 335 ttt gat ttc cgg gtg aaa ggt gta acc
agc att tca gct gac acc cat 1056 Phe Asp Phe Arg Val Lys Gly Val
Thr Ser Ile Ser Ala Asp Thr His 340 345 350 aag tat ggc tat gcc cca
aaa ggc tca tca ttg gtg ttg tat agt gac 1104 Lys Tyr Gly Tyr Ala
Pro Lys Gly Ser Ser Leu Val Leu Tyr Ser Asp 355 360 365 aag aag tac
agg aac tat cag ttc ttc gtc gat aca gat tgg cag ggt 1152 Lys Lys
Tyr Arg Asn Tyr Gln Phe Phe Val Asp Thr Asp Trp Gln Gly 370 375 380
ggc atc tat gct tcc cca acc atc gca ggc tca cgg cct ggt ggc att
1200 Gly Ile Tyr Ala Ser Pro Thr Ile Ala Gly Ser Arg Pro Gly Gly
Ile 385 390 395 400 agc gca gcc tgt tgg gct gcc ttg atg cac ttc ggt
gag aac ggc tat 1248 Ser Ala Ala Cys Trp Ala Ala Leu Met His Phe
Gly Glu Asn Gly Tyr 405 410 415 gtt gaa gct acc aaa cag atc atc aaa
act gct cgc ttc ctc aag tca 1296 Val Glu Ala Thr Lys Gln Ile Ile
Lys Thr Ala Arg Phe Leu Lys Ser 420 425 430 gaa ctg gaa aat atc aaa
ggc atc ttt gtt ttt ggg aat ccc caa ttg 1344 Glu Leu Glu Asn Ile
Lys Gly Ile Phe Val Phe Gly Asn Pro Gln Leu 435 440 445 tca ctc att
gct ctg gga tcc cgt gat ttt gac atc tac cga cta tca 1392 Ser Leu
Ile Ala Leu Gly Ser Arg Asp Phe Asp Ile Tyr Arg Leu Ser 450 455 460
aac ctg atg act gct aag ggg tgg aac ttg aac cag ttg cag ttc cca
1440 Asn Leu Met Thr Ala Lys Gly Trp Asn Leu Asn Gln Leu Gln Phe
Pro 465 470 475 480 ccc agt att cat ttc tgc atc aca tta cta cac gcc
cgg aaa cga gta 1488 Pro Ser Ile His Phe Cys Ile Thr Leu Leu His
Ala Arg Lys Arg Val 485 490 495 gct ata caa ttc cta aag gac att cga
gaa tct gtc act caa atc atg 1536 Ala Ile Gln Phe Leu Lys Asp Ile
Arg Glu Ser Val Thr Gln Ile Met 500 505 510 aag aat cct aaa gcg aag
acc aca gga atg ggt gcc atc tat gcc atg 1584 Lys Asn Pro Lys Ala
Lys Thr Thr Gly Met Gly Ala Ile Tyr Ala Met 515 520 525 gcc cag aca
act gtt gac agg aat atg gtt gca gaa ttg tcc tca gtc 1632 Ala Gln
Thr Thr Val Asp Arg Asn Met Val Ala Glu Leu Ser Ser Val 530 535 540
ttc ttg gac agc ttg tac agc acc gac act gtc acc cag ggc agc cag
1680 Phe Leu Asp Ser Leu Tyr Ser Thr Asp Thr Val Thr Gln Gly Ser
Gln 545 550 555 560 atg aat ggt tct cca aaa ccc cac tga 1707 Met
Asn Gly Ser Pro Lys Pro His * 565 8 568 PRT Homo sapiens 8 Met Pro
Ser Thr Asp Leu Leu Met Leu Lys Ala Phe Glu Pro Tyr Leu 1 5 10 15
Glu Ile Leu Glu Val Tyr Ser Thr Lys Ala Lys Asn Tyr Val Asn Gly 20
25 30 His Cys Thr Lys Tyr Glu Pro Trp Gln Leu Ile Ala Trp Ser Val
Val 35 40 45 Trp Thr Leu Leu Ile Val Trp Gly Tyr Glu Phe Val Phe
Gln Pro Glu 50 55 60 Ser Leu Trp Ser Arg Phe Lys Lys Lys Cys Phe
Lys Leu Thr Arg Lys 65 70 75 80 Met Pro Ile Ile Gly Arg Lys Ile Gln
Asp Lys Leu Asn Lys Thr Lys 85 90 95 Asp Asp Ile Ser Lys Asn Met
Ser Phe Leu Lys Val Asp Lys Glu Tyr 100 105 110 Val Lys Ala Leu Pro
Ser Gln Gly Leu Ser Ser Ser Ala Val Leu Glu 115 120 125 Lys Leu Lys
Glu Tyr Ser Ser Met Asp Ala Phe Trp Gln Glu Gly Arg 130 135 140 Ala
Ser Gly Thr Val Tyr Ser Gly Glu Glu Lys Leu Thr Glu Leu Leu 145 150
155 160 Val Lys Ala Tyr Gly Asp Phe Ala Trp Ser Asn Pro Leu His Pro
Asp 165 170 175 Ile Phe Pro Gly Leu Arg Lys Ile Glu Ala Glu Ile Val
Arg Ile Ala 180 185 190 Cys Ser Leu Phe Asn Gly Gly Pro Asp Ser Cys
Gly Cys Val Thr Ser 195 200 205 Gly Gly Thr Glu Ser Ile Leu Met Ala
Cys Lys Ala Cys Arg Asp Leu 210 215 220 Ala Phe Glu Lys Gly Ile Lys
Thr Pro Glu Ile Val Ala Pro Gln Ser 225 230 235 240 Ala His Ala Ala
Phe Asn Lys Ala Ala Ser Tyr Phe Gly Met Lys Ile 245 250 255 Val Arg
Val Pro Leu Thr Lys Met Met Glu Val Asp Val Arg Ala Met 260 265 270
Arg Arg Ala Ile Ser Arg Asn Thr Ala Met Leu Val Cys Ser Thr Pro 275
280 285 Gln Phe Pro His Gly Val Ile Asp Pro Val Pro Glu Val Ala Lys
Leu 290 295 300 Ala Val Lys Tyr Lys Ile Pro Leu His Val Asp Ala Cys
Leu Gly Gly 305 310 315 320 Phe Leu Ile Val Phe Met Glu Lys Ala Gly
Tyr Pro Leu Glu His Pro 325 330 335 Phe Asp Phe Arg Val Lys Gly Val
Thr Ser Ile Ser Ala Asp Thr His 340 345 350 Lys Tyr Gly Tyr Ala Pro
Lys Gly Ser Ser Leu Val Leu Tyr Ser Asp 355 360 365 Lys Lys Tyr Arg
Asn Tyr Gln Phe Phe Val Asp Thr Asp Trp Gln Gly 370 375 380 Gly Ile
Tyr Ala Ser Pro Thr Ile Ala Gly Ser Arg Pro Gly Gly Ile 385 390 395
400 Ser Ala Ala Cys Trp Ala Ala Leu Met His Phe Gly Glu Asn Gly Tyr
405 410 415 Val Glu Ala Thr Lys Gln Ile Ile Lys Thr Ala Arg Phe Leu
Lys Ser 420 425 430 Glu Leu Glu Asn Ile Lys Gly Ile Phe Val Phe Gly
Asn Pro Gln Leu 435 440 445 Ser Leu Ile Ala Leu Gly Ser Arg Asp Phe
Asp Ile Tyr Arg Leu Ser 450 455 460 Asn Leu Met Thr Ala Lys Gly Trp
Asn Leu Asn Gln Leu Gln Phe Pro 465 470 475 480 Pro Ser Ile His Phe
Cys Ile Thr Leu Leu His Ala Arg Lys Arg Val 485 490 495 Ala Ile Gln
Phe Leu Lys Asp Ile Arg Glu Ser Val Thr Gln Ile Met 500 505 510 Lys
Asn Pro Lys Ala Lys Thr Thr Gly Met Gly Ala Ile Tyr Ala Met 515 520
