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HUMAN SYNTHETASES
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of synthetases
and to the use
of these sequences in the diagnosis, treatment, and prevention of immune,
neuronal, and reproductive
disorders, and cell prol.iferative disorders including cancer.
l0 BACKGROUND OF THE INVENTION
A large number of cellular biosynthetic intermediary metabolism processes
involve
intermolecular transfer of carbon atom-containing substrates (carbon
substrates). Examples of such
reactions include the tricarboxylic acid cycle, synthesis of fatty acids and
long-chain phospholipids,
synthesis of alcohols and aldehydes, synthesis of intermediary metabolites,
and reactions involved in
15 the amino acid degradation pathways. Many of these reactions are catalyzed
by synthetases (also
called ligases), which catalyze the formation of a bond between two substrate
molecules. Some of
these reactions require input of energy, usually in the form of conversion of
ATP to either ADP or
AMP and pyrophosphate. Synthetases are named for the products of the reaction
they catalyze and
are involved in such processes as metabolism and the synthesis of
macromolecules.
20 LiQases forming carbon-oxygen bonds
Proteins make up more than half of the total dry mass of a cell. The synthesis
of proteins is
central to cell maintenance, growth, and development. Synthesis occurs on
ribosomes and depends
on the cooperative interaction of several classes of RNA molecules. The
process begins with
transcription of the genetic code contained within the DNA to form messenger
RNA (mRNA). The
25 mRNA moves in steps through a ribosome and the nucleotide sequence of the
mRNA is translated
into a corresponding sequence of amino acids to construct a distinct protein
chain.
The aminoacyl-transfer RNA (tRNA) synthetases are important RNA-associated
enzymes
with roles in translation. Protein biosynthesis depends on each amino acid
forming a linkage with the
appropriate tRNA. The aminoacyl-tRNA synthetases are responsible for the
activation and correct
30 attachment of an amino acid with its cognate tRNA. The 20 aminoacyl-tRNA
synthetase enzymes
can be divided into two structural classes, and each class is characterized by
a distinctive topology of
the catalytic domain. Class I enzymes contain a catalytic domain based on the
nucleotide-binding
Rossman 'fold'. Class II enzymes contain a central catalytic domain, which
consists of a
seven-stranded antiparallel I3-sheet motif, as well as N- and C- terminal
regulatory domains. Class II
35 enzymes are separated into two groups based on the heterodimeric or
homodimeric structure of the
enzyme; the latter group is further subdivided by the structure of the N- and
C-terminal regulatory
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domains (Hartlein, M. and Cusack, S. ( 1995) J. Mol. Evol. 40:519-530).
Autoantibodies against
aminoacyl-tRNAs are generated by patients with dermatomyositis and
polymyositis, and correlate
strongly with complicating interstitial lung disease (ILD). These antibodies
appear to be generated in
response to viral infection, and coxsackie virus has been used to induce
experimental viral myositis in
animals (Friedman, A.W. et al. (1996) Semin. Arthritis Rheum. 26:459-467). A
synthetase homolog
has been shown to be expressed in chronic myeloid leukemia (CML). A
phenylalanine-tRNA
synthetase homolog has been found to be tumor-selective and expressed in a
cell cycle stage- and
differentiation-dependent fashion in an acute-phase human CML cell line (Sen,
S. et al. ( 1997) Proc.
Nat). Acad. Sci.USA 94:6164-6169).
L~yases forming carbon-sulfur bonds (Acid-thiol 1i ag ses)
In many cases, a carbon substrate is derived from a small molecule containing
at least two
carbon atoms. The carbon substrate is often covalently bound to a larger
molecule which acts as a
carbon substrate carrier molecule within the cell. In the biosynthetic
mechanisms described above,
the carrier molecule is coenzyme A. Coenzyme A (CoA) is structurally related
to derivatives of the
nucleotide ADP and consists of 4'-phosphopantetheine linked via a
phosphodiester bond to the alpha
phosphate group of adenosine 3',5'-bisphosphate. The terminal thiol group of
4'-phosphopantetheine
acts as the site for carbon substrate bond formation. The predominant carbon
substrates which utilize
CoA as a carrier molecule during biosynthesis and intermediary metabolism in
the cell are acetyl,
succinyl, and propionyl moieties, collectively referred to as acyl groups.
Other carbon substrates
include enoyl lipid, which acts as a fatty acid oxidation intermediate, and
carnitine, which acts as an
acetyl-CoA flux regulator) mitochondria) acyl group transfer protein. Acyl-CoA
and acetyl-CoA are
synthesized in the cell by acyl-CoA synthetase and acetyl-CoA synthetase,
respectively.
Activation of fatty acids is mediated by at least three forms of acyl-CoA
synthetase activity:
i) acetyl-CoA synthetase, which activates acetate and several other low
molecular weight carboxylic
acids and is found in muscle mitochondria and the cytosol of other tissues;
ii) medium-chain
acyl-CoA synthetase, which activates fatty acids containing between four and
eleven carbon atoms
(predominantly from dietary sources), and is present only in liver
mitochondria; and iii) acyl CoA
synthetase, which is specific for long chain fatty acids with between six and
twenty carbon atoms,
and is found in microsomes and the mitochondria. Proteins associated with acyl-
CoA synthetase
activity have been identified from many sources including bacteria, yeast,
plants, mouse, and man.
The activity of acyl-CoA synthetase may be modulated by phosphorylation of the
enzyme by
cAMP-dependent protein kinase. The COL4A5 (collagen, type IV, alpha-5)
chromosomal region
found deleted in 2 patients with Alport syndrome, elliptocytosis, and mental
retardation (Piccini, M.
et al. (1998) Genomics 47: 350-358) is contiguous with the region containing
long-chain acyl-CoA
synthetase 4 (FACL4). Therefore, it has been suggested (Piccini supra) that
the absence of FACL4
may be involved in the development of mental retardation and other phenotypes
associated with
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Alport syndrome in these patients.
Li~ases formin; carbon-nitrogen bonds
A key representative of the amide synthases is the enzyme glutamine synthetase
(glutamate-
ammonia ligase) that catalyzes the amination of glutamic acid to glutamine by
ammonia using the
energy of ATP hydrolysis. Glutamine is the primary source for the amino group
in various amide
transfer reactions involved in de novo pyrimidine nucleotide synthesis and in
purine and pyrimidine
ribonucleotide interconversions, as well as the conversion of aspartate to
asparagine. Overexpression
of glutamine synthetase has been observed in primary liver cancer (Christa, L.
et al. ( 1994) Gastroent.
106:1312-1320).
Cyclo-ligases and other carbon-nitrogen ligases comprise various enzymes and
enzyme
complexes that participate in the de novo pathways to purine and pyrimidine
biosynthesis. Because
these pathways are critical to the synthesis of nucleotides for replication of
both RNA and DNA,
many of these enzymes have been the targets of clinical agents for the
treatment of cell proliferative
disorders such as cancer and infectious diseases.
Purine biosynthesis occurs de novo from the amino acids glycine and glutamine,
and other
small molecules. Three of the key reactions in this process are catalyzed by a
trifunctional enzyme
composed of glycinamide-ribonucleotide synthetase (GARS), aminoimidazole
ribonucleotide
synthetase (AIRS), and glycinamide ribonucleotide transformylase (GART).
Together these three
enzymes combine ribosylamine phosphate with glycine to yield phosphoribosyl
aminoimidazole, a
precursor to both adenylate and guanylate nucleotides. This trifunctional
protein has been implicated
in the pathology of Downs syndrome (Aimi, J. et al. ( 1990) Nucleic Acid Res.
18:6665-6672).
Adenylosuccinate synthetase catalyzes a later step in purine biosynthesis that
converts inosinic acid
to adenylosuccinate, a key step on the path to ATP synthesis. This enzyme is
also similar to another
carbon-nitrogen ligase, argininosuccinate synthetase, that catalyzes a similar
reaction in the urea
cycle (Powell, S.M. et al. (1992) FEBS Lett. 303:4-10).
Like the de novo biosynthesis of purines, de novo synthesis of the pyrimidine
nucleotides
uridylate and cytidylate also arises from a common precursor, in this instance
the nucleotide
orotidylate derived from orotate and phosphoribosyl pyrophosphate (PPRP).
Again a trifunctional
enzyme comprising three carbon-nitrogen ligases plays a key role in the
process. In this case the
enzymes aspartate transcarbamylase (ATCase), carbamyl phosphate synthetase II,
and dihydroorotase
(DHOase) are encoded by a single gene called CAD. Together these three enzymes
combine the
initial reactants in pyrimidine biosynthesis, glutamine, CO,, and ATP to form
dihydroorotate, the
precursor to orotate and orotidylate (Iwahana, H. et al. ( 1996) Biochem.
Biophys. Res. Commun.
219:249-255). Further steps then lead to the synthesis of uridine nucleotides
from orotidylate.
Cytidine nucleotides are derived from uridine-5'-triphosphate (UTP) by the
amidation of UTP using
glutamine as the amino donor and the enzyme CTP synthetase. Regulatory
mutations in the human
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CTP synthetase are believed to confer multi-drug resistance to agents widely
used in cancer therapy
(Yamauchi, M. et al. ( 1990) EMBO J. 9:2095-2099).
uses forming carbon-carbon bonds
Ligases in this group are represented by the carboxylases acetyl-CoA
carboxylase and
pyruvate carboxylase. Acetyl-CoA carboxylase is a complex which includes a
biotin carboxyl carrier
protein, biotin carboxylase, and a carboxyl transferase made up of two alpha
and two beta subunits.
This complex catalyzes the carboxylation of Acetyl-CoA from CO, and H~O using
the energy of ATP
hydrolysis (PRINTS document PR01069). Acetyl-CoA carboxylase is the rate-
limiting step in the
biogenesis of long-chain fatty acids. Two isoforms of Acetyl-CoA carboxylase,
types I and types II,
are expressed in humans in a tissue-specific manner (Ha, J. et al. (1994) Eur.
J. Biochem. 219:297-
306). Pyruvate carboxylase is a nuclear-encoded mitochondria) enzyme that
catalyzes the conversion
of pyruvate to oxaloacetate, a key intermediate in the citric acid cycle.
Li~ases forming.phosphoric ester bonds
Ligases in this group are represented by the DNA ligases involved in both DNA
replication
IS and repair. DNA ligases seal phosphodiester bonds between two adjacent
nucleotides in a DNA
chain using the energy from ATP hydrolysis to first activate the free 5'-
phosphate of one nucleotide
and then react it with the 3'-OH group of the adjacent nucleotide. This
resealing reaction is used in
both DNA replication to join small DNA fragments called "Okazaki" fragments
that are transiently
formed in the process of replicating new DNA, and in DNA repair. DNA repair is
the process by
which accidental base changes, such as those produced by oxidative damage,
hydrolytic attack, or
uncontrolled methylation of DNA, are corrected before replication or
transcription of the DNA can
occur. Bloom's syndrome is an inherited human disease in which individuals are
partially deficient
in DNA ligation and consequently have an increased incidence of cancer
(Alberts, B. et al. ( 1994)
The Molecular Biology of the Cell, Garland Publishing Inc., New York, NY, p.
247).
The discovery of new synthetases and the polynucleotides encoding them
satisfies a need in
the art by providing new compositions which are useful in the diagnosis,
prevention, and treatment of
immune, neuronal, and reproductive disorders, and cell proliferative disorders
including cancer.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, human synthetases, referred to
collectively as
"SYNT" and individually as "SYNT-I," "SYNT-2," "SYNT-3," "SYNT-4,""SYNT-5,"
"SYNT-6,"
"SYNT-7," "SYNT-8," "SYNT-9,""SYNT-10," "SYNT-11," "SYNT-12," "SYNT-13," "SYNT-
14,"
and "SYNT-15." In one aspect, the invention provides an isolated polypeptide
comprising an amino
acid sequence selected from the group consisting of a) an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-15, b) a naturally occurring amino acid
sequence having at least
90% sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID
4
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NO:1-15, c) a biologically active fragment of an amino acid sequence selected
from the group
consisting of SEQ >D NO:1-15, and d) an immunogenic fragment of an amino acid
sequence selected
from the group consisting of SEQ ID NO:1-15. In one alternative, the invention
provides an isolated
polypeptide comprising the amino acid sequence of SEQ ID NO:1-15.