525 Ala Gln Thr Thr Val Asp Arg Asn Met Val Ala Glu Leu Ser Ser Val
530 535 540 Phe Leu Asp Ser Leu Tyr Ser Thr Asp Thr Val Thr Gln Gly
Ser Gln 545 550 555 560 Met Asn Gly Ser Pro Lys Pro His 565 9 1467
DNA Homo sapiens CDS (1)...(1467) 9 atg cct agc aca gac ctt ctg atg
ttg aag gcc ttt gag ccc tac tta 48 Met Pro Ser Thr Asp Leu Leu Met
Leu Lys Ala Phe Glu Pro Tyr Leu 1 5 10 15 gag att ttg gaa gta tac
tcc aca aaa gcc aag aat tat gta aat gga 96 Glu Ile Leu Glu Val Tyr
Ser Thr Lys Ala Lys Asn Tyr Val Asn Gly 20 25 30 cat tgc acc aag
tat gag ccc tgg cag cta att gca tgg agt gtc gtg 144 His Cys Thr Lys
Tyr Glu Pro Trp Gln Leu Ile Ala Trp Ser Val Val 35 40 45 tgg acc
ctg ctg ata gtc tgg gga tat gag ttt gtc ttc cag cca gag 192 Trp Thr
Leu Leu Ile Val Trp Gly Tyr Glu Phe Val Phe Gln Pro Glu 50 55 60
agt tta tgg tca agg ttt aaa aag aaa tgt ttt aag ctc acc agg aag 240
Ser Leu Trp Ser Arg Phe Lys Lys Lys Cys Phe Lys Leu Thr Arg Lys 65
70 75 80 atg ccc att att ggt cgt aag att caa gac aag ttg aac aag
acc aag 288 Met Pro Ile Ile Gly Arg Lys Ile Gln Asp Lys Leu Asn Lys
Thr Lys 85 90 95 gat gat att agc aag aac atg tca ttc ctg aaa gtg
gac aaa gag tat 336 Asp Asp Ile Ser Lys Asn Met Ser Phe Leu Lys Val
Asp Lys Glu Tyr 100 105 110 gtg aaa gct tta ccc tcc cag ggt ctg agc
tca tct gct gtt ttg gag 384 Val Lys Ala Leu Pro Ser Gln Gly Leu Ser
Ser Ser Ala Val Leu Glu 115 120 125 aaa ctt aag gag tac agc tct atg
gac gcc ttc tgg caa gag ggg aga 432 Lys Leu Lys Glu Tyr Ser Ser Met
Asp Ala Phe Trp Gln Glu Gly Arg 130 135 140 gcc tct gga aca gtg tac
agt ggg gag gag aag ctc act gag ctc ctt 480 Ala Ser Gly Thr Val Tyr
Ser Gly Glu Glu Lys Leu Thr Glu Leu Leu 145 150 155 160 gtg aag gct
tat gga gat ttt gca tgg agt aac ccc ctg cat cca gat 528 Val Lys Ala
Tyr Gly Asp Phe Ala Trp Ser Asn Pro Leu His Pro Asp 165 170 175 atc
ttc cca gga cta cgc aag ata gag gca gaa att gtg agg ata gct 576 Ile
Phe Pro Gly Leu Arg Lys Ile Glu Ala Glu Ile Val Arg Ile Ala 180 185
190 tgt tcc ctg ttc aat ggg gga cca gat tcg tgt gga tgt gtg act tct
624 Cys Ser Leu Phe Asn Gly Gly Pro Asp Ser Cys Gly Cys Val Thr Ser
195 200 205 ggg gga aca gaa agc ata ctc atg gcc tgc aaa gca tgt cgg
gat ctg 672 Gly Gly Thr Glu Ser Ile Leu Met Ala Cys Lys Ala Cys Arg
Asp Leu 210 215 220 gcc ttt gag aag ggg atc aaa act cca gaa att gtg
gct ccc caa agt 720 Ala Phe Glu Lys Gly Ile Lys Thr Pro Glu Ile Val
Ala Pro Gln Ser 225 230 235 240 gcc cat gct gca ttt aac aaa gca gcc
agt tac ttt ggg atg aag att 768 Ala His Ala Ala Phe Asn Lys Ala Ala
Ser Tyr Phe Gly Met Lys Ile 245 250 255 gtg cgg gtc cca ttg acg aag
atg atg gag gtg gat gtg agg gca atg 816 Val Arg Val Pro Leu Thr Lys
Met Met Glu Val Asp Val Arg Ala Met 260 265 270 aga aga gct atc tcc
agg aac act gcc atg ctc gtc tgt tct acc cca 864 Arg Arg Ala Ile Ser
Arg Asn Thr Ala Met Leu Val Cys Ser Thr Pro 275 280 285 cag ttt cct
cat ggt gta ata gat cct gtc cct gaa gtg gcc aag ctg 912 Gln Phe Pro
His Gly Val Ile Asp Pro Val Pro Glu Val Ala Lys Leu 290 295 300 gct
gtc aaa tac aaa ata ccc ctt cat gtc gac gct tgt ctg gga ggc 960 Ala
Val Lys Tyr Lys Ile Pro Leu His Val Asp Ala Cys Leu Gly Gly 305 310
315 320 ttc ctc atc gtc ttt atg gag aaa gca gga tac cca ctg gag cac
cca 1008 Phe Leu Ile Val Phe Met Glu Lys Ala Gly Tyr Pro Leu Glu
His Pro 325 330 335 ttt gat ttc cgg gtg aaa ggt gta acc agc att tca
gct gac acc cat 1056 Phe Asp Phe Arg Val Lys Gly Val Thr Ser Ile
Ser Ala Asp Thr His 340 345 350 aag ctg gaa aat atc aaa ggc atc ttt
gtt ttt ggg aat ccc caa ttg 1104 Lys Leu Glu Asn Ile Lys Gly Ile
Phe Val Phe Gly Asn Pro Gln Leu 355 360 365 tca ctc att gct ctg gga
tcc cgt gat ttt gac atc tac cga cta tca 1152 Ser Leu Ile Ala Leu
Gly Ser Arg Asp Phe Asp Ile Tyr Arg Leu Ser 370 375 380 aac ctg atg
act gct aag ggg tgg aac ttg aac cag ttg cag ttc cca 1200 Asn Leu
Met Thr Ala Lys Gly Trp Asn Leu Asn Gln Leu Gln Phe Pro 385 390 395
400 ccc agt att cat ttc tgc atc aca tta cta cac gcc cgg aaa cga gta
1248 Pro Ser Ile His Phe Cys Ile Thr Leu Leu His Ala Arg Lys Arg
Val 405 410 415 gct ata caa ttc cta aag gac att cga gaa tct gtc act
caa atc atg 1296 Ala Ile Gln Phe Leu Lys Asp Ile Arg Glu Ser Val
Thr Gln Ile Met 420 425 430 aag aat cct aaa gcg aag acc aca gga atg
ggt gcc atc tat gcc atg 1344 Lys Asn Pro Lys Ala Lys Thr Thr Gly
Met Gly Ala Ile Tyr Ala Met 435 440 445 gcc cag aca act gtt gac agg
aat atg gtt gca gaa ttg tcc tca gtc 1392 Ala Gln Thr Thr Val Asp
Arg Asn Met Val Ala Glu Leu Ser Ser Val 450 455 460 ttc ttg gac agc
ttg tac agc acc gac act gtc acc cag ggc agc cag 1440 Phe Leu Asp
Ser Leu Tyr Ser Thr Asp Thr Val Thr Gln Gly Ser Gln 465 470 475 480
atg aat ggt tct cca aaa ccc cac tga 1467 Met Asn Gly Ser Pro Lys
Pro His * 485 10 488 PRT Homo sapiens 10 Met Pro Ser Thr Asp Leu