The invention further provides an isolated polynucleotide encoding a
polypeptide comprising
an amino acid sequence selected from the group consisting of a) an amino acid
sequence selected
from the group consisting of SEQ ID NO:1-15, b) a naturally occurnng amino
acid sequence having
at least 90% sequence identity to an amino acid sequence selected from the
group consisting of SEQ
ID NO:I-15, c) a biologically active fragment of an amino acid sequence
selected from the group
consisting of SEQ ID NO:l-15, and d) an immunogenic fragment of an amino acid
sequence selected
from the group consisting of SEQ ID NO:1-15. In one alternative, the
polynucleotide encodes a
polypeptide selected from the group consisting of SEQ ID NO:1-15. In another
alternative, the
polynucleotide is selected from the group consisting of SEQ ID N0:16-30.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide comprising
an amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-15, b) a naturally occurring amino acid sequence
having at least 90%
sequence identity to an amino acid sequence selected from the group consisting
of SEQ ID NO:1-15,
c) a biologically active fragment of an amino acid sequence selected from the
group consisting of
SEQ ID NO:1-15, and d) an immunogenic fragment of an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-15. In one alternative, the invention provides
a cell transformed
with the recombinant polynucleotide. In another alternative, the invention
provides a transgenic
organism comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide comprising an
amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID NO:I-15, b) a naturally occurring amino acid sequence
having at least 90%
sequence identity to an amino acid sequence selected from the group consisting
of SEQ ID NO: I-I5,
c) a biologically active fragment of an amino acid sequence selected from the
group consisting of
SEQ ID NO:1-15, and d) an immunogenic fragment of an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-15. The method comprises a) culturing a cell
under conditions
suitable for expression of the polypeptide, wherein said cell is transformed
with a recombinant
polynucleotide comprising a promoter sequence operably linked to a
polynucleotide encoding the
polypeptide, and b) recovering the polypeptide so expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide comprising an amino acid sequence selected from the group
consisting of a) an amino
acid sequence selected from the group consisting of SEQ ID NO:1-15, b) a
naturally occurring amino
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acid sequence having at least 90% sequence identity to an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-15, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-15, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-15.
The invention further provides an isolated polynucleotide comprising a
polynucleotide
sequence selected from the group consisting of a) a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:16-30, b) a naturally occurring polynucleotide
sequence having at least
90% sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:16-30, c) a polynucleotide sequence complementary to a), d) a
polynucleotide sequence
complementary to b), and e) an RNA equivalent of a)-d). In one alternative,
the polynucleotide
comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a
sample, said target polynucleotide having a sequence of a polynucleotide
comprising a polynucleotide
sequence selected from the group consisting of a) a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:16-30, b) a naturally occurring polynucleotide
sequence having at least
90% sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ 1D
N0:16-30, c) a polynucleotide sequence complementary to a), d) a
polynucleotide sequence
complementary to b), and e) an RNA equivalent of a)-d). The method comprises
a) hybridizing the
sample with a probe comprising at least 20 contiguous nucleotides comprising a
sequence
complementary to said target polynucleotide in the sample, and which probe
specifically hybridizes to
said target polynucleotide, under conditions whereby a hybridization complex
is formed between said
probe and said target polynucleotide or fragments thereof, and b) detecting
the presence or absence of
said hybridization complex, and optionally, if present, the amount thereof. In
one alternative, the
probe comprises at least 60 contiguous nucleotides.
The invention further provides a method for detecting a target polynucleotide
in a sample,
said target polynucleotide having a sequence of a polynucleotide comprising a
polynucleotide
sequence selected from the group consisting of a) a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:16-30, b) a naturally occurring polynucleotide
sequence having at least
90% sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:16-30, c) a polynucleotide sequence complementary to a), d) a
polynucleotide sequence
complementary to b), and e) an RNA equivalent of a)-d). The method comprises
a) amplifying said
target polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b)
detecting the presence or absence of said amplified target polynucleotide or
fragment thereof, and,
optionally, if present, the amount thereof.
The invention further provides a pharmaceutical composition comprising an
effective amount
of a polypeptide comprising an amino acid sequence selected from the group
consisting of a) an
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amino acid sequence selected from the group consisting of SEQ ID NO:1-15, b) a
naturally occurring
amino acid sequence having at least 90% sequence identity to an amino acid
sequence selected from
the group consisting of SEQ ID NO:1-15, c) a biologically active fragment of
an amino acid sequence
selected from the group consisting of SEQ ID NO:1-15, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-15, and a
pharmaceutically
acceptable excipient. In one embodiment, the pharmaceutical composition
comprises an amino acid
sequence selected from the group consisting of SEQ ID NO:1-15. The invention
additionally
provides a method of treating a disease or condition associated with decreased
expression of
functional SYNT, comprising administering to a patient in need of such
treatment the pharmaceutical
composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide comprising an amino acid sequence selected from the
group consisting of a)
an amino acid sequence selected from the group consisting of SEQ )D NO:1-15,
b) a naturally
occurring amino acid sequence having at least 90% sequence identity to an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-15, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO: I-15, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-15. The
method comprises a) exposing a sample comprising the polypeptide to a
compound, and b) detecting
agonist activity in the sample. In one alternative, the invention provides a
pharmaceutical
composition comprising an agonist compound identified by the method and a
pharmaceutically
acceptable excipient. In another alternative, the invention provides a method
of treating a disease or
condition associated with decreased expression of functional SYNT, comprising
administering to a
patient in need of such treatment the pharmaceutical composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide comprising an amino acid sequence selected from
the group consisting
of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-
15, b) a naturally
occurring amino acid sequence having at least 90% sequence identity to an
amino acid sequence
selected from the group consisting of SEQ ID NO: I-15, c) a biologically
active fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-15, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
117 NO:1-15. The
method comprises a) exposing a sample comprising the polypeptide to a
compound, and b) detecting
antagonist activity in the sample. In one alternative, the invention provides
a pharmaceutical
composition comprising an antagonist compound identified by the method and a
pharmaceutically
acceptable excipient. In another alternative, the invention provides a method
of treating a disease or
condition associated with overexpression of functional SYNT, comprising
administering to a patient
in need of such treatment the pharmaceutical composition.
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The invention further provides a method of screening for a compound that
specifically binds
to a polypeptide comprising an amino acid sequence selected from the group
consisting of a) an
amino acid sequence selected from the group consisting of SEQ ID NO:1-15, b) a
naturally occurring
amino acid sequence having at least 90% sequence identity to an amino acid
sequence selected from
the group consisting of SEQ ID NO:1-15, c) a biologically active fragment of
an amino acid sequence
selected from the group consisting of SEQ ID NO:1-15, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:I-15. The method
comprises a)
combining the polypeptide with at least one test compound under suitable
conditions, and b)
detecting binding of the polypeptide to the test compound, thereby identifying
a compound that
specifically binds to the polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide comprising an amino acid sequence selected from the
group consisting of a)
an amino acid sequence selected from the group consisting of SEQ ID NO:I-I5,
b) a naturally
occurnng amino acid sequence having at least 90% sequence identity to an amino
acid sequence
selected from the group consisting of SEQ ID NO:1-15, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-15, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-15. The
method comprises a) combining the polypeptide with at least one test compound
under conditions
permissive for the activity of the polypeptide, b) assessing the activity of
the polypeptide in the
presence of the test compound, and c) comparing the activity of the
polypeptide in the presence of the
test compound with the activity of the polypeptide in the absence of the test
compound, wherein a
change in the activity of the polypeptide in the presence of the test compound
is indicative of a
compound that modulates the activity of the polypeptide.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
sequence selected from the group consisting of SEQ ID N0:16-30, the method
comprising a)
exposing a sample comprising the target polynucleotide to a compound, and b)
detecting altered
expression of the target polynucleotide.
The invention further provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound;
b) hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide comprising a polynucleotide
sequence selected from the
group consisting of i) a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:16-30, ii) a naturally occurring polynucleotide sequence having at least
90% sequence identity to
a polynucleotide sequence selected from the group consisting of SEQ ID N0:16-
30, iii) a
polynucleotide sequence complementary to i), iv) a polynucleotide sequence
complementary to ii),
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and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions
whereby a specific
hybridization complex is formed between said probe and a target polynucleotide
in the biological
sample, said target polynucleotide comprising a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:16-30, ii) a naturally occurring polynucleotide
sequence having at least
90% sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:16-30, iii) a polynucleotide sequence complementary to i), iv) a
polynucleotide sequence
complementary to ii), and v) an RNA equivalent of i)-iv). Alternatively, the
target polynucleotide
comprises a fragment of the above polynucleotide sequence; c) quantifying the
amount of
hybridization complex; and d) comparing the amount of hybridization complex in
the treated
biological sample with the amount of hybridization complex in an untreated
biological sample,
wherein a difference in the amount of hybridization complex in the treated
biological sample is
indicative of toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ
ID NOs),
clone identification numbers (clone IDs), cDNA libraries, and cDNA fragments
used to assemble full-
length sequences encoding SYNT.
Table 2 shows features of each polypeptide sequence, including potential
motifs, homologous
sequences, and methods, algorithms, and searchable databases used for analysis
of SYNT.
Table 3 shows selected fragments of each nucleic acid sequence; the tissue-
specific
expression patterns of each nucleic acid sequence as determined by northern
analysis; diseases,
disorders, or conditions associated with these tissues; and the vector into
which each cDNA was
cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which
cDNA clones
encoding SYNT were isolated.
Table 5 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold
parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which
will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
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and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods are now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing
the cell lines, protocols, reagents and vectors which are reported in the
publications and which might
be used in connection with the invention. Nothing herein is to be construed as
an admission that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
DEFINITIONS
"SYNT" refers to the amino acid sequences of substantially purified SYNT
obtained from
any species, particularly a mammalian species, including bovine, ovine,
porcine, murine, equine, and
human, and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
SYNT. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of SYNT either by
directly interacting with
SYNT or by acting on components of the biological pathway in which SYNT
participates.
An "allelic variant" is an alternative form of the gene encoding SYNT. Allelic
variants may
result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in
polypeptides whose structure or function may or may not be altered. A gene may
have none, one, or
many allelic variants of its naturally occurring form. Common mutational
changes which give rise to
allelic variants are generally ascribed to natural deletions, additions, or
substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the
others, one or more times
in a given sequence.
"Altered" nucleic acid sequences encoding SYNT include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as SYNT or a
polypeptide with at least one functional characteristic of SYNT. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe
of the polynucleotide encoding SYNT, and improper or unexpected hybridization
to allelic variants,
with a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding
SYNT. The encoded protein may also be "altered," and may contain deletions,
insertions, or
substitutions of amino acid residues which produce a silent change and result
in a functionally
equivalent SYNT. Deliberate amino acid substitutions may be made on the basis
of similarity in
CA 02380317 2002-O1-21
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polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the
amphipathic nature of the
residues, as long as the biological or immunological activity of SYNT is
retained. For example,
negatively charged amino acids may include aspartic acid and glutamic acid,
and positively charged
amino acids may include lysine and arginine. Amino acids with uncharged polar
side chains having
similar hydrophilicity values may include: asparagine and glutamine; and
serine and threonine.
Amino acids with uncharged side chains having similar hydrophilicity values
may include: leucine,
isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or synthetic
molecules. Where "amino acid sequence" is recited to refer to a sequence of a
naturally occurring
protein molecule, "amino acid sequence" and like terms are not meant to limit
the amino acid
sequence to the complete native amino acid sequence associated with the
recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well
known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of SYNT. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of SYNT either by
directly interacting with SYNT or by acting on components of the biological
pathway in which SYNT
participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')=, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind SYNT polypeptides can be prepared using intact
polypeptides or using
fragments containing small peptides of interest as the immunizing antigen. The
polypeptide or
oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit)
can be derived from the
translation of RNA, or synthesized chemically, and can be conjugated to a
earner protein if desired.
Commonly used carriers that are chemically coupled to peptides include bovine
serum albumin,
thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is
then used to immunize
the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures
on the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
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(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA;
RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone
linkages such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
designation "positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic SYNT, or of
any oligopeptide thereof,
to induce a specific immune response in appropriate animals or cells and to
bind with specific
antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising
a given amino acid sequence" refer broadly to any composition containing the
given polynucleotide
or amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding SYNT or fragments of
SYNT may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCI), detergents
(e.g., sodium dodecyl
sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk,
salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to repeated
DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit
(PE Biosystems,
Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which
has been assembled from
one or more overlapping cDNA, EST, or genomic DNA fragments using a computer
program for
fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison
WI) or Phrap
(University of Washington, Seattle WA). Some sequences have been both extended
and assembled to
produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of
the protein is conserved and not significantly changed by such substitutions.
The table below shows
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amino acids which may be substituted for an original amino acid in a protein
and which are regarded
as conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide sequence can include, for example,
replacement of
hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative
polynucleotide encodes a
polypeptide which retains at least one biological or immunological function of
the natural molecule.
A derivative polypeptide is one modified by glycosylation, pegylation, or any
similar process that
retains at least one biological or immunological function of the polypeptide
from which it was
derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
A "fragment" is a unique portion of SYNT or the polynucleotide encoding SYNT
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example,
a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10,
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15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 25% or 50% of a polypeptide)
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ ID N0:16-30 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:16-30, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:16-30 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID N0:16-30 from related polynucleotide sequences. The precise length of a
fragment of SEQ
ID N0:16-30 and the region of SEQ ID N0:16-30 to which the fragment
corresponds are routinely
determinable by one of ordinary skill in the art based on the intended purpose
for the fragment.
A fragment of SEQ ID NO:1-15 is encoded by a fragment of SEQ ID N0:16-30. A
fragment
of SEQ ID NO:1-15 comprises a region of unique amino acid sequence that
specifically identifies
SEQ ID NO:1-15. For example, a fragment of SEQ ID NO:1-15 is useful as an
immunogenic peptide
for the development of antibodies that specifically recognize SEQ ID NO:I-15.
The precise length of
a fragment of SEQ ID NO:1-15 and the region of SEQ ID NO:1-15 to which the
fragment
corresponds are routinely determinable by one of ordinary skill in the art
based on the intended
purpose for the fragment.
A "full-length" polynucleotide sequence is one containing at least a
translation initiation
codon (e.g., methionine) followed by an open reading frame and a translation
termination codon. A
"full-length" polynucleotide sequence encodes a "full-length" polypeptide
sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between
two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide
sequences, refer
to the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps
in the sequences being compared in order to optimize alignment between two
sequences, and
therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. (1992) CABIOS
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8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The
"weighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
is provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment
Search Tool (BLAST) (Altschul, S.F. et al. ( 1990) J. Mol. Biol. 215:403-410),
which is available
from several sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence
analysis programs including "blastn," that is used to align a known
polynucleotide sequence with
other polynucleotide sequences from a variety of databases. Also available is
a tool called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html.
The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: -2
Open Gap: S and Extension Gap: 2 penalties
Gap x drop-off:' S0
Expect: 10
Word Size: I I
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length
supported by the sequences shown herein, in the tables, figures, or Sequence
Listing, may be used to
describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
CA 02380317 2002-O1-21
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The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some
alignment methods take into account conservative amino acid substitutions.
Such conservative
substitutions, explained in more detail above, generally preserve the charge
and hydrophobicity at the
site of substitution, thus preserving the structure (and therefore function)
of the polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=l, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version
2Ø12 (Apr-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Open Gap: II and Extension Gap: I penalties
Gap x drop-off.' SO
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ ID number, or may be measured over
a shorter length, for
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
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"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the
stringency of the hybridization process, with more stringent conditions
allowing less non-specific
binding, i.e., binding between pairs of nucleic acid strands that are not
perfectly matched. Permissive
conditions for annealing of nucleic acid sequences are routinely determinable
by one of ordinary skill
in the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
1 % (w/v) SDS, and about 100 pg/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook, J. et al.,
1989, Molecular Cloning: A Laboratory Manual, 2"'' ed., vol. 1-3, Cold Spring
Harbor Press,
Plainview NY; specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present
invention include wash conditions of 68°C in the presence of about 0.2
x SSC and about 0.1 % SDS,
for 1 hour. Alternatively, temperatures of about 65°C, 60°C,
55°C, or 42°C may be used. SSC
concentration may be varied from about 0.1 to 2 x SSC, with SDS being present
at about 0.1 %.
Typically, blocking reagents are used to block non-specific hybridization.
Such blocking reagents
include, for instance, sheared and denatured salmon sperm DNA at about 100-200
~tg/ml. Organic
solvent, such as formamide at a concentration of about 35-50% v/v, may also be
used under particular
circumstances, such as for RNA:DNA hybridizations. Useful variations on these
wash conditions
will be readily apparent to those of ordinary skill in the art. Hybridization,
particularly under high
stringency conditions, may be suggestive of evolutionary similarity between
the nucleotides. Such
similarity is strongly indicative of a similar role for the nucleotides and
their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
17
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to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
An "irnmunogenic fragment" is a polypeptide or oligopeptide fragment of SYNT
which is
capable of eliciting an immune response when introduced into a living
organism, for example, a
mammal. The term "immunogenic fragment" also includes any polypeptide or
oligopeptide fragment
of SYNT which is useful in any of the antibody production methods disclosed
herein or known in the
art.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides,
polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of SYNT. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
biological, functional, or irnmunological properties of SYNT.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop
transcript
elongation, and may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an SYNT may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in
the art. These processes may occur synthetically or biochemically. Biochemical
modifications will
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vary by cell type depending on the enzymatic milieu of SYNT.
"Probe" refers to nucleic acid sequences encoding SYNT, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents,
and enzymes.
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be
annealed to a target
polynucleotide by complementary base-pairing. The primer may then be extended
along the target
DNA strand by a DNA polymerase enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence, e.g., by the polymerase chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, 2'~
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et a1.,1987, Current
Protocols in Molecular
Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al., 1990, PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to
5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer
selection programs have incorporated additional features for expanded
capabilities. For example, the
PrimOU primer selection program (available to the public from the Genome
Center at University of
Texas South West Medical Center, Dallas TX) is capable of choosing specific
primers from
megabase sequences and is thus useful for designing primers on a genome-wide
scope. The Primer3
primer selection program (available to the public from the Whitehead
Institute/MIT Center for
Genome Research, Cambridge MA) allows the user to input a "mispriming
library," in which
sequences to avoid as primer binding sites are user-specified. Primer3 is
useful, in particular, for the
selection of oligonucleotides for microarrays. (The source code for the latter
two primer selection
programs may also be obtained from their respective sources and modified to
meet the user's specific
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needs.) The PrimeGen program (available to the public from the UK Human Genome
Mapping
Project Resource Centre, Cambridge UK) designs primers based on multiple
sequence alignments,
thereby allowing selection of primers that hybridize to either the most
conserved or least conserved
regions of aligned nucleic acid sequences. Hence, this program is useful for
identification of both
unique and conserved oligonucleotides and polynucleotide fragments. The
oligonucleotides and
polynucleotide fragments identified by any of the above selection methods are
useful in hybridization
technologies, for example, as PCR or sequencing primers, microarray elements,
or specific probes to
identify fully or partially complementary polynucleotides in a sample of
nucleic acids. Methods of
oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter
sequence. Such a recombinant nucleic acid may be part of a vector that is
used, for example, to
transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions
(UTRs). Regulatory elements interact with host or viral proteins which control
transcription,
translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of the
nitrogenous base thymine are replaced with uracil, and the sugar backbone is
composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing nucleic
acids encoding SYNT, or fragments thereof, or SYNT itself, may comprise a
bodily fluid; an extract
from a cell, chromosome, organelle, or membrane isolated from a cell; a cell;
genomic DNA, RNA, or
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cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular
structure of the protein, e.g., the antigenic determinant or epitope,
recognized by the binding
molecule. For example, if an antibody is specific for epitope "A," the
presence of a polypeptide
comprising the epitope A, or the presence of free unlabeled A, in a reaction
containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the
antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 75% free, and most preferably at least 90% free from other
components with which
they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides
by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" refers to the collective pattern of gene expression by a
particular cell
type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into
a recipient
cell. Transformation may occur under natural or artificial conditions
according to various methods
well known in the art, and may rely on any known method for the insertion of
foreign nucleic acid
sequences into a prokaryotic or eukaryotic host cell. The method for
transformation is selected based
on the type of host cell being transformed and may include, but is not limited
to, bacteriophage or
viral infection, electroporation, heat shock, lipofection, and particle
bombardment. The term
"transformed" cells includes stably transformed cells in which the inserted
DNA is capable of
replication either as an autonomously replicating plasmid or as part of the
host chromosome, as well
as transiently transformed cells which express the inserted DNA or RNA for
limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not
limited to
animals and plants, in which one or more of the cells of the organism contains
heterologous nucleic
acid introduced by way of human intervention, such as by transgenic techniques
well known in the
art. The nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor
of the cell, by way of deliberate genetic manipulation, such as by
microinjection or by infection with
a recombinant virus. The term genetic manipulation does not include classical
cross-breeding, or in
vitro fertilization, but rather is directed to the introduction of a
recombinant DNA molecule. The
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transgenic organisms contemplated in accordance with the present invention
include bacteria,
cyanobacteria, fungi, plants, and animals. The isolated DNA of the present
invention can be
introduced into the host by methods known in the art, for example infection,
transfection,
transformation or transconjugation. Techniques for transfernng the DNA of the
present invention
into such organisms are widely known and provided in references such as
Sambrook et al. ( 1989),
supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
95% or at least 98% or
greater sequence identity over a certain defined length. A variant may be
described as, for example,
an "allelic" (as defined above), "splice," "species," or "polymorphic"
variant. A splice variant may
have significant identity to a reference molecule, but will generally have a
greater or lesser number of
IS polynucleotides due to alternative splicing of exons during mRNA
processing. The corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotide sequences that vary
from one species to
another. The resulting polypeptides generally will have significant amino acid
identity relative to
each other. A polymorphic variant is a variation in the polynucleotide
sequence of a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least
98% or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human synthetases (SYNT), the
polynucleotides encoding SYNT, and the use of these compositions for the
diagnosis, treatment, or
prevention of immune, neuronal, and reproductive disorders, and cell
proliferative disorders including
cancer.
Table 1 lists the Incyte clones used to assemble full length nucleotide
sequences encoding
SYNT. Columns 1 and 2 show the sequence identification numbers (SEQ >D NOs) of
the polypeptide
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and nucleotide sequences, respectively. Column 3 shows the clone IDs of the
Incyte clones in which
nucleic acids encoding each SYNT were identified, and column 4 shows the cDNA
libraries from
which these clones were isolated. Column 5 shows Incyte clones and their
corresponding cDNA
libraries. Clones for which cDNA libraries are not indicated were derived from
pooled cDNA
libraries. In some cases, GenBank sequence identifiers are also shown in
column 5. The Incyte clones
and GenBank cDNA sequences, where indicated, in column 5 were used to assemble
the consensus
nucleotide sequence of each SYNT and are useful as fragments in hybridization
technologies.
The columns of Table 2 show various properties of each of the polypeptides of
the invention:
column 1 references the SEQ ID NO; column 2 shows the number of amino acid
residues in each
polypeptide; column 3 shows potential phosphorylation sites; column 4 shows
potential glycosylation
sites; column 5 shows the amino acid residues comprising signature sequences
and motifs; column 6
shows homologous sequences as identified by BLAST analysis; and column 7 shows
analytical
methods and in some cases, searchable databases to which the analytical
methods were applied. The
methods of column 7 were used to characterize each polypeptide through
sequence homology and
protein motifs.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or
conditions
associated with nucleotide sequences encoding SYNT. The first column of Table
3 lists the
nucleotide SEQ ID NOs. Column 2 lists fragments of the nucleotide sequences of
column 1. These
fragments are useful, for example, in hybridization or amplification
technologies to identify SEQ ID
N0:16, SEQ ID N0:17, SEQ ID N0:18, SEQ ID N0:19 SEQ ID N0:20, SEQ ID N0:21,
SEQ )D
N0:22, SEQ ID N0:23, SEQ ID N0:25, SEQ ID N0:26, SEQ ID N0:27, SEQ ID N0:29,
and SEQ
ID N0:30 and to distinguish between SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:18,
SEQ ID
N0:19 SEQ ID N0:20, SEQ >D N0:21, SEQ ID N0:22, SEQ ID N0:23, SEQ ID N0:25,
SEQ ID
N0:26, SEQ ID N0:27, SEQ ID N0:29, and SEQ ID N0:30 and related polynucleotide
sequences.