Leu Met Leu Lys Ala Phe Glu Pro Tyr Leu 1 5 10 15 Glu Ile Leu Glu
Val Tyr Ser Thr Lys Ala Lys Asn Tyr Val Asn Gly 20 25 30 His Cys
Thr Lys Tyr Glu Pro Trp Gln Leu Ile Ala Trp Ser Val Val 35 40 45
Trp Thr Leu Leu Ile Val Trp Gly Tyr Glu Phe Val Phe Gln Pro Glu 50
55 60 Ser Leu Trp Ser Arg Phe Lys Lys Lys Cys Phe Lys Leu Thr Arg
Lys 65 70 75 80 Met Pro Ile Ile Gly Arg Lys Ile Gln Asp Lys Leu Asn
Lys Thr Lys 85 90 95 Asp Asp Ile Ser Lys Asn Met Ser Phe Leu Lys
Val Asp Lys Glu Tyr 100 105 110 Val Lys Ala Leu Pro Ser Gln Gly Leu
Ser Ser Ser Ala Val Leu Glu 115 120 125 Lys Leu Lys Glu Tyr Ser Ser
Met Asp Ala Phe Trp Gln Glu Gly Arg 130 135 140 Ala Ser Gly Thr Val
Tyr Ser Gly Glu Glu Lys Leu Thr Glu Leu Leu 145 150 155 160 Val Lys
Ala Tyr Gly Asp Phe Ala Trp Ser Asn Pro Leu His Pro Asp 165 170 175
Ile Phe Pro Gly Leu Arg Lys Ile Glu Ala Glu Ile Val Arg Ile Ala 180
185 190 Cys Ser Leu Phe Asn Gly Gly Pro Asp Ser Cys Gly Cys Val Thr
Ser 195 200 205 Gly Gly Thr Glu Ser Ile Leu Met Ala Cys Lys Ala Cys
Arg Asp Leu 210 215 220 Ala Phe Glu Lys Gly Ile Lys Thr Pro Glu Ile
Val Ala Pro Gln Ser 225 230 235 240 Ala His Ala Ala Phe Asn Lys Ala
Ala Ser Tyr Phe Gly Met Lys Ile 245 250 255 Val Arg Val Pro Leu Thr
Lys Met Met Glu Val Asp Val Arg Ala Met 260 265 270 Arg Arg Ala Ile
Ser Arg Asn Thr Ala Met Leu Val Cys Ser Thr Pro 275 280 285 Gln Phe
Pro His Gly Val Ile Asp Pro Val Pro Glu Val Ala Lys Leu 290 295 300
Ala Val Lys Tyr Lys Ile Pro Leu His Val Asp Ala Cys Leu Gly Gly 305
310 315 320 Phe Leu Ile Val Phe Met Glu Lys Ala Gly Tyr Pro Leu Glu
His Pro 325 330 335 Phe Asp Phe Arg Val Lys Gly Val Thr Ser Ile Ser
Ala Asp Thr His 340 345 350 Lys Leu Glu Asn Ile Lys Gly Ile Phe Val
Phe Gly Asn Pro Gln Leu 355 360 365 Ser Leu Ile Ala Leu Gly Ser Arg
Asp Phe Asp Ile Tyr Arg Leu Ser 370 375 380 Asn Leu Met Thr Ala Lys
Gly Trp Asn Leu Asn Gln Leu Gln Phe Pro 385 390 395 400 Pro Ser Ile
His Phe Cys Ile Thr Leu Leu His Ala Arg Lys Arg Val 405 410 415 Ala
Ile Gln Phe Leu Lys Asp Ile Arg Glu Ser Val Thr Gln Ile Met 420 425
430 Lys Asn Pro Lys Ala Lys Thr Thr Gly Met Gly Ala Ile Tyr Ala Met
435 440 445 Ala Gln Thr Thr Val Asp Arg Asn Met Val Ala Glu Leu Ser
Ser Val 450 455 460 Phe Leu Asp Ser Leu Tyr Ser Thr Asp Thr Val Thr
Gln Gly Ser Gln 465 470 475 480 Met Asn Gly Ser Pro Lys Pro His 485
11 552 PRT C. elegans 11 Met Asp Ser Val Lys His Thr Thr Glu Ile
Ile Val Asp Leu Thr Lys 1 5 10 15 Met His Tyr His Met Ile Asn Asp
Arg Leu Ser Arg Tyr Asp Pro Val 20 25 30 Val Leu Val Leu Ala Ala
Phe Gly Gly Thr Leu Val Tyr Thr Lys Val 35 40 45 Val His Leu Tyr
Arg Lys Ser Glu Asp Pro Ile Leu Lys Arg Met Gly 50 55 60 Ala Tyr
Val Phe Ser Leu Leu Arg Lys Leu Pro Ala Val Arg Asp Lys 65 70 75 80
Ile Glu Lys Glu Leu Ala Ala Glu Lys Pro Lys Leu Ile Glu Ser Ile 85
90 95 His Lys Asp Asp Lys
Asp Lys Gln Phe Ile Ser Thr Leu Pro Ile Ala 100 105 110 Pro Leu Ser
Gln Asp Ser Ile Met Glu Leu Ala Lys Lys Tyr Glu Asp 115 120 125 Tyr
Asn Thr Phe Asn Ile Asp Gly Gly Arg Val Ser Gly Ala Val Tyr 130 135
140 Thr Asp Arg His Ala Glu His Ile Asn Leu Leu Gly Lys Ile Tyr Glu
145 150 155 160 Lys Tyr Ala Phe Ser Asn Pro Leu His Pro Asp Val Phe
Pro Gly Ala 165 170 175 Arg Lys Met Glu Ala Glu Leu Ile Arg Met Val
Leu Asn Leu Tyr Asn 180 185 190 Gly Pro Glu Asp Ser Ser Gly Ser Val
Thr Ser Gly Gly Thr Glu Ser 195 200 205 Ile Ile Met Ala Cys Phe Ser
Tyr Arg Asn Arg Ala His Ser Leu Gly 210 215 220 Ile Glu His Pro Val
Ile Leu Ala Cys Lys Thr Ala His Ala Ala Phe 225 230 235 240 Asp Lys
Ala Ala His Leu Cys Gly Met Arg Leu Arg His Val Pro Val 245 250 255
Asp Ser Asp Asn Arg Val Asp Leu Lys Glu Met Glu Arg Leu Ile Asp 260
265 270 Ser Asn Val Cys Met Leu Val Gly Ser Ala Pro Asn Phe Pro Ser
Gly 275 280 285 Thr Ile Asp Pro Ile Pro Glu Ile Ala Lys Leu Gly Lys
Lys Tyr Gly 290 295 300 Ile Pro Val His Val Asp Ala Cys Leu Gly Gly
Phe Met Ile Pro Phe 305 310 315 320 Met Asn Asp Ala Gly Tyr Leu Ile
Pro Val Phe Asp Phe Arg Asn Pro 325 330 335 Gly Val Thr Ser Ile Ser
Cys Asp Thr His Lys Tyr Gly Cys Thr Pro 340 345 350 Lys Gly Ser Ser
Ile Val Met Tyr Arg Ser Lys Glu Leu His His Phe 355 360 365 Gln Tyr
Phe Ser Val Ala Asp Trp Cys Gly Gly Ile Tyr Ala Thr Pro 370 375 380
Thr Ile Ala Gly Ser Arg Ala Gly Ala Asn Thr Ala Val Ala Trp Ala 385
390 395 400 Thr Leu Leu Ser Phe Gly Arg Asp Glu Tyr Val Arg Arg Cys
Ala Gln 405 410 415 Ile Val Lys His Thr Arg Met Leu Ala Glu Lys Ile
Glu Lys Ile Lys 420 425 430 Trp Ile Lys Pro Tyr Gly Lys Ser Asp Val
Ser Leu Val Ala Phe Ser 435 440 445 Gly Asn Gly Val Asn Ile Tyr Glu
Val Ser Asp Lys Met Met Lys Leu 450 455 460 Gly Trp Asn Leu Asn Thr
Leu Gln Asn Pro Ala Ala Ile His Ile Cys 465 470 475 480 Leu Thr Ile
Asn Gln Ala Asn Glu Glu Val Val Asn Ala Phe Ala Val 485 490 495 Asp