The polypeptides encoded by these fragments are useful, for example, as
immunogenic peptides.
Column 3 lists tissue categories which express SYNT as a fraction of total
tissues expressing SYNT.
Column 4 lists diseases, disorders, or conditions associated with those
tissues expressing SYNT as a
fraction of total tissues expressing SYNT. Column 5 lists the vectors used to
subclone each cDNA
library.
The columns of Table 4 show descriptions of the tissues used to construct the
cDNA libraries
from which cDNA clones encoding SYNT were isolated. Column 1 references the
nucleotide SEQ
ID NOs, column 2 shows the cDNA libraries from which these clones were
isolated, and column 3
shows the tissue origins and other descriptive information relevant to the
cDNA libraries in column 2.
SEQ ID NO: 16 maps to chromosome 5 within the interval from 147.10 to 150.00
centiMorgans. SEQ ID NO: 17 maps to chromosome 10 within the interval from
137.60 to 139.20
centiMorgans. This interval also contains gene MXI1, a member of the MYC
family. SEQ ID NO:
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18 maps to chromosome 2 within the interval from 228.80 to 230.10
centiMorgans. This interval also
contains a gene for a proto-oncogene encoding a tyrosine-protein kinase. SEQ
ID N0:21 maps to
chromosome 5 within the interval from 172.6 to 184.7 centiMorgans. SEQ ID
N0:24 maps to
chromosome 2 within the interval from 118.0 to 127.4 centiMorgans. SEQ ID
N0:26 maps to
chromosome 3 within the interval from 157.4 to 162.0 centiMorgans. SEQ ID
N0:27 maps to
chromosome 12 within the interval from 97.1 to 116.6 centiMorgans. SEQ ID
N0:28 maps to
chromosome 4 within the interval from 77.3 to 99.2 centiMorgans and to
chromosome 5 within the
intervals from 79.2 to 92.3 centiMorgans, from 116.3 to 127.9 centiMorgans,
and from 157.6 to 163.0
centiMorgans. SEQ ID N0:29 maps to chromosome 1 within the interval from 242.5
to 258.7
centiMorgans and to chromosome 19 within the interval from 69.9 to 104.9
centiMorgans. SEQ ID
N0:30 maps to chromosome 1 within the interval from 57.2 to 57.5 centiMorgans.
The invention also encompasses SYNT variants. A preferred SYNT variant is one
which has
at least about 80%, or alternatively at least about 90%, or even at least
about 95% amino acid
sequence identity to the SYNT amino acid sequence, and which contains at least
one functional or
structural characteristic of SYNT.
The invention also encompasses polynucleotides which encode SYNT. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:16-30, which encodes SYNT. The
polynucleotide
sequences of SEQ ID N0:16-30, as presented in the Sequence Listing, embrace
the equivalent RNA
sequences, wherein occurrences of the nitrogenous base thymine are replaced
with uracil, and the
sugar backbone is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding
SYNT. In
particular, such a variant polynucleotide sequence will have at least about
70%, or alternatively at
least about 85%, or even at least about 95% polynucleotide sequence identity
to the polynucleotide
sequence encoding SYNT. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID
N0:16-30 which has at least about 70%, or alternatively at least about 85%, or
even at least about
95% polynucleotide sequence identity to a nucleic acid sequence selected from
the group consisting
of SEQ ID N0:16-30. Any one of the polynucleotide variants described above can
encode an amino
acid sequence which contains at least one functional or structural
characteristic of SYNT.
It will be appreciated by those skilled in the art that as a result of the
degeneracy of the
genetic code, a multitude of polynucleotide sequences encoding SYNT, some
bearing minimal
similarity to the polynucleotide sequences of any known and naturally
occurring gene, may be
produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
sequence that could be made by selecting combinations based on possible codon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
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polynucleotide sequence of naturally occurring SYNT, and all such variations
are to be considered as
being specifically disclosed.
Although nucleotide sequences which encode SYNT and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally occurring SYNT under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding SYNT or
its derivatives possessing a substantially different codon usage, e.g.,
inclusion of non-naturally
occurring codons. Codons may be selected to increase the rate at which
expression of the peptide
occurs in a particular prokaryotic or eukaryotic host in accordance with the
frequency with which
particular codons are utilized by the host. Other reasons for substantially
altering the nucleotide
sequence encoding SYNT and its derivatives without altering the encoded amino
acid sequences
include the production of RNA transcripts having more desirable properties,
such as a greater
half-life, than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode SYNT
and
SYNT derivatives, or fragments thereof, entirely by synthetic chemistry. After
production, the
synthetic sequence may be inserted into any of the many available expression
vectors and cell
systems using reagents well known in the art. Moreover, synthetic chemistry
may be used to
introduce mutations into a sequence encoding SYNT or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
N0:16-30 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger ( 1987) Methods Enzymol. 152:399-407; Kimmel, A.R. ( 1987) Methods
Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash
conditions, are described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(PE
Biosystems, Foster City CA), thermostable T7 polymerase (Amersham Pharmacia
Biotech,
Piscataway NJ), or combinations of polymerases and proofreading exonucleases
such as those found
in the ELONGASE amplification system (Life Technologies, Gaithersburg MD).
Preferably,
sequence preparation is automated with machines such as the MICROLAB 2200
liquid transfer
system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA)
and ABI
CATALYST 800 thermal cycler (PE Biosystems). Sequencing is then carried out
using either the
ABI 373 or 377 DNA sequencing system (PE Biosystems), the MEGABACE 1000 DNA
sequencing
system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art.
The resulting
sequences are analyzed using a variety of algorithms which are well known in
the art. (See, e.g.,
Ausubel, F.M. (1997) Short Protocols in Molecular BioloQV, John Wiley & Sons,
New York NY, unit
CA 02380317 2002-O1-21
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7.7: Meyers, R.A. ( 1995) Molecular BioloQV and Biotechnology, Wiley VCH, New
York NY, pp.
856-853.)
The nucleic acid sequences encoding SYNT may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. ( 1993) PCR Methods
Applic. 2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unknown
sequence from a circularized template. The template is derived from
restriction fragments comprising
a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et
al. ( 1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments
adjacent to known sequences in human and yeast artificial chromosome DNA.
(See, e.g., Lagerstrom,
M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme
digestions and ligations may be used to insert an engineered double-stranded
sequence into a region
IS of unknown sequence before performing PCR. Other methods which may be used
to retrieve
unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991)
Nucleic Acids Res.
19:3055-3060). Additionally, one may use PCR, nested primers, and
PROMOTERFINDER libraries
(Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need
to screen libraries
and is useful in finding intron/exon junctions. For all PCR-based methods,
primers may be designed
using commercially available software, such as OLIGO 4.06 Primer Analysis
software (National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30
nucleotides in
length, to have a GC content of about 50% or more, and to anneal to the
template at temperatures of
about 68°C to 72°C.
When screening for full-length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence
into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, PE Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
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which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof
which encode SYNT may be cloned in recombinant DNA molecules that direct
expression of SYNT,
or fragments or functional equivalents thereof, in appropriate host cells. Due
to the inherent
degeneracy of the genetic code, other DNA sequences which encode substantially
the same or a
functionally equivalent amino acid sequence may be produced and used to
express SYNT.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter SYNT-encoding sequences for a variety of
purposes including, but
not limited to, modification of the cloning, processing, and/or expression of
the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction
sites, alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
Number
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. ( 1996) Nat. Biotechnol. 14:315-
319) to alter or
improve the biological properties of SYNT, such as its biological or enzymatic
activity or its ability
to bind to other molecules or compounds. DNA shuffling is a process by which a
library of gene
variants is produced using PCR-mediated recombination of gene fragments. The
library is then
subjected to selection or screening procedures that identify those gene
variants with the desired
properties. These preferred variants may then be pooled and further subjected
to recursive rounds of
DNA shuffling and selection/screening. Thus, genetic diversity is created
through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single
gene containing random
point mutations may be recombined, screened, and then reshuffled until the
desired properties are
optimized. Alternatively, fragments of a given gene may be recombined with
fragments of
homologous genes in the same gene family, either from the same or different
species, thereby
maximizing the genetic diversity of multiple naturally occurring genes in a
directed and controllable
manner.
In another embodiment, sequences encoding SYNT may be synthesized, in whole or
in part,
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
al. (1980) Nucleic Acids
Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.)
Alternatively, SYNT itself or a fragment thereof may be synthesized using
chemical methods. For
example, peptide synthesis can be performed using various solution-phase or
solid-phase techniques.
(See, e.g., Creighton, T. ( 1984) Proteins, Structures and Molecular
Properties, WH Freeman, New
York NY, pp. 55-60; and Roberge, J.Y. et al. ( 1995) Science 269:202-204.)
Automated synthesis
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may be achieved using the ABI 431A peptide synthesizer (PE Biosystems).
Additionally, the amino
acid sequence of SYNT, or any part thereof, may be altered during direct
synthesis and/or combined
with sequences from other proteins, or any part thereof, to produce a variant
polypeptide or a
polypeptide having a sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier ( 1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by
sequencing. (See, e.g., Creighton, sera, pp. 28-53.)
In order to express a biologically active SYNT, the nucleotide sequences
encoding SYNT or
derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which contains
the necessary elements for transcriptional and translational control of the
inserted coding sequence in
a suitable host. These elements include regulatory sequences, such as
enhancers, constitutive and
inducible promoters, and 5' and 3' untranslated regions in the vector and in
polynucleotide sequences
encoding SYNT. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
SYNT. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding SYNT and its initiation codon and upstream regulatory
sequences are inserted
into the appropriate expression vector, no additional transcriptional or
translational control signals
may be needed. However, in cases where only coding sequence, or a fragment
thereof, is inserted,
exogenous translational control signals including an in-frame ATG initiation
codon should be
provided by the vector. Exogenous translational elements and initiation codons
may be of various
origins, both natural and synthetic. The efficiency of expression may be
enhanced by the inclusion of
enhancers appropriate for the particular host cell system used. (See, e.g.,
Scharf, D. et al. ( 1994)
Results Probl. Cell Differ. 20:125-162.)
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding SYNT and appropriate transcriptional and
translational
control elements. These methods include in vitro recombinant DNA techniques,
synthetic techniques,
and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. ( 1989)
Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-
17; Ausubel, F.M. et
al. ( 1995) Current Protocols in Molecular BioloQV, John Wiley & Sons, New
York NY, ch. 9, 13, and
16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding SYNT. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV,
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or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti
or pBR322 plasmids); or
animal cell systems. (See, e.g., Sambrook, supra; Ausubel, su ra; Van Heeke,
G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Bitter, G.A. et al. (1987) Methods
Enzymol. 153:516-544;
Scorer, C.A. et al. (1994) Bio/Technology 12:181-184; Engelhard, E.K. et al.
(1994) Proc. Natl.
Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-
1945; Takamatsu,
N. (1987) EMBO J. 6:307-311; Coruzzi, G. et al. ( 1984) EMBO J. 3:1671-1680;
Brogue, R. et al.
(1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105; The
McGraw Hill Yearbook of Science and Technology ( 1992) McGraw Hill, New York
NY, pp.
191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-
3659: and Harrington,
J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors derived from
retroviruses,
adenoviruses, or herpes or vaccinia viruses, or from various bacterial
plasmids, may be used for
delivery of nucleotide sequences to the targeted organ, tissue, or cell
population. (See, e.g., Di
Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al., (1993)
Proc. Natl. Acad. Sci.
USA 90(13):6340-6344; Butler, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al.
( 1994) Mol. Immunol. 31 (3):219-226; and Verma, LM. and N. Somia ( 1997)
Nature 389:239-242.)
The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding SYNT. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding SYNT can be
achieved using a
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORT1
plasmid (Life Technologies). Ligation of sequences encoding SYNT into the
vector's multiple
cloning site disrupts the lacZ gene, allowing a colorimetric screening
procedure for identification of
transformed bacteria containing recombinant molecules. In addition, these
vectors may be useful for
in vitro transcription, dideoxy sequencing, single strand rescue with helper
phage, and creation of
nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M.