Leu Glu Lys Ile Cys Glu Glu Leu Ala Ala Lys Gly Glu Gln Lys 500 505
510 Ala Asp Ser Gly Met Ala Ala Met Tyr Gly Met Ala Ala Gln Val Pro
515 520 525 Lys Ser Val Val Asp Glu Val Ile Ala Leu Tyr Ile Asp Ala
Thr Tyr 530 535 540 Ser Ala Pro Pro Ser Thr Ser Asn 545 550 12 3162
DNA C. elegans 12 atggattcgg ttaagcacac aaccgaaatt attgtcgact
tgacaaaaat gcactatcac 60 atgataaatg ataggtgaat tttaaacaaa
aattagatat ttggaaatta ctaattcaag 120 attttcagac tttctcggta
tgatccggtt gttctagtgt tggccgcttt tgggggtacc 180 cttgtctata
caaaagtcgt ccatttgtac cgaaaaagcg aggatccaat tttgaaacgg 240
caagtgtttt cttgcgaatt ttagaaatat caaaatgaaa ttttcagcat gggagcttat
300 gtattctcac ttcttcgaaa acttccagct gttcgggata aaatcgaaaa
agagctggct 360 gctgagaagc caaagcttat tgaatcgatt cataaggatg
ataaggacaa gcaattcatt 420 tccagtttgt ttgaacattt attaattaac
caattcatta attctatttt tcagctcttc 480 ccatcgctcc attatctcag
gactcaatta tggaactggc gaaaaaatat gaggattaca 540 acacatttaa
cattgacgga ggacgagtat ctggagcggt ttatactgat cgtcatgctg 600
aacacattaa tttgcttgga aaggtttaga aattctagaa tttttcaaaa tcttagctct
660 caaatatatt ctcttgtaaa tagctacata gtatatcctg tagggaagct
ttgaatccaa 720 ttcagatcag gggcgacaaa cgattttttc cggcaaatcg
gcaaatcgcc ggaatggaaa 780 tttcctgcaa atcggcaaat tgccggaatg
gaaatttcct gcaagttggc aaattgacgg 840 aattgaaatt tccggcaaac
cgacaaattt ccgtaattaa aatttcctgc aaaccggcga 900 attggcggaa
ttgaaatttc ctgcaaaccg gcaaattgcc gtaattgaaa tttcctgcaa 960
accggcaaat tgccggaatt gaaatttccg gcaaaccggc aaatcggctg aattgaaatt
1020 tcctgcaaac cggcaaattg cggtaattga aatttcctgc aaaccggtca
gttgccgatt 1080 tgcctttgcc tgaaaaacgg cgattgccag aaatattcgg
caaattgtgg ttttgcacat 1140 ttttctggaa atttcaggca aaattgtacg
catcctatga atatccctat taacatcttt 1200 tttgaaaagt cagtaaatta
tatgaaaata tctaaagaaa acggggaaaa tatttcaaag 1260 aggcacagtt
ttatgtgttt ccgtcatcta aatagtccct ctaaacattt ccggcaaatc 1320
tgatatccgg caaacggcaa atcgggatat tgccggaatt taaaatttgc cgaacttgtc
1380 gacaaaaaaa atgcgccttg aatccgattc agatattcaa aaattgaatt
ttggacgttt 1440 tagaaatcat ttagtttgtc aattttcaag aaatttctag
aaaattggat ggtttccgcc 1500 aagaaatatt agctacatga aaataatttt
gaaactagac atttcttaaa ataaaaattg 1560 ccatctttta tatccagatt
tacgaaaagt atgcgttctc gaatcccctc caccctgacg 1620 tctttccggg
agctcgtaaa atggaggcag aacttattcg aatggttctg aacctgtata 1680
atggaccaga agattctagt ggaagtgtaa cttctggtgg tactgaaagt attattatgg
1740 catgcttttc gtatcggtaa gcatttattc aactcttaaa attcaatttt
gcaaactcta 1800 cagaaatcgt gcacactctc ttggcattga acatccagtt
attttggcat gtaaaacagc 1860 tcacgcggca tttgataagg ccgcccatct
atgcggaatg cgtcttcgcc acgttccagt 1920 tgattcggat aatcgtgtcg
atttaaaaga aatggagaga ctaattgatt cgaatgtttg 1980 tatgttggtt
ggctcagcgc ctaacttccc atcaggcaca attgatccaa ttccggaaat 2040
tgctaaggta ctggaaattc ccgcctcaat atcgcggaaa aaatagagaa atgactgaac
2100 aaaattacat tgtgagcggg aactctaatt gaattcagca aaaatacgat
acttttttct 2160 aacttaaaat aatttttaaa aaaactcaca gatgctagtc
caaaaaatgg ccttttttga 2220 ttacttaatc gaacgtttac actttcagct
cggcaaaaag tatggaatcc cggtccacgt 2280 ggacgcatgt cttggtggat
tcatgattcc atttatgaat gacgccggat acctgattcc 2340 tgtattcgat
ttcagaaatc ccggtgttac atctatttcg tgtgatactc ataaggttgg 2400
atacagttct atccattttt ttccttcaat tcaaaatctt tcagtacgga tgcacaccga
2460 aaggttcatc gattgtcatg tatcgttcca aggaacttca tcacttccag
tatttctcgg 2520 ttgccgattg gtgtggaggc atctatgcca ccccgactat
tgcaggtttg aagaatgttt 2580 tagtagcttc aatagaatca aagagatccc
ttaggatccc gagctggagc caacactgcc 2640 gtcgcctggg ccacactttt
atccttcggt cgagacgaat atgttcgaag atgtgctcaa 2700 attgtgaagc
atacacgaat gctggccgag aaaattgaga aaatcaaatg gatcaagcct 2760
tatggaaaat cggatgtttc attggtggcg ttctccggaa atggtgtgaa tatctacgaa
2820 gtttctgaca aaatgatgaa gctcggatgg aatttgaaca ctctgcagaa
tccagcggcg 2880 tatgtttatc aattttatga gttatcagct tgctaaattt
tttgtttcag aatccacatt 2940 tgtttgacaa tcaatcaagc gaacgaggaa
gttgtgaatg cgttcgccgt cgaccttgag 3000 aagatttgtg aagaactcgc
tgcaaaaggt gaacaaaaag ctgacagtgg aatggctgcg 3060 atgtatggaa
tggctgcgca agtaccaaaa tcagtagtgg acgaggttat cgctctgtac 3120
attgacgcaa cttattcagc tccaccttca acttctaatt aa 3162 13 34 DNA
Artificial Sequence primer 13 gaggaattca tggattcggt taagcacaca accg
34 14 33 DNA Artificial Sequence primer 14 agcctcgagt taattagaag
ttgaaggtgg agc 33 15 1638 DNA Drosophila melanogaster 15 atgcgtccgt
tctccggcag cgattgcctt aagcccgtca ccgagggcat caaccgggcg 60
ttcggcgcca aggagccctg gcaggtggcc accatcacgg ccaccacggt gctgggaggc
120 gtctggctct ggactgtgat ctgccaggat gaaaatcttt acattcgtgg
caagcgtcag 180 ttctttaagt ttgccaagaa gattccagcc gtgcgtcgtc
aggtggagac tgaattggcc 240 aaggccaaaa acgacttcga gacggaaatc
aaaaagagca acgcccacct tacctactcg 300 gaaactctgc ccgagaaggg
actcagcaag gaggagatcc tccgactggt ggatgagcac 360 ctgaagactg
gtcactacaa ctggcgtgat ggtcgtgtat ctggcgcggt ctacggctac 420