Schuster ( 1989) J. Biol.
Chem. 264:5503-5509.) When large quantities of SYNT are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of SYNT may be used.
For example, vectors
containing the strong, inducible T5 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of SYNT. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharom~ces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable
integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel,
1995, supra; Bitter, s_ upra; and Scorer, supra.)
Plant systems may also be used for expression of SYNT. Transcription of
sequences
encoding SYNT may be driven viral promoters, e.g., the 35S and 19S promoters
of CaMV used alone
29
CA 02380317 2002-O1-21
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or in combination with the omega leader sequence from TMV (Takamatsu, N. (
1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used. (See, e.g., Coruzzi, supra; Broglie, supra; and Winter,
supra.) These
constructs can be introduced into plant cells by direct DNA transformation or
pathogen-mediated
transfection. (See, e.g., The McGraw Hill Yearbook of Science and TechnoloQV (
1992) McGraw
Hill, New York NY, pp. 191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding SYNT
may be ligated into
an adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential E 1 or E3 region of the viral genome
may be used to obtain
infective virus which expresses SYNT in host cells. (See, e.g., Logan, J. and
T. Shenk ( 1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalian host
cells. SV40 or EBV-
based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. ( 1997) Nat. Genet.
15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression
of SYNT in cell lines is preferred. For example, sequences encoding SYNT can
be transformed into
cell lines using expression vectors which may contain viral origins of
replication and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media
before being switched to selective media. The purpose of the selectable marker
is to confer resistance
to a selective agent, and its presence allows growth and recovery of cells
which successfully express
the introduced sequences. Resistant clones of stably transformed cells may be
propagated using
tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk and apr cells, respectively.
(See, e.g., Wigler, M. et
al. ( 1977) Cell 11:223-232; Lowy, I. et al. ( 1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic,
or herbicide resistance can be used as the basis for selection. For example,
dhfr confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. ( 1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. ( 1981 )
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hisD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan ( 1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech), f3 glucuronidase and its substrate f3-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
confirmed. For example, if the
sequence encoding SYNT is inserted within a marker gene sequence, transformed
cells containing
sequences encoding SYNT can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding SYNT under the
control of a single
promoter. Expression of the marker gene in response to induction or selection
usually indicates
expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding SYNT
and that express
SYNT may be identified by a variety of procedures known to those of skill in
the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations,
PCR
amplification, and protein bioassay or immunoassay techniques which include
membrane, solution, or
chip based technologies for the detection and/or quantification of nucleic
acid or protein sequences.
Immunological methods for detecting and measuring the expression of SYNT using
either
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques
include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),
and
fluorescence activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on SYNT is
preferred, but a
competitive binding assay may be employed. These and other assays are well
known in the art. (See,
e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN,
Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Immunolo~y> Greene
Pub. Associates and
Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical
Protocols, Humana
Press, Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled
hybridization or PCR probes for detecting sequences related to polynucleotides
encoding SYNT
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide.
Alternatively, the sequences encoding SYNT, or any fragments thereof, may be
cloned into a vector
for the production of an mRNA probe. Such vectors are known in the art, are
commercially available,
and may be used to synthesize RNA probes in vitro by addition of an
appropriate RNA polymerase
31
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such as T7, T3, or SP6 and labeled nucleotides. These procedures may be
conducted using a variety
of commercially available kits, such as those provided by Amersham Pharmacia
Biotech, Promega
(Madison WI), and US Biochemical. Suitable reporter molecules or labels which
may be used for
ease of detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic
agents, as well as substrates, cofactors, inhibitors, magnetic particles, and
the like.
Host cells transformed with nucleotide sequences encoding SYNT may be cultured
under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the sequence
and/or the vector used. As will be understood by those of skill in the art,
expression vectors
containing polynucleotides which encode SYNT may be designed to contain signal
sequences which
direct secretion of SYNT through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of
the polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro" or
"pro" form of the protein may also be used to specify protein targeting,
folding, and/or activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are
available from the
American Type Culture Collection (ATCC, Manassas VA) and may be chosen to
ensure the correct
modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding SYNT may be ligated to a heterologous sequence resulting in
translation of a
fusion protein in any of the aforementioned host systems. For example, a
chimeric SYNT protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of SYNT activity.
Heterologous protein and
peptide moieties may also facilitate purification of fusion proteins using
commercially available
affinity matrices. Such moieties include, but are not limited to, glutathione
S-transferase (GST),
maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide
(CBP), 6-His, FLAG,
c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification of their
cognate fusion proteins on immobilized glutathione, maltose, phenylarsine
oxide, calmodulin, and
metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable
immunoaffinity
purification of fusion proteins using commercially available monoclonal and
polyclonal antibodies
that specifically recognize these epitope tags. A fusion protein may also be
engineered to contain a
proteolytic cleavage site located between the SYNT encoding sequence and the
heterologous protein
sequence, so that SYNT may be cleaved away from the heterologous moiety
following purification.
Methods for fusion protein expression and purification are discussed in
Ausubel ( 1995, supra, ch. 10).
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A variety of commercially available kits may also be used to facilitate
expression and purification of
fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled SYNT may
be achieved in
vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These
systems couple transcription and translation of protein-coding sequences
operably associated with the
T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
precursor, for example, 35S-methionine.
SYNT of the present invention or fragments thereof may be used to screen for
compounds
that specifically bind to SYNT. At least one and up to a plurality of test
compounds may be screened
for specific binding to SYNT. Examples of test compounds include antibodies,
oligonucleotides,
proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the
natural ligand of
SYNT, e.g., a ligand or fragment thereof, a natural substrate, a structural or
functional mimetic, or a
natural binding partner. (See, Coligan, J.E. et al. (1991) Current Protocols
in Immunology 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which SYNT
binds, or to at least a fragment of the receptor, e.g., the ligand binding
site. In either case, the
compound can be rationally designed using known techniques. In one embodiment,
screening for
these compounds involves producing appropriate cells which express SYNT,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or
E. coli. Cells expressing SYNT or cell membrane fractions which contain SYNT
are then contacted
with a test compound and binding, stimulation, or inhibition of activity of
either SYNT or the
compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide,
wherein binding is
detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable
label. For example,
the assay may comprise the steps of combining at least one test compound with
SYNT, either in
solution or affixed to a solid support, and detecting the binding of SYNT to
the compound.
Alternatively, the assay may detect or measure binding of a test compound in
the presence of a
labeled competitor. Additionally, the assay may be carried out using cell-free
preparations, chemical
libraries, or natural product mixtures, and the test compounds) may be free in
solution or affixed to a
solid support.
SYNT of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of SYNT. Such compounds may include agonists,
antagonists, or partial or
inverse agonists. In one embodiment, an assay is performed under conditions
permissive for SYNT
activity, wherein SYNT is combined with at least one test compound, and the
activity of SYNT in the
presence of a test compound is compared with the activity of SYNT in the
absence of the test
compound. A change in the activity of SYNT in the presence of the test
compound is indicative of a
33
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compound that modulates the activity of SYNT. Alternatively, a test compound
is combined with an
in vitro or cell-free system comprising SYNT under conditions suitable for
SYNT activity, and the
assay is performed. In either of these assays, a test compound which modulates
the activity of SYNT
may do so indirectly and need not come in direct contact with the test
compound. At least one and up
to a plurality of test compounds may be screened.
In another embodiment, polynucleotides encoding SYNT or their mammalian
homologs may
be "knocked out" in an animal model system using homologous recombination in
embryonic stem
(ES) cells. Such techniques are well known in the art and are useful for the
generation of animal
models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent
No. 5,767,337.) For
example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from
the early mouse
embryo and grown in culture. The ES cells are transformed with a vector
containing the gene of
interest disrupted by a marker gene, e.g., the neomycin phosphotransferase
gene (neo; Capecchi,
M.R. (1989) Science 244:1288-1292). The vector integrates into the
corresponding region of the host
genome by homologous recombination. Alternatively, homologous recombination
takes place using
the Cre-IoxP system to knockout a gene of interest in a tissue- or
developmental stage-specific
manner (March, J.D. ( 1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (
1997) Nucleic Acids
Res. 25:4323-4330). Transformed ES cells are identified and microinjected into
mouse cell
blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are
surgically transferred
to pseudopregnant dams, and the resulting chimeric progeny are genotyped and
bred to produce
heterozygous or homozygous strains. Transgenic animals thus generated may be
tested with potential
therapeutic or toxic agents.
Polynucleotides encoding SYNT may also be manipulated in vitro in ES cells
derived from
human blastocysts. Human ES cells have the potential to differentiate into at
least eight separate cell
lineages including endoderm, mesoderm, and ectodermal cell types. These cell
lineages differentiate
into, for example, neural cells, hematopoietic lineages, and cardiomyocytes
(Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding SYNT can also be used to create "knockin" humanized
animals
(pigs) or transgenic animals (mice or rats) to model human disease. With
knockin technology, a
region of a polynucleotide encoding SYNT is injected into animal ES cells, and
the injected sequence
integrates into the animal cell genome. Transformed cells are injected into
blastulae, and the
blastulae are implanted as described above. Transgenic progeny or inbred lines
are studied and
treated with potential pharmaceutical agents to obtain information on
treatment of a human disease.
Alternatively, a mammal inbred to overexpress SYNT, e.g., by secreting SYNT in
its milk, may also
serve as a convenient source of that protein (Janne, J. et al. ( 1998)
Biotechnol. Annu. Rev. 4:55-74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists
34
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between regions of SYNT and human synthetases. In addition, the expression of
SYNT is closely
associated with hematopoietic/immune, cancerous, proliferating, inflamed,
immune, nervous,
gastrointestinal and reproductive tissues. Therefore, SYNT appears to play a
role in an immune
disorder such as inflammation, actinic keratosis, acquired immunodeficiency
syndrome (AIDS),
Addison's disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis,
anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic
anemia, autoimmune
thyroiditis, bronchitis, bursitis, cholecystitis, cirrhosis, contact
dermatitis, Crohn's disease, atopic
dermatitis, dermatomyositis, diabetes mellitus, emphysema, erythroblastosis
fetalis, erythema
nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout,
Graves' disease,
Hashimoto's thyroiditis, paroxysmal nocturnal hemoglobinuria, hepatitis,
hypereosinophilia, irritable
bowel syndrome, episodic lymphopenia with lymphocytotoxins, mixed connective
tissue disease
(MCTD), multiple sclerosis, myasthenia gravis, myocardial or pericardial
inflammation,
myelofibrosis, osteoarthritis, osteoporosis, pancreatitis, polycythemia vera,
polymyositis, psoriasis,
Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome,
systemic anaphylaxis,
systemic lupus erythematosus, systemic sclerosis, primary thrombocythemia,
thrombocytopenic
purpura, ulcerative colitis, uveitis, Werner syndrome, complications of
cancer, hemodialysis, and
extracorporeal circulation, trauma, viral, bacterial, fungal, parasitic,
protozoal, and helminthic
infections, and hematopoietic cancer including lymphoma, leukemia, and
myeloma; a neuronal
disorder, such as akathesia, Alzheimer's disease, amnesia, amyotrophic lateral
sclerosis, bipolar
disorder, catatonia, cerebral neoplasms, dementia, depression, diabetic
neuropathy, Down's syndrome,
tardive dyskinesia, dystonias, epilepsy, Huntington's disease, peripheral
neuropathy, multiple
sclerosis, neurofibromatosis, Parkinson's disease, paranoid psychoses,
postherpetic neuralgia,
schizophrenia, and Tourette's disorder; a reproductive disorder, such as a
disorder of prolactin
production, infertility, including tubal disease, ovulatory defects, and
endometriosis, a disruption of
the estrous cycle, a disruption of the menstrual cycle, polycystic ovary
syndrome, ovarian
hyperstimulation syndrome, an endometrial or ovarian tumor, a uterine fibroid,
autoimmune
disorders, an ectopic pregnancy, and teratogenesis; cancer of the breast,
fibrocystic breast disease,
and galactorrhea; a disruption of spermatogenesis, abnormal sperm physiology,
cancer of the testis,
cancer of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's
disease, impotence,
carcinoma of the male breast, and gynecomastia; and a cell proliferative
disorder, such as actinic
keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis,
mixed connective tissue
disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria,
polycythemia vera, psoriasis,
primary thrombocythemia, and cancers including adenocarcinoma, leukemia,
lymphoma, melanoma,
myeloma, sarcoma, teratocarcinoma, and, in particular, a cancer of the adrenal
gland, bladder, bone,
bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal
tract, heart, kidney, liver,
lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands,
skin, spleen, testis,
CA 02380317 2002-O1-21
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thymus, thyroid, and uterus.
In another embodiment, a vector capable of expressing SYNT or a fragment or
derivative
thereof may be administered to a subject to treat or prevent a disorder
associated with decreased
expression or activity of SYNT including, but not limited to, those described
above.
In a further embodiment, a pharmaceutical composition comprising a
substantially purified
SYNT in conjunction with a suitable pharmaceutical carrier may be administered
to a subject to treat
or prevent a disorder associated with decreased expression or activity of SYNT
including, but not
limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of SYNT
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of SYNT including, but not limited to, those listed above.
In a further embodiment, an antagonist of SYNT may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of SYNT.
Examples of such
disorders include, but are not limited to, those immune, neuronal,
reproductive, and cell proliferative
disorders described above. In one aspect, an antibody which specifically binds
SYNT may be used
directly as an antagonist or indirectly as a targeting or delivery mechanism
for bringing a
pharmaceutical agent to cells or tissues which express SYNT.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding SYNT may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of SYNT including, but not limited to, those
described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be administered in combination with
other appropriate
therapeutic agents. Selection of the appropriate agents for use in combination
therapy may be made
by one of ordinary skill in the art, according to conventional pharmaceutical
principles. The
combination of therapeutic agents may act synergistically to effect the
treatment or prevention of the
various disorders described above. Using this approach, one may be able to
achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the potential for
adverse side effects.
An antagonist of SYNT may be produced using methods which are generally known
in the
art. In particular, purified SYNT may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind SYNT.
Antibodies to SYNT may
also be generated using methods that are well known in the art. Such
antibodies may include, but are
not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies,
Fab fragments, and
fragments produced by a Fab expression library. Neutralizing antibodies (i.e.,
those which inhibit
dimer formation) are generally preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, humans,
and others may be immunized by injection with SYNT or with any fragment or
oligopeptide thereof
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WO 01/07628 PCT/US00/19980
which has immunogenic properties. Depending on the host species, various
adjuvants may be used to
increase immunological response. Such adjuvants include, but are not limited
to, Freund's, mineral
gels such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among
adjuvants used in
humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are
especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
SYNT have an amino acid sequence consisting of at least about 5 amino acids,
and generally will
consist of at least about 10 amino acids. It is also preferable that these
oligopeptides, peptides, or
fragments are identical to a portion of the amino acid sequence of the natural
protein. Short stretches
of SYNT amino acids may be fused with those of another protein, such as KLH,
and antibodies to the
chimeric molecule may be produced.
Monoclonal antibodies to SYNT may be prepared using any technique which
provides for the
production of antibody molecules by continuous cell lines in culture. These
include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma technique, and
the EBV-hybridoma
technique. (See, e.g., Kohler, G. et al. ( 1975) Nature 256:495-497; Kozbor,
D. et al. ( 1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. ( 1983) Proc. Natl. Acad. Sci.
USA 80:2026-2030; and
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. ( 1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and Takeda,
S. et al. ( 1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the art, to produce
SYNT-specific single
chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be
generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g.,
Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte
population or by screening immunoglobulin libraries or panels of highly
specific binding reagents as
disclosed in the literature. (See, e.g., Orlandi, R. et al. ( 1989) Proc.
Natl. Acad. Sci. USA
86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for SYNT may also be
generated.
For example, such fragments include, but are not limited to, F(ab')~ fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab')2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.)
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Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
SYNT and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies
reactive to two non-interfering SYNT epitopes is generally used, but a
competitive binding assay may
also be employed (Pound, s_u~ra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay
techniques may be used to assess the affinity of antibodies for SYNT. Affinity
is expressed as an
association constant, Ka, which is defined as the molar concentration of SYNT-
antibody complex
divided by the molar concentrations of free antigen and free antibody under
equilibrium conditions.
The K~ determined for a preparation of polyclonal antibodies, which are
heterogeneous in their
affinities for multiple SYNT epitopes, represents the average affinity, or
avidity, of the antibodies for
SYNT. The K~ determined for a preparation of monoclonal antibodies, which are
monospecific for a
particular SYNT epitope, represents a true measure of affinity. High-affinity
antibody preparations
with K~ ranging from about 109 to 10'2 L/mole are preferred for use in
immunoassays in which the
SYNT-antibody complex must withstand rigorous manipulations. Low-affinity
antibody preparations
with K~ ranging from about 106 to 10' L/mole are preferred for use in
immunopurification and similar
procedures which ultimately require dissociation of SYNT, preferably in active
form, from the
antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL
Press, Washington DC;
Liddell, J.E. and A. Cryer ( 1991) A Practical Guide to Monoclonal Antibodies,
John Wiley & Sons,
New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
determine the quality and suitability of such preparations for certain
downstream applications. For
example, a polyclonal antibody preparation containing at least I-2 mg specific
antibody/ml,
preferably 5-10 mg specific antibody/ml, is generally employed in procedures
requiring precipitation
of SYNT-antibody complexes. Procedures for evaluating antibody specificity,
titer, and avidity, and
guidelines for antibody quality and usage in various applications, are
generally available. (See, e.g.,
Catty, supra, and Coligan et al., supra.)
In another embodiment of the invention, the polynucleotides encoding SYNT, or
any
fragment or complement thereof, may be used for therapeutic purposes. In one
aspect, modifications
of gene expression can be achieved by designing complementary sequences or
antisense molecules
(DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory
regions of the gene
encoding SYNT. Such technology is well known in the art, and antisense
oligonucleotides or larger
fragments can be designed from various locations along the coding or control
regions of sequences
encoding SYNT. (See, e.g., Agrawal, S., ed. ( 1996) Antisense Therapeutics,
Humana Press Inc.,
38
CA 02380317 2002-O1-21
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Totawa NJ.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. ( 1998) J. Allergy Clin. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. ( 1995)
9( 13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. ( 1990) Blood
76:271; Ausubel, supra; Uckert, W. and W. Walther ( 1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et
al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al. (1997)
Nucleic Acids Res.
25( 14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding SYNT may be
used for
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease
characterized by X
linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672),
severe combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. ( 1995) Science 270:475-480; Bordignon, C. et al. ( 1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. ( 1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and Somia, N. (1997) Nature
389:239-242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites
(e.g., against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore, D.
( 1988) Nature 335:395-396; Poeschla, E. et al. ( 1996) Proc. Natl. Acad. Sci.
USA. 93:11395-11399),
hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans
and Paracoccidioides
brasiliensis; and protozoan parasites such as Plasmodium falciparum and
Trypanosoma cruzi). In the
case where a genetic deficiency in SYNT expression or regulation causes
disease, the expression of
SYNT from an appropriate population of transduced cells may alleviate the
clinical manifestations
caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by
deficiencies in
SYNT are treated by constructing mammalian expression vectors encoding SYNT
and introducing
these vectors by mechanical means into SYNT-deficient cells. Mechanical
transfer technologies for
use with cells in vivo or ex vitro include (i) direct DNA microinjection into
individual cells, (ii)
39
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ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv)
receptor-mediated gene
transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (
1993) Annu. Rev.
Biochem. 62:191-217; Ivics, Z. ( 1997) Cell 91:501-510; Boulay, J-L. and H.
Recipon ( 1998) Curr.
Opin. Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of SYNT include,
but are not
limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen,
Carlsbad CA),
PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF,
PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). SYNT may be
expressed
using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV),
Rous sarcoma virus
(RSV), SV40 virus, thymidine kinase (TK), or ~i-actin genes), (ii) an
inducible promoter (e.g., the
tetracycline-regulated promoter (Gossen, M. and H. Bujard ( 1992) Proc. Natl.
Acad. Sci. USA
89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V.
and H.M. Blau (1998)
Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen)); the
ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the
FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible
promoter (Rossi, F.M.V.
and H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native
promoter of the endogenous
gene encoding SYNT from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KTT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. Eb ( 1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
( 1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of
these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to SYNT expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding SYNT under the control of an independent promoter or
the retrovirus long
terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and
(iii) a Rev-responsive
element (RRE) along with additional retrovirus cis-acting RNA sequences and
coding sequences
required for efficient vector propagation. Retrovirus vectors (e.g., PFB and
PFBNEO) are
commercially available (Stratagene) and are based on published data (Riviere,
I. et al. ( 1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The
vector is propagated in
an appropriate vector producing cell line (VPCL) that expresses an envelope
gene with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller ( 1988) J. Virol. 62:3802-3806; Dull, T. et al. ( 1998) J. Virol.
72:8463-8471; Zufferey, R.
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
et al. (1998) J. Virol. 72:9873-9880). U.S. Patent Number 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant")
discloses a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by
reference. Propagation of retrovirus vectors, transduction of a population of
cells (e.g., CD4+ T-
cells), and the return of transduced cells to a patient are procedures well
known to persons skilled in
the art of gene therapy and have been well documented (Ranga, U. et al. (
1997) J. Virol. 71:7020-
7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J.
Virol. 71:4707-4716;
Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997)
Blood 89:2283-
2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding SYNT to cells which have one or more genetic
abnormalities with respect
to the expression of SYNT. The construction and packaging of adenovirus-based
vectors are well
known to those with ordinary skill in the art. Replication defective
adenovirus vectors have proven to
be versatile for importing genes encoding immunoregulatory proteins into
intact islets in the pancreas
(Csete, M.E. et al. ( 1995) Transplantation 27:263-268). Potentially useful
adenoviral vectors are
described in U.S. Patent Number 5,707,618 to Armentano ("Adenovirus vectors
for gene therapy"),
hereby incorporated by reference. For adenoviral vectors, see also Antinozzi,
P.A. et al. ( 1999)
Annu. Rev. Nutr. 19:511-544; and Verma, LM. and N. Somia ( 1997) Nature
18:389:239-242, both
incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding SYNT to target cells which have one or more genetic
abnormalities with
respect to the expression of SYNT. The use of herpes simplex virus (HSV)-based
vectors may be
especially valuable for introducing SYNT to cells of the central nervous
system, for which HSV has a
tropism. The construction and packaging of herpes-based vectors are well known
to those with
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
type 1-based vector has
been used to deliver a reporter gene to the eyes of primates (Liu, X. et al.
(1999) Exp. Eye
Res.169:385-395). The construction of a HSV-1 virus vector has also been
disclosed in detail in U.S.
Patent Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is
hereby incorporated by reference. U.S. Patent Number 5,804,413 teaches the use
of recombinant
HSV d92 which consists of a genome containing at least one exogenous gene to
be transferred to a
cell under the control of the appropriate promoter for purposes including
human gene therapy. Also
taught by this patent are the construction and use of recombinant HSV strains
deleted for ICP4, ICP27
and ICP22. For HSV vectors, see also Goins, W.F. et al. ( 1999) J. Virol.
73:519-532 and Xu, H. et al.
(1994) Dev. Biol. 163:152-161, hereby incorporated by reference. The
manipulation of cloned
herpesvirus sequences, the generation of recombinant virus following the
transfection of multiple
plasmids containing different segments of the large herpesvirus genomes, the
growth and propagation
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of herpesvirus, and the infection of cells with herpesvirus are techniques
well known to those of
ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding SYNT to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based
on the SFV genome (Garoff, H. and K.-J. Li ( 1998) Curr. Opin. Biotech. 9:464-
469). During
alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full-length
genomic RNA,
resulting in the overproduction of capsid proteins relative to the viral
proteins with enzymatic activity
(e.g., protease and polymerase). Similarly, inserting the coding sequence for
SYNT into the
alphavirus genome in place of the capsid-coding region results in the
production of a large number of
SYNT-coding RNAs and the synthesis of high levels of SYNT in vector transduced
cells. While
alphavirus infection is typically associated with cell lysis within a few
days, the ability to establish a
persistent infection in hamster normal kidney cells (BHK-21) with a variant of
Sindbis virus (SIN)
indicates that the lytic replication of alphaviruses can be altered to suit
the needs of the gene therapy
application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host
range of alphaviruses will
allow the introduction of SYNT into a variety of cell types. The specific
transduction of a subset of
cells in a population may require the sorting of cells prior to transduction.