aagcctgatc tggtggagct cgtcactgaa gtgtacggca aggcctccta caccaatccc
480 ttgcacgcag atcttttccc gggagtttgc aaaatggagg cggaggtagt
gcgcatggca 540 tgcaacctgt tccatggaaa ctcagccagc tgtggaacca
tgaccaccgg cggcaccgaa 600 tccattgtaa tggccatgaa ggcgtacagg
gatttcgcta gagagtacaa gggaatcacc 660 aggccaaaca tcgtggtgcc
taagacggtc cacgcggcct tcgacaaggg cggtcagtac 720 tttaatatcc
acgtgcgatc cgtggatgta gatccggaga cctacgaagt ggacattaag 780
aagttcaaac gtgccattaa caggaacacg attctgctgg ttgggtctgc tccgaacttc
840 ccctatggaa ccatcgatga catcgaagct atcgccgctt tgggcgttaa
gtacgacatt 900 cccgtgcacg tggacgcctg cctgggcagc tttgtggtgg
ccttggtccg caacgccggc 960 tataagctgc gtcccttcga ctttgaggtc
aagggagtga ccagtatctc cgctgatacc 1020 cacaagtatg gtttcgcgcc
caagggatca tcggtgatcc tttactcgga caagaagtac 1080 aaggaccatc
agttcactgt gactactgac tggcctggcg gcgtgtatgg ttctcccaca 1140
gtcaacggtt cccgtgccgg aggtattatc gccgcctgct gggctaccat gatgagcttt
1200 ggctatgatg gttatctgga agccactaag cgcattgtgg atacggcgcg
ctatatcgag 1260 aggggcgttc gcgacatcga tggcatcttt atctttggca
agccagctac ttcagtgatt 1320 gccctgggtt ccaatgtgtt tgacattttc
cggctatcgg attcgctgtg caaactgggc 1380 tggaacctaa atgcgctgca
gtttccatct ggtatccacc tgtgcgtgac ggacatgcac 1440 acacagcccg
gagtcgcgga taaattcatt gccgatgtgc gcagctgtac ggcggagatc 1500
atgaaggatc ccggccagcc cgtcgttgga aagatggctc tttacggcat ggcacagagc
1560 atacccgacc gttcggtgat cggagaagtg actcgcctat tcctgcactc
catgtactac 1620 actcccagcc agaaatag 1638 16 545 PRT Drosophila
melanogaster 16 Met Arg Pro Phe Ser Gly Ser Asp Cys Leu Lys Pro Val
Thr Glu Gly 1 5 10 15 Ile Asn Arg Ala Phe Gly Ala Lys Glu Pro Trp
Gln Val Ala Thr Ile 20 25 30 Thr Ala Thr Thr Val Leu Gly Gly Val
Trp Leu Trp Thr Val Ile Cys 35 40 45 Gln Asp Glu Asn Leu Tyr Ile
Arg Gly Lys Arg Gln Phe Phe Lys Phe 50 55 60 Ala Lys Lys Ile Pro
Ala Val Arg Arg Gln Val Glu Thr Glu Leu Ala 65 70 75 80 Lys Ala Lys
Asn Asp Phe Glu Thr Glu Ile Lys Lys Ser Asn Ala His 85 90 95 Leu
Thr Tyr Ser Glu Thr Leu Pro Glu Lys Gly Leu Ser Lys Glu Glu 100 105
110 Ile Leu Arg Leu Val Asp Glu His Leu Lys Thr Gly His Tyr Asn Trp
115 120 125 Arg Asp Gly Arg Val Ser Gly Ala Val Tyr Gly Tyr Lys Pro
Asp Leu 130 135 140 Val Glu Leu Val Thr Glu Val Tyr Gly Lys Ala Ser
Tyr Thr Asn Pro 145 150 155 160 Leu His Ala Asp Leu Phe Pro Gly Val
Cys Lys Met Glu Ala Glu Val 165 170 175 Val Arg Met Ala Cys Asn Leu
Phe His Gly Asn Ser Ala Ser Cys Gly 180 185 190 Thr Met Thr Thr Gly
Gly Thr Glu Ser Ile Val Met Ala Met Lys Ala 195 200 205 Tyr Arg Asp
Phe Ala Arg Glu Tyr Lys Gly Ile Thr Arg Pro Asn Ile 210 215 220 Val
Val Pro Lys Thr Val His Ala Ala Phe Asp Lys Gly Gly Gln Tyr 225 230
235 240 Phe Asn Ile His Val Arg Ser Val Asp Val Asp Pro Glu Thr Tyr
Glu 245 250 255 Val Asp Ile Lys Lys Phe Lys Arg Ala Ile Asn Arg Asn
Thr Ile Leu 260 265 270 Leu Val Gly Ser Ala Pro Asn Phe Pro Tyr Gly
Thr Ile Asp Asp Ile 275 280 285 Glu Ala Ile Ala Ala Leu Gly Val Lys
Tyr Asp Ile Pro Val His Val 290 295 300 Asp Ala Cys Leu Gly Ser Phe
Val Val Ala Leu Val Arg Asn Ala Gly 305 310 315 320 Tyr Lys Leu Arg
Pro Phe Asp Phe Glu Val Lys Gly Val Thr Ser Ile 325 330 335 Ser Ala
Asp Thr His Lys Tyr Gly Phe Ala Pro Lys Gly Ser Ser Val 340 345 350
Ile Leu Tyr Ser Asp Lys Lys Tyr Lys Asp His Gln Phe Thr Val Thr 355
360 365 Thr Asp Trp Pro Gly Gly Val Tyr Gly Ser Pro Thr Val Asn Gly
Ser 370 375 380 Arg Ala Gly Gly Ile Ile Ala Ala Cys Trp Ala Thr Met
Met Ser Phe 385 390 395 400 Gly Tyr Asp Gly Tyr Leu Glu Ala Thr Lys
Arg Ile Val Asp Thr Ala 405 410 415 Arg Tyr Ile Glu Arg Gly Val Arg
Asp Ile Asp Gly Ile Phe Ile Phe 420 425 430 Gly Lys Pro Ala Thr Ser
Val Ile Ala Leu Gly Ser Asn Val Phe Asp 435 440 445 Ile Phe Arg Leu
Ser Asp Ser Leu Cys Lys Leu Gly Trp Asn Leu Asn 450 455 460 Ala Leu
Gln Phe Pro Ser Gly Ile His Leu Cys Val Thr Asp Met His 465 470 475
480 Thr Gln Pro Gly Val Ala Asp Lys Phe Ile Ala Asp Val Arg Ser Cys
485 490 495 Thr Ala Glu Ile Met Lys Asp Pro Gly Gln Pro Val Val Gly
Lys Met 500 505 510 Ala Leu Tyr Gly Met Ala Gln Ser Ile Pro Asp Arg
Ser Val Ile Gly 515 520 525 Glu Val Thr Arg Leu Phe Leu His Ser Met
Tyr Tyr Thr Pro Ser Gln 530 535 540 Lys 545 17 1707 DNA Homo
sapiens CDS (1)...(1707) 17 atg cct agc aca gac ctt ctg atg ttg aag
gcc ttt gag ccc tac tta 48 Met Pro Ser Thr Asp Leu Leu Met Leu Lys
Ala Phe Glu Pro Tyr Leu 1 5 10 15 gag att ttg gaa gta tac tcc aca
aaa gcc aag aat tat gta aat gga 96 Glu Ile Leu Glu Val Tyr Ser Thr
Lys Ala Lys Asn Tyr Val Asn Gly 20 25 30 cat tgc acc aag tat gag
ccc tgg cag cta att gca tgg agt gtc gtg 144 His Cys Thr Lys Tyr Glu
Pro Trp Gln Leu Ile Ala Trp Ser Val Val 35 40 45 tgg acc ctg ctg
ata gtc tgg gga tat gag ttt gtc ttc cag cca gag 192 Trp Thr Leu Leu
Ile Val Trp Gly Tyr Glu Phe Val Phe Gln Pro Glu 50 55 60 agt tta
tgg tca agg ttt aaa aag aaa tgt ttt aag ctc acc agg aag 240 Ser Leu
Trp Ser Arg Phe Lys Lys Lys Cys Phe Lys Leu Thr Arg Lys 65 70 75 80
atg ccc att att ggt cgt aag att caa gac aag ttg aac aag acc aag 288
Met Pro Ile Ile Gly Arg Lys Ile Gln Asp Lys Leu Asn Lys Thr Lys 85
90 95 gat gat att agc aag aac atg tca ttc ctg aaa gtg gac aaa gag
tat 336 Asp Asp Ile Ser Lys Asn Met Ser Phe Leu Lys Val Asp Lys Glu
Tyr 100 105 110 gtg aaa gct tta ccc tcc cag ggt ctg agc tca tct gct
gtt ttg gag 384 Val Lys Ala Leu Pro Ser Gln Gly Leu Ser Ser Ser Ala
Val Leu Glu 115 120 125 aaa ctt aag gag tac agc tct atg gac gcc ttc
tgg caa gag ggg aga 432 Lys Leu Lys Glu Tyr Ser Ser Met Asp Ala Phe
Trp Gln Glu Gly Arg 130 135 140 gcc tct gga aca gtg tac agt ggg gag
gag aag ctc act gag ctc ctt 480 Ala Ser Gly Thr Val Tyr Ser Gly Glu
Glu Lys Leu Thr Glu Leu Leu 145 150 155 160 gtg aag gct tat gga gat
ttt gca tgg agt aac ccc ctg cat cca gat 528 Val Lys Ala Tyr Gly Asp
Phe Ala Trp Ser Asn Pro Leu His Pro Asp 165 170 175 atc ttc cca gga
cta cgc aag ata gag gca gaa att gtg agg ata gct 576 Ile Phe Pro Gly
Leu Arg Lys Ile Glu Ala Glu Ile Val Arg Ile Ala 180 185 190 tgt tcc
ctg ttc aat ggg gga cca gat tcg tgt gga tgt gtg act tct 624 Cys Ser
Leu Phe Asn Gly Gly Pro Asp Ser Cys Gly Cys Val Thr Ser 195 200 205
ggg gga aca gaa agc ata ctc atg gcc tgc aaa gca tat cgg gat ctg 672
Gly Gly Thr Glu Ser Ile Leu Met Ala Cys Lys Ala Tyr Arg Asp Leu 210
215 220 gcc ttt gag aag ggg atc aaa act cca gaa att gtg gct ccc caa
agt 720 Ala Phe Glu Lys Gly Ile Lys Thr Pro Glu Ile Val Ala Pro Gln
Ser 225 230 235 240 gcc cat gct gca ttt aac aaa gca gcc agt tac ttt
ggg atg aag att 768 Ala His Ala Ala Phe Asn Lys Ala Ala Ser Tyr Phe
Gly Met Lys Ile 245 250 255 gtg cgg gtc cca ttg acg aag atg atg gag
gtg gat gtg agg gca atg 816 Val Arg Val Pro Leu Thr Lys Met Met Glu
Val Asp Val Arg Ala Met 260 265 270 aga aga gct atc tcc agg aac act
gcc atg ctc gtc tgt tct acc cca 864 Arg Arg Ala Ile Ser Arg Asn Thr
Ala Met Leu Val Cys Ser Thr Pro 275 280 285 cag ttt cct cat ggt gta
ata gat cct gtc cct gaa gtg gcc aag ctg 912 Gln Phe Pro His Gly Val
Ile Asp Pro Val Pro Glu Val Ala Lys Leu 290 295 300 gct gtc aaa tac
aaa ata ccc ctt cat gtc gac gct tgt ctg gga ggc 960 Ala Val Lys Tyr
Lys Ile Pro Leu His Val Asp Ala Cys Leu Gly Gly 305 310 315 320 ttc
ctc atc gtc ttt atg gag aaa gca gga tac cca ctg gag cac cca 1008
Phe Leu Ile Val Phe Met Glu Lys Ala Gly Tyr Pro Leu Glu His Pro 325
330 335 ttt gat ttc cgg gtg aaa ggt gta acc agc att tca gct gac acc
cat 1056 Phe Asp Phe Arg Val Lys Gly Val Thr Ser Ile Ser Ala Asp
Thr His 340 345 350 aag tat ggc tat gcc cca aaa ggc tca tca ttg gtg
ttg tat agt gac 1104 Lys Tyr Gly Tyr Ala Pro Lys Gly Ser Ser Leu
Val Leu Tyr Ser Asp 355 360 365 aag aag tac agg aac tat cag ttc ttc
gtc gat aca gat tgg cag ggt 1152 Lys Lys Tyr Arg Asn Tyr Gln Phe
Phe Val Asp Thr Asp Trp Gln Gly 370
375 380 ggc atc tat gct tcc cca acc atc gca ggc tca cgg cct ggt ggc
att 1200 Gly Ile Tyr Ala Ser Pro Thr Ile Ala Gly Ser Arg Pro Gly
Gly Ile 385 390 395 400 agc gca gcc tgt tgg gct gcc ttg atg cac ttc
ggt gag aac ggc tat 1248 Ser Ala Ala Cys Trp Ala Ala Leu Met His
Phe Gly Glu Asn Gly Tyr 405 410 415 gtt gaa gct acc aaa cag atc atc
aaa act gct cgc ttc ctc aag tca 1296 Val Glu Ala Thr Lys Gln Ile
Ile Lys Thr Ala Arg Phe Leu Lys Ser 420 425 430 gaa ctg gaa aat atc
aaa ggc atc ttt gtt ttt ggg aat ccc caa ttg 1344 Glu Leu Glu Asn
Ile Lys Gly Ile Phe Val Phe Gly Asn Pro Gln Leu 435 440 445 tca gtc
att gct ctg gga tcc cgt gat ttt gac atc tac cga cta tca 1392 Ser
Val Ile Ala Leu Gly Ser Arg Asp Phe Asp Ile Tyr Arg Leu Ser 450 455
460 aac ctg atg act gct aag ggg tgg aac ttg aac cag ttg cag ttc cca
1440 Asn Leu Met Thr Ala Lys Gly Trp Asn Leu Asn Gln Leu Gln Phe
Pro 465 470 475 480 ccc agt att cat ttc tgc atc aca tta cta cac gcc
cgg aaa cga gta 1488 Pro Ser Ile His Phe Cys Ile Thr Leu Leu His
Ala Arg Lys Arg Val 485 490 495 gct ata caa ttc cta aag gac att cga
gaa tct gtc act caa atc atg 1536 Ala Ile Gln Phe Leu Lys Asp Ile
Arg Glu Ser Val Thr Gln Ile Met 500 505 510 aag aat cct aaa gcg aag
acc aca gga atg ggt gcc atc tat ggc atg 1584 Lys Asn Pro Lys Ala
Lys Thr Thr Gly Met Gly Ala Ile Tyr Gly Met 515 520 525 gcc cag aca
act gtt gac agg aat atg gtt gca gaa ttg tcc tca gtc 1632 Ala Gln
Thr Thr Val Asp Arg Asn Met Val Ala Glu Leu Ser Ser Val 530 535 540
ttc ttg gac agc ttg tac agc acc gac act gtc acc cag ggc agc cag
1680 Phe Leu Asp Ser Leu Tyr Ser Thr Asp Thr Val Thr Gln Gly Ser
Gln 545 550 555 560 atg aat ggt tct cca aaa ccc cac tga 1707 Met
Asn Gly Ser Pro Lys Pro His * 565 18 568 PRT Homo sapiens 18 Met
Pro Ser Thr Asp Leu Leu Met Leu Lys Ala Phe Glu Pro Tyr Leu 1 5 10