The methods of
manipulating infectious cDNA clones of alphaviruses, performing alphavirus
cDNA and RNA
transfections, and performing alphavirus infections, are well known to those
with ordinary skill in the
art.
Oligonucleotides derived from the transcription initiation site, e.g., between
about positions
-10 and +10 from the start site, may also be employed to inhibit gene
expression. Similarly,
inhibition can be achieved using triple helix base-pairing methodology. Triple
helix pairing is useful
because it causes inhibition of the ability of the double helix to open
sufficiently for the binding of
polymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances using
triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et
al. ( 1994) in Huber, B.E.
and B.I. Carr, Molecular and Immunoloy'c Approaches, Futura Publishing, Mt.
Kisco NY, pp. 163-
177.) A complementary sequence or antisense molecule may also be designed to
block translation of
mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding SYNT.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
42
CA 02380317 2002-O1-21
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scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared
by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA
sequences encoding SYNT. Such DNA sequences may be incorporated into a wide
variety of vectors
with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these
cDNA constructs
that synthesize complementary RNA, constitutively or inducibly, can be
introduced into cell lines,
cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half-
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3'
ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather
than phosphodiesterase
linkages within the backbone of the molecule. This concept is inherent in the
production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as inosine,
queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly
modified forms of adenine,
cytidine, guanine, thymine, and uridine which are not as easily recognized by
endogenous
endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding SYNT. Compounds
which may be effective in altering expression of a specific polynucleotide may
include, but are not
limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming
oligonucleotides,
transcription factors and other polypeptide transcriptional regulators, and
non-macromolecular
chemical entities which are capable of interacting with specific
polynucleotide sequences. Effective
compounds may alter polynucleotide expression by acting as either inhibitors
or promoters of
polynucleotide expression. Thus, in the treatment of disorders associated with
increased SYNT
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding SYNT may be therapeutically useful, and in the treament of disorders
associated with
decreased SYNT expression or activity, a compound which specifically promotes
expression of the
polynucleotide encoding SYNT may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for
effectiveness in
altering expression of a specific polynucleotide. A test compound may be
obtained by any method
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commonly known in the art, including chemical modification of a compound known
to be effective in
altering polynucleotide expression; selection from an existing, commercially-
available or proprietary
library of naturally-occurring or non-natural chemical compounds; rational
design of a compound
based on chemical and/or structural properties of the target polynucleotide;
and selection from a
library of chemical compounds created combinatorially or randomly. A sample
comprising a
polynucleotide encoding SYNT is exposed to at least one test compound thus
obtained. The sample
may comprise, for example, an intact or permeabilized cell, or an in vitro
cell-free or reconstituted
biochemical system. Alterations in the expression of a polynucleotide encoding
SYNT are assayed
by any method commonly known in the art. Typically, the expression of a
specific nucleotide is
detected by hybridization with a probe having a nucleotide sequence
complementary to the sequence
of the polynucleotide encoding SYNT. The amount of hybridization may be
quantified, thus forming
the basis for a comparison of the expression of the polynucleotide both with
and without exposure to
one or more test compounds. Detection of a change in the expression of a
polynucleotide exposed to
a test compound indicates that the test compound is effective in altering the
expression of the
polynucleotide. A screen for a compound effective in altering expression of a
specific polynucleotide
can be carried out, for example, using a Schizosaccharomyces pombe gene
expression system
(Atkins, D. et al. ( 1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al.
(2000) Nucleic Acids Res.
28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000)
Biochem. Biophys. Res.
Commun. 268:8-13). A particular embodiment of the present invention involves
screening a
combinatorial library of oligonucleotides (such as deoxyribonucleotides,
ribonucleotides, peptide
nucleic acids, and modified oligonucleotides) for antisense activity against a
specific polynucleotide
sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5.686,242; Bruice, T.W.
et al. (2000) U.S.
Patent No. 6,022,691 ).
Many methods for introducing vectors into cells or tissues are available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. ( 1997) Nat.
Biotechnol. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
pharmaceutical
composition which generally comprises an active ingredient formulated with a
pharmaceutically
acceptable excipient. Excipients may include, for example, sugars, starches,
celluloses, gums, and
proteins. Various formulations are commonly known and are thoroughly discussed
in the latest
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edition of Remin~ton's Pharmaceutical Sciences (Maack Publishing, Easton PA).
Such
pharmaceutical compositions may consist of SYNT, antibodies to SYNT, and
mimetics, agonists,
antagonists, or inhibitors of SYNT.
The pharmaceutical compositions utilized in this invention may be administered
by any
number of routes including, but not limited to, oral, intravenous,
intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, pulmonary, transdermal,
subcutaneous, intraperitoneal,
intranasal, enteral, topical, sublingual, or rectal means.
Pharmaceutical compositions for pulmonary administration may be prepared in
liquid or dry
powder form. These compositions are generally aerosolized immediately prior to
inhalation by the
patient. In the case of small molecules (e.g. traditional low molecular weight
organic drugs), aerosol
delivery of fast-acting formulations is well-known in the art. In the case of
macromolecules (e.g.
larger peptides and proteins), recent developments in the field of pulmonary
delivery via the alveolar
region of the lung have enabled the practical delivery of drugs such as
insulin to blood circulation
(see, e.g., Patton, J.S. et al., U.S. Patent No. 5,997,848). Pulmonary
delivery has the advantage of
administration without needle injection, and obviates the need for potentially
toxic penetration
enhancers.
Pharmaceutical compositions suitable for use in the invention include
compositions wherein
the active ingredients are contained in an effective amount to achieve the
intended purpose. The
determination of an effective dose is well within the capability of those
skilled in the art.
Specialized forms of pharmaceutical compositions may be prepared for direct
intracellular
delivery of macromolecules comprising SYNT or fragments thereof. For example,
liposome
preparations containing a cell-impermeable macromolecule may promote cell
fusion and intracellular
delivery of the macromolecule. Alternatively, SYNT or a fragment thereof may
be joined to a short
cationic N-terminal portion from the HN Tat-I protein. Fusion proteins thus
generated have been
found to transduce into the cells of all tissues, including the brain, in a
mouse model system
(Schwarze, S.R. et al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs,
monkeys, or pigs. An animal model may also be used to determine the
appropriate concentration
range and route of administration. Such information can then be used to
determine useful doses and
routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example SYNT
or fragments thereof, antibodies of SYNT, and agonists, antagonists or
inhibitors of SYNT, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by
standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDSO (the dose
CA 02380317 2002-O1-21
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lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDSo/EDSO ratio.
Pharmaceutical compositions
which exhibit large therapeutic indices are preferred. The data obtained from
cell culture assays and
animal studies are used to formulate a range of dosage for human use. The
dosage contained in such
compositions is preferably within a range of circulating concentrations that
includes the EDSo with
little or no toxicity. The dosage varies within this range depending upon the
dosage form employed,
the sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and
response to therapy. Long-acting pharmaceutical compositions may be
administered every 3 to 4
days, every week, or biweekly depending on the half-life and clearance rate of
the particular
formulation.
Normal dosage amounts may vary from about 0.1 ,ug to 100,000 ,ug, up to a
total dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind SYNT may be used for
the
diagnosis of disorders characterized by expression of SYNT, or in assays to
monitor patients being
treated with SYNT or agonists, antagonists, or inhibitors of SYNT. Antibodies
useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic assays
for SYNT include methods which utilize the antibody and a label to detect SYNT
in human body
fluids or in extracts of cells or tissues. The antibodies may be used with or
without modification, and
may be labeled by covalent or non-covalent attachment of a reporter molecule.
A wide variety of
reporter molecules, several of which are described above, are known in the art
and may be used.
A variety of protocols for measuring SYNT, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
SYNT expression. Normal
or standard values for SYNT expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibody to SYNT under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of SYNT
expressed in
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subject, control, and disease samples from biopsied tissues are compared with
the standard values.
Deviation between standard and subject values establishes the parameters for
diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding SYNT may
be used for
diagnostic purposes. The polynucleotides which may be used include
oligonucleotide sequences,
complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used
to detect
and quantify gene expression in biopsied tissues in which expression of SYNT
may be correlated
with disease. The diagnostic assay may be used to determine absence, presence,
and excess
expression of SYNT, and to monitor regulation of SYNT levels during
therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding SYNT or closely related
molecules may be used
to identify nucleic acid sequences which encode SYNT. The specificity of the
probe, whether it is
made from a highly specific region, e.g., the 5'regulatory region, or from a
less specific region, e.g., a
conserved motif, and the stringency of the hybridization or amplification will
determine whether the
probe identifies only naturally occurring sequences encoding SYNT, allelic
variants, or related
sequences.
Probes may also be used for the detection of related sequences, and may have
at least 50%
sequence identity to any of the SYNT encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:16-30 or from
genomic sequences including promoters, enhancers, and introns of the SYNT
gene.
Means for producing specific hybridization probes for DNAs encoding SYNT
include the
cloning of polynucleotide sequences encoding SYNT or SYNT derivatives into
vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and may
be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as 3'P or 35S,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding SYNT may be used for the diagnosis of
disorders
associated with expression of SYNT. Examples of such disorders include, but
are not limited to, an
immune disorder such as inflammation, actinic keratosis, acquired
immunodeficiency syndrome
(AIDS), Addison's disease, adult respiratory distress syndrome, allergies,
ankylosing spondylitis,
amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune
hemolytic anemia,
autoimmune thyroiditis, bronchitis, bursitis, cholecystitis, cirrhosis,
contact dermatitis, Crohn's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema,
erythroblastosis fetalis,
erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's
syndrome, gout, Graves'
disease, Hashimoto's thyroiditis, paroxysmal nocturnal hemoglobinuria,
hepatitis, hypereosinophilia,
irritable bowel syndrome, episodic lymphopenia with lymphocytotoxins, mixed
connective tissue
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disease (MCTD), multiple sclerosis, myasthenia gravis, myocardial or
pericardial inflammation,
myelofibrosis, osteoarthritis, osteoporosis, pancreatitis, polycythemia vera,
polymyositis, psoriasis,
Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome,
systemic anaphylaxis,
systemic lupus erythematosus, systemic sclerosis, primary thrombocythemia,
thrombocytopenic
purpura, ulcerative colitis, uveitis, Werner syndrome, complications of
cancer, hemodialysis, and
extracorporeal circulation, trauma, viral, bacterial, fungal, parasitic,
protozoal, and helminthic
infections, and hematopoietic cancer including lymphoma, leukemia, and
myeloma; a neuronal
disorder, such as akathesia, Alzheimeis disease, amnesia, amyotrophic lateral
sclerosis, bipolar
disorder, catatonia, cerebral neoplasms, dementia, depression, diabetic
neuropathy, Down's syndrome,
tardive dyskinesia, dystonias, epilepsy, Huntington's disease, peripheral
neuropathy, multiple
sclerosis, neurofibromatosis, Parkinson's disease, paranoid psychoses,
postherpetic neuralgia,
schizophrenia, and Tourette's disorder; a reproductive disorder, such as a
disorder of prolactin
production, infertility, including tubal disease, ovulatory defects, and
endometriosis, a disruption of
the estrous cycle, a disruption of the menstrual cycle, polycystic ovary
syndrome, ovarian
hyperstimulation syndrome, an endometrial or ovarian tumor, a uterine fibroid,
autoimmune
disorders, an ectopic pregnancy, and teratogenesis; cancer of the breast,
fibrocystic breast disease,
and galactorrhea; a disruption of spermatogenesis, abnormal sperm physiology,
cancer of the testis,
cancer of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's
disease, impotence,
carcinoma of the male breast, and gynecomastia; and a cell proliferative
disorder, such as actinic
keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis,
mixed connective tissue
disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria,
polycythemia vera, psoriasis,
primary thrombocythemia, and cancers including adenocarcinoma, leukemia,
lymphoma, melanoma,
myeloma, sarcoma, teratocarcinoma, and, in particular, a cancer of the adrenal
gland, bladder, bone,
bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal
tract, heart, kidney, liver,
lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands,
skin, spleen, testis,
thymus, thyroid, and uterus. The polynucleotide sequences encoding SYNT may be
used in Southern
or northern analysis, dot blot, or other membrane-based technologies; in PCR
technologies; in
dipstick, pin, and multiformat ELISA-like assays; and in microarrays utilizing
fluids or tissues from
patients to detect altered SYNT expression. Such qualitative or quantitative
methods are well known
in the art.