15 Glu Ile Leu Glu Val Tyr Ser Thr Lys Ala Lys Asn Tyr Val Asn Gly
20 25 30 His Cys Thr Lys Tyr Glu Pro Trp Gln Leu Ile Ala Trp Ser
Val Val 35 40 45 Trp Thr Leu Leu Ile Val Trp Gly Tyr Glu Phe Val
Phe Gln Pro Glu 50 55 60 Ser Leu Trp Ser Arg Phe Lys Lys Lys Cys
Phe Lys Leu Thr Arg Lys 65 70 75 80 Met Pro Ile Ile Gly Arg Lys Ile
Gln Asp Lys Leu Asn Lys Thr Lys 85 90 95 Asp Asp Ile Ser Lys Asn
Met Ser Phe Leu Lys Val Asp Lys Glu Tyr 100 105 110 Val Lys Ala Leu
Pro Ser Gln Gly Leu Ser Ser Ser Ala Val Leu Glu 115 120 125 Lys Leu
Lys Glu Tyr Ser Ser Met Asp Ala Phe Trp Gln Glu Gly Arg 130 135 140
Ala Ser Gly Thr Val Tyr Ser Gly Glu Glu Lys Leu Thr Glu Leu Leu 145
150 155 160 Val Lys Ala Tyr Gly Asp Phe Ala Trp Ser Asn Pro Leu His
Pro Asp 165 170 175 Ile Phe Pro Gly Leu Arg Lys Ile Glu Ala Glu Ile
Val Arg Ile Ala 180 185 190 Cys Ser Leu Phe Asn Gly Gly Pro Asp Ser
Cys Gly Cys Val Thr Ser 195 200 205 Gly Gly Thr Glu Ser Ile Leu Met
Ala Cys Lys Ala Tyr Arg Asp Leu 210 215 220 Ala Phe Glu Lys Gly Ile
Lys Thr Pro Glu Ile Val Ala Pro Gln Ser 225 230 235 240 Ala His Ala
Ala Phe Asn Lys Ala Ala Ser Tyr Phe Gly Met Lys Ile 245 250 255 Val
Arg Val Pro Leu Thr Lys Met Met Glu Val Asp Val Arg Ala Met 260 265
270 Arg Arg Ala Ile Ser Arg Asn Thr Ala Met Leu Val Cys Ser Thr Pro
275 280 285 Gln Phe Pro His Gly Val Ile Asp Pro Val Pro Glu Val Ala
Lys Leu 290 295 300 Ala Val Lys Tyr Lys Ile Pro Leu His Val Asp Ala
Cys Leu Gly Gly 305 310 315 320 Phe Leu Ile Val Phe Met Glu Lys Ala
Gly Tyr Pro Leu Glu His Pro 325 330 335 Phe Asp Phe Arg Val Lys Gly
Val Thr Ser Ile Ser Ala Asp Thr His 340 345 350 Lys Tyr Gly Tyr Ala
Pro Lys Gly Ser Ser Leu Val Leu Tyr Ser Asp 355 360 365 Lys Lys Tyr
Arg Asn Tyr Gln Phe Phe Val Asp Thr Asp Trp Gln Gly 370 375 380 Gly
Ile Tyr Ala Ser Pro Thr Ile Ala Gly Ser Arg Pro Gly Gly Ile 385 390
395 400 Ser Ala Ala Cys Trp Ala Ala Leu Met His Phe Gly Glu Asn Gly
Tyr 405 410 415 Val Glu Ala Thr Lys Gln Ile Ile Lys Thr Ala Arg Phe
Leu Lys Ser 420 425 430 Glu Leu Glu Asn Ile Lys Gly Ile Phe Val Phe
Gly Asn Pro Gln Leu 435 440 445 Ser Val Ile Ala Leu Gly Ser Arg Asp
Phe Asp Ile Tyr Arg Leu Ser 450 455 460 Asn Leu Met Thr Ala Lys Gly
Trp Asn Leu Asn Gln Leu Gln Phe Pro 465 470 475 480 Pro Ser Ile His
Phe Cys Ile Thr Leu Leu His Ala Arg Lys Arg Val 485 490 495 Ala Ile
Gln Phe Leu Lys Asp Ile Arg Glu Ser Val Thr Gln Ile Met 500 505 510
Lys Asn Pro Lys Ala Lys Thr Thr Gly Met Gly Ala Ile Tyr Gly Met 515
520 525 Ala Gln Thr Thr Val Asp Arg Asn Met Val Ala Glu Leu Ser Ser
Val 530 535 540 Phe Leu Asp Ser Leu Tyr Ser Thr Asp Thr Val Thr Gln
Gly Ser Gln 545 550 555 560 Met Asn Gly Ser Pro Lys Pro His 565 19
490 PRT Drosophila melanogaster 19 Phe Arg Ser Ser Asn Asp Tyr Gly
Val Asn Leu Gln Thr Ala Glu Met 1 5 10 15 Trp His His Thr Ile Arg
Lys His Lys Arg Gly Asn Gly Ser Ser Ser 20 25 30 Pro Ala Asp Cys
Gly Lys Gln Leu Leu Ile Leu Leu Asn Pro Lys Ser 35 40 45 Gly Ser
Gly Lys Gly Arg Glu Leu Phe Gln Lys Gln Val Ala Pro Leu 50 55 60
Leu Thr Glu Ala Glu Val Gln Tyr Asp Leu Gln Ile Thr Thr His Pro 65
70 75 80 Gln Tyr Ala Lys Glu Phe Val Arg Thr Arg Arg Asp Leu Leu
Thr Arg 85 90 95 Tyr Ser Gly Ile Val Val Ala Ser Gly Asp Gly Leu
Phe Tyr Glu Val 100 105 110 Leu Asn Gly Leu Met Glu Arg Met Asp Trp
Arg Arg Ala Cys Arg Glu 115 120 125 Leu Pro Leu Gly Ile Ile Pro Cys
Gly Ser Gly Asn Gly Leu Ala Lys 130 135 140 Ser Val Ala His His Cys
Asn Glu Pro Tyr Glu Pro Lys Pro Ile Leu 145 150 155 160 His Ala Thr
Leu Thr Cys Met Ala Gly Lys Ser Thr Pro Met Asp Val 165 170 175 Val
Arg Val Glu Leu Ala Thr Arg Asp Lys His Phe Val Met Tyr Ser 180 185
190 Phe Leu Ser Val Gly Trp Gly Leu Ile Ala Asp Ile Asp Ile Glu Ser
195 200 205 Glu Arg Leu Arg Ser Ile Gly Ala Gln Arg Phe Thr Leu Trp
Ala Ile 210 215 220 Lys Arg Leu Ile Gly Leu Arg Ser Tyr Lys Gly Arg
Val Ser Tyr Leu 225 230 235 240 Leu Gly Lys Gly Lys Lys Glu Pro Pro
Val Glu Ala Ala Arg Glu Leu 245 250 255 Pro Ala Glu Ser Thr Ala Ala
Gly Ile Arg Ser Ser Leu Pro Leu Asn 260 265 270 Ala Gly Glu Phe His
Asp Leu Pro Glu Glu Glu Glu Gly Glu Ala Val 275 280 285 Leu Asp Gly
Glu Gln Phe Ala Asp Ala Ile Ser Leu Asp Arg Ser Val 290 295 300 Tyr
Arg Gln His Ala Asp Ser Trp His Ser Ala Met Ser Arg Arg Thr 305 310
315 320 Ala Tyr Tyr Ser Leu Gly Gly Pro Ser Met Arg Ser Asn Arg Ser
Arg 325 330 335 Met Ser Ile Ser Gln Arg Ile Glu Ala Ala Asn Ala Glu
Phe Ala Glu 340 345 350 Arg Val Pro Thr Gly Thr Ile Pro Pro Leu Gln
Met Pro Leu Leu Ser 355 360 365 Ser Asp Gly Trp Ile Cys Glu Asp Gly
Asp Phe Val Met Val His Ala 370 375 380 Ala Tyr Thr Thr His Leu Ser
Ser Asp Val Phe Phe Ala Pro Glu Ser 385 390 395 400 Arg Leu Asp Asp
Gly Leu Ile Tyr Leu Val Ile Ile Arg Arg Gly Val 405 410 415 Ser Arg
His Gln Leu Leu Asn Phe Met Leu Asn Leu Asn Ala Gly Thr 420 425 430
His Leu Pro Ile Gly Glu Asp Pro Phe Ile Lys Val Val Pro Cys Arg 435
440 445 Ala Phe Arg Ile Glu Pro Ser Ser Ser Asp Gly Ile Leu Val Val
Asp 450 455 460 Gly Glu Arg Val Glu Tyr Gly Pro Ile Gln Ala Glu Val
Met Pro Gly 465 470 475 480 Leu Ile Asn Val Met Thr Thr Ser Gly Gln
485 490 20 524 PRT Drosophila melanogaster 20 Phe Arg Ser Phe Asp
Thr Phe Glu Asp Asn Met Arg Glu Ala Asp Arg 1 5 10 15 Trp Tyr Arg
Ser Leu Arg Trp Gln Leu His Arg Thr Leu Glu Glu Ile 20 25 30 Phe
Val Ala Pro Thr Val Asp Glu Arg Arg Arg Arg Val Leu Val Leu 35 40
45 Leu Asn Pro Lys Ser Gly Ser Gly Asp Ala Arg Glu Val Phe Asn Met
50 55 60 His Val Thr Pro Val Leu Asn Glu Ala Glu Val Pro Tyr Asp
Leu Tyr 65 70 75 80 Val Thr Lys His Ser Asn Phe Ala Ile Glu Phe Leu
Ser Thr Arg Cys 85 90 95 Leu Asp Ala Trp Cys Cys Val Val Ala Val
Gly Gly Asp Gly Leu Phe 100 105 110 His Glu Ile Val Asn Gly Leu Leu
Gln Arg Gln Asp Trp Ala His Val 115 120 125 Leu Pro His Leu Ala Leu
Gly Ile Ile Pro Cys Gly Ser Gly Asn Gly 130 135 140 Leu Ala Arg Ser
Ile Ala His Cys Tyr Asn Lys Pro Val Leu Gly Ala 145 150 155 160 Ala
Leu Thr Val Ile Ser Gly Arg Ser Ser Pro Met Asp Val Val Arg 165 170
175 Val Gln Leu Gln Ser Arg Ser Leu Tyr Ser Phe Leu Ser Ile Gly Trp
180 185 190 Gly Leu Ile Ser Asp Val Asp Ile Glu Ser Glu Arg Ile Arg
Met Leu 195 200 205 Gly Tyr Gln Arg Phe Thr Val Trp Thr Leu Tyr Arg
Leu Val Asn Leu 210 215 220 Arg Thr Tyr Asn Gly Arg Ile Ser Tyr Leu
Leu Thr Asp His Glu Val 225 230 235 240 Ser Ser Thr His Ser Ala Thr
Gly Tyr Ala Ala Gln Arg Arg Met Gln 245 250 255 Ser Ser Arg Ser Cys
Asn Thr His Ile Asp Met Leu Asn Gly Pro Ala 260 265 270 Pro Ile Tyr
His Ser Ser Ala Glu Tyr Leu Pro Gln Glu Phe Ala Asp 275 280 285 Val
Ile Ser Leu Glu Thr Ser Ile Asn Gln Ser Phe Arg Ser Arg Cys 290 295
300 Asp Ser Trp Leu Ser Gly Gly Ser Arg Arg Ser Phe Tyr Tyr Ser Ile
305 310 315 320 Ser Glu Ser Ile Tyr His Ser Leu Ala Asp Glu Ser Glu
Phe Ala Gly 325 330 335 Leu Ala Ala Ala Ser Leu Glu Asn Arg Gln Gln
Asn Tyr Gly Pro Ala 340 345 350 Ser Glu Leu Pro Asp Leu Asn Glu Pro
Leu Ser Glu Asp Gln Gly Trp 355 360 365 Leu Val Glu Glu Gly Glu Phe
Val Met Met His Ala Val Tyr Gln Thr 370 375 380 His Leu Gly Ile Asp
Cys His Phe Ala Pro Lys Ala Gln Leu Asn Asp 385 390 395 400 Gly Thr
Ile Tyr Leu Ile Leu Ile Arg Ala Gly Ile Ser Arg Pro His 405 410 415
Leu Leu Ser Phe Leu Tyr Asn Met Ser Ser Gly Thr His Leu Pro Glu 420
425 430 Ser His Asp Asp His Val Lys Val Leu Pro Val Arg Ala Phe Arg
Leu 435 440 445 Glu Pro Tyr Asp Asn His Gly Ile Ile Thr Val Asp Gly
Glu Arg Val 450 455 460 Glu Phe Gly Pro Leu Gln Ala Glu Val Leu Pro
Gly Ile Ala Arg Val 465 470 475 480 Met Val Pro Asn Val Ser Thr Phe
Arg Phe Gln Ser Ala Thr Leu Gln 485 490 495 His Gly Ile Pro Val Cys
Ile Pro Val Arg Lys Arg Phe Val Leu Tyr 500 505 510 Asn Met Ser Ser
Glu Glu Leu Ala Pro Ile Asn Glu 515 520 21 368 PRT Homo sapiens 21
Val Leu Val Leu Leu Asn Pro Arg Gly Gly Lys Gly Lys Ala Leu Gln 1 5
10 15 Leu Phe Arg Ser His Val Gln Pro Leu Leu Ala Glu Ala Glu Ile
Ser 20 25 30 Phe Thr Leu Met Leu Thr Glu Arg Arg Asn His Ala Arg
Glu Leu Val 35 40 45 Arg Ser Glu Glu Leu Gly Arg Trp Asp Ala Leu
Val Val Met Ser Gly 50 55 60 Asp Gly Leu Met His Glu Val Val Asn
Gly Leu Met Glu Arg Pro Asp 65 70 75 80 Trp Glu Thr Ala Ile Gln Lys
Pro Leu Cys Ser Leu Pro Ala Gly Ser 85 90 95 Gly Asn Ala Leu Ala
Ala Ser Leu Asn His Tyr Ala Gly Tyr Glu Gln 100 105 110 Val Thr Asn
Glu Asp Leu Leu Thr Asn Cys Thr Leu Leu Leu Cys Arg 115 120 125 Arg
Leu Leu Ser Pro Met Asn Leu Leu Ser Leu His Thr Ala Ser Gly 130 135
140 Leu Arg Leu Phe Ser Val Leu Ser Leu Ala Trp Gly Phe Ile Ala Asp
145 150 155 160 Val Asp Leu Glu Ser Glu Lys Tyr Arg Arg Leu Gly Glu
Met Arg Phe 165 170 175 Thr Leu Gly Thr Phe Leu Arg Leu Ala Ala Leu
Arg Thr Tyr Arg Gly 180 185 190 Arg Leu Ala Tyr Leu Pro Val Gly Arg
Val Gly Ser Lys Thr Pro Ala 195 200 205 Ser Pro Val Val Val Gln Gln
Gly Pro Val Asp Ala His Leu Val Pro 210 215 220 Leu Glu Glu Pro Val
Pro Ser His Trp Thr Val Val Pro Asp Glu Asp 225 230 235 240 Phe Val
Leu Val Leu Ala Leu Leu His Ser His Leu Gly Ser Glu Met 245 250 255
Phe Ala Ala Pro Met Gly Arg Cys Ala Ala Gly Val Met His Leu Phe 260
265 270 Tyr Val Arg Ala Gly Val Ser Arg Ala Met Leu Leu Arg Leu Phe
Leu 275 280 285 Ala Met Glu Lys Gly Arg His Met Glu Tyr Glu Cys Pro
Tyr Leu Val 290 295 300 Tyr Val Pro Val Val Ala Phe Arg Leu Glu Pro
Lys Asp Gly Lys Gly 305 310 315 320 Val Phe Ala Val Asp Gly Glu Leu
Met Val Ser Glu Ala Val Gln Gly 325 330 335 Gln Val His Pro Asn Tyr
Phe Trp Met Val Ser Gly Cys Val Glu Pro 340 345 350 Pro Pro Ser Trp
Lys Pro Gln Gln Met Pro Pro Pro Glu Glu Pro Leu 355 360 365
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