In a particular aspect, the nucleotide sequences encoding SYNT may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding SYNT may be labeled by standard methods and added to a
fluid or tissue sample
from a patient under conditions suitable for the formation of hybridization
complexes. After a
suitable incubation period, the sample is washed and the signal is quantified
and compared with a
standard value. If the amount of signal in the patient sample is significantly
altered in comparison to
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a control sample then the presence of altered levels of nucleotide sequences
encoding SYNT in the
sample indicates the presence of the associated disorder. Such assays may also
be used to evaluate
the efficacy of a particular therapeutic treatment regimen in animal studies,
in clinical trials, or to
monitor the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with
expression of
SYNT, a normal or standard profile for expression is established. This may be
accomplished by
combining body fluids or cell extracts taken from normal subjects, either
animal or human, with a
sequence, or a fragment thereof, encoding SYNT, under conditions suitable for
hybridization or
amplification. Standard hybridization may be quantified by comparing the
values obtained from
normal subjects with values from an experiment in which a known amount of a
substantially purified
polynucleotide is used. Standard values obtained in this manner may be
compared with values
obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard
values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the
development of the disease, or may provide a means for detecting the disease
prior to the appearance
of actual clinical symptoms. A more definitive diagnosis of this type may
allow health professionals
to employ preventative measures or aggressive treatment earlier thereby
preventing the development
or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding SYNT
may involve the use of PCR. These oligomers may be chemically synthesized,
generated
enzymatically, or produced in vitro. Oligomers will preferably contain a
fragment of a polynucleotide
encoding SYNT, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
SYNT, and will be employed under optimized conditions for identification of a
specific gene or
condition. Oligomers may also be employed under less stringent conditions for
detection or
quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding SYNT may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are
substitutions, insertions and deletions that are a frequent cause of inherited
or acquired genetic
disease in humans. Methods of SNP detection include, but are not limited to,
single-stranded
conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In
SSCP,
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oligonucleotide primers derived from the polynucleotide sequences encoding
SYNT are used to
amplify DNA using the polymerase chain reaction (PCR). The DNA may be derived,
for example,
from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
SNPs in the DNA cause
differences in the secondary and tertiary structures of PCR products in single-
stranded form, and
these differences are detectable using gel electrophoresis in non-denaturing
gels. In fSCCP, the
oligonucleotide primers are fluorescently labeled, which allows detection of
the amplimers in high-
throughput equipment such as DNA sequencing machines. Additionally, sequence
database analysis
methods, termed in silico SNP (isSNP), are capable of identifying
polymorphisms by comparing the
sequence of individual overlapping DNA fragments which assemble into a common
consensus
sequence. These computer-based methods filter out sequence variations due to
laboratory preparation
of DNA and sequencing errors using statistical models and automated analyses
of DNA sequence
chromatograms. In the alternative, SNPs may be detected and characterized by
mass spectrometry
using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San
Diego CA).
Methods which may also be used to quantify the expression of SYNT include
radiolabeling
or biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. ( 1993) J. Immunol. Methods
159:235-244; Duplaa, C.
et al. ( 1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives
rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described in Seilhamer, J.J. et al.,
"Comparative Gene Transcript
Analysis," U.S. Patent No. 5,840,484, incorporated herein by reference. The
microarray may also be
used to identify genetic variants, mutations, and polymorphisms. This
information may be used to
determine gene function, to understand the genetic basis of a disorder, to
diagnose a disorder, to
monitor progression/regression of disease as a function of gene expression,
and to develop and
monitor the activities of therapeutic agents in the treatment of disease. In
particular, this information
may be used to develop a pharmacogenomic profile of a patient in order to
select the most appropriate
and effective treatment regimen for that patient. For example, therapeutic
agents which are highly
effective and display the fewest side effects may be selected for a patient
based on his/her
pharmacogenomic profile.
In another embodiment, antibodies specific for SYNT, or SYNT or fragments
thereof may be
used as elements on a microarray. The microarray may be used to monitor or
measure protein-protein
interactions, drug-target interactions, and gene expression profiles, as
described above.
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A particular embodiment relates to the use of the polynucleotides of the
present invention to
generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern of
gene expression by a particular tissue or cell type. Global gene expression
patterns are analyzed by
quantifying the number of expressed genes and their relative abundance under
given conditions and at
a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent Number
5,840,484, expressly incorporated by reference herein.) Thus a transcript
image may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines,
biopsies, or other biological samples. The transcript image may thus reflect
gene expression in vivo,
as in the case of a tissue or biopsy sample, or in vitro, as in the case of a
cell line.
Transcript images which profile the expression of the polynucleotides of the
present
invention may also be used in conjunction with in vitro model systems and
preclinical evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and
toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S.
and N.L. Anderson
(2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference
herein). If a test
compound has a signature similar to that of a compound with known toxicity, it
is likely to share
those toxic properties. These fingerprints or signatures are most useful and
refined when they contain
expression information from a large number of genes and gene families.
Ideally, a genome-wide
measurement of expression provides the highest quality signature. Even genes
whose expression is
not altered by any tested compounds are important as well, as the levels of
expression of these genes
are used to normalize the rest of the expression data. The normalization
procedure is useful for
comparison of expression data after treatment with different compounds. While
the assignment of
gene function to elements of a toxicant signature aids in interpretation of
toxicity mechanisms,
knowledge of gene function is not necessary for the statistical matching of
signatures which leads to
prediction of toxicity. (See, for example, Press Release 00-02 from the
National Institute of
Environmental Health Sciences, released February 29, 2000, available at
http:/lwww.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is important and
desirable in
toxicological screening using toxicant signatures to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological
sample containing nucleic acids with the test compound. Nucleic acids that are
expressed in the
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treated biological sample are hybridized with one or more probes specific to
the polynucleotides of
the present invention. so that transcript levels corresponding to the
polynucleotides of the present
invention may be quantified. The transcript levels in the treated biological
sample are compared with
levels in an untreated biological sample. Differences in the transcript levels
between the two samples
are indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a
proteome can be subjected individually to further analysis. Proteome
expression patterns, or profiles,
are analyzed by quantifying the number of expressed proteins and their
relative abundance under
given conditions and at a given time. A profile of a cell's proteome may thus
be generated by
separating and analyzing the polypeptides of a particular tissue or cell type.
In one embodiment, the
separation is achieved using two-dimensional gel electrophoresis, in which
proteins from a sample are
separated by isoelectric focusing in the first dimension, and then according
to molecular weight by
sodium dodecyl sulfate slab gel electrophoresis in the second dimension
(Steiner and Anderson,
su~a). The proteins are visualized in the gel as discrete and uniquely
positioned spots, typically by
staining the gel with an agent such as Coomassie Blue or silver or fluorescent
stains. The optical
density of each protein spot is generally proportional to the level of the
protein in the sample. The
optical densities of equivalently positioned protein spots from different
samples, for example, from
biological samples either treated or untreated with a test compound or
therapeutic agent, are
compared to identify any changes in protein spot density related to the
treatment. The proteins in the
spots are partially sequenced using, for example, standard methods employing
chemical or enzymatic
cleavage followed by mass spectrometry. The identity of the protein in a spot
may be determined by
comparing its partial sequence, preferably of at least 5 contiguous amino acid
residues, to the
polypeptide sequences of the present invention. In some cases, further
sequence data may be
obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for SYNT
to quantify the
levels of SYNT expression. In one embodiment, the antibodies are used as
elements on a microarray,
and protein expression levels are quantified by exposing the microarray to the
sample and detecting
the levels of protein bound to each array element (Lueking, A. et al. (1999)
Anal. Biochem. 270:103-
111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be
performed by a
variety of methods known in the art, for example, by reacting the proteins in
the sample with a thiol-
or amino-reactive fluorescent compound and detecting the amount of
fluorescence bound at each
array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
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correlation between transcript and protein abundances for some proteins in
some tissues (Anderson,
N.L. and J. Seilhamer ( 1997) Electrophoresis 18:533-537), so proteome
toxicant signatures may be
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to
rapid degradation of mRNA, so proteomic profiling may be more reliable and
informative in such
cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
each protein is compared to the amount of the corresponding protein in an
untreated biological
sample. A difference in the amount of protein between the two samples is
indicative of a toxic
response to the test compound in the treated sample. Individual proteins are
identified by sequencing
the amino acid residues of the individual proteins and comparing these partial
sequences to the
polypeptides of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are
incubated with antibodies specific to the polypeptides of the present
invention. The amount of
protein recognized by the antibodies is quantified. The amount of protein in
the treated biological
sample is compared with the amount in an untreated biological sample. A
difference in the amount of
protein between the two samples is indicative of a toxic response to the test
compound in the treated
sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-
2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types
of microarrays are
well known and thoroughly described in DNA Microarrays: A Practical Approach,
M. Schena, ed.
( 1999) Oxford University Press, London, hereby expressly incorporated by
reference.
In another embodiment of the invention, nucleic acid sequences encoding SYNT
may be used
to generate hybridization probes useful in mapping the naturally occurring
genomic sequence. Either
coding or noncoding sequences may be used, and in some instances, noncoding
sequences may be
preferable over coding sequences. Fdr example, conservation of a coding
sequence among members
of a multi-gene family may potentially cause undesired cross hybridization
during chromosomal
mapping. The sequences may be mapped to a particular chromosome, to a specific
region of a
chromosome, or to artificial chromosome constructions, e.g., human artificial
chromosomes (HACs),
yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs),
bacterial P1
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constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. ( 1997) Nat.
Genet. 15:345-355; Price, C.M. ( 1993) Blood Rev. 7:127-134; and Trask, B.J. (
1991 ) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop
genetic linkage maps, for example, which correlate the inheritance of a
disease state with the
inheritance of a particular chromosome region or restriction fragment length
polymorphism (RFLP).
(See, e.g., Lander, E.S. and D. Botstein ( 1986) Proc. Natl. Acad. Sci. USA
83:7353-7357.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic
map data. (See, e.g., Heinz-Ulrich, et al. ( 1995) in Meyers, supra, pp. 965-
968.) Examples of genetic
map data can be found in various scientific journals or at the Online
Mendelian Inheritance in Man
(OMIM) World Wide Web site. Correlation between the location of the gene
encoding SYNT on a
physical map and a specific disorder, or a predisposition to a specific
disorder, may help define the
region of DNA associated with that disorder and thus may further positional
cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
may reveal associated markers even if the exact chromosomal locus is not
known. This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely
localized by genetic linkage to a particular genomic region, e.g., ataxia-
telangiectasia to 1 1q22-23,
any sequences mapping to that area may represent associated or regulatory
genes for further
investigation. (See, e.g., Gatti, R.A. et al. ( 1988) Nature 336:577-580.) The
nucleotide sequence of
the instant invention may also be used to detect differences in the
chromosomal location due to
translocation, inversion, etc., among normal, carrier, or affected
individuals.
In another embodiment of the invention, SYNT, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used for screening libraries of compounds in any
of a variety of drug
screening techniques. The fragment employed in such screening may be free in
solution, affixed to a
solid support, borne on a cell surface, or located intracellularly. The
formation of binding complexes
between SYNT and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. ( 1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with SYNT, or
fragments thereof,
and washed. Bound SYNT is then detected by methods well known in the art.
Purified SYNT can
also be coated directly onto plates for use in the aforementioned drug
screening techniques.
Alternatively, non-neutralizing antibodies can be used to capture the peptide
and immobilize it on a
solid support.
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In another embodiment, one may use competitive drug screening assays in which
neutralizing
antibodies capable of binding SYNT specifically compete with a test compound
for binding SYNT.
In this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with SYNT.
In additional embodiments, the nucleotide sequences which encode SYNT may be
used in
any molecular biology techniques that have yet to be developed, provided the
new techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following preferred specific
embodiments are, therefore, to be construed as merely illustrative, and not
limitative of the remainder
of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below, in
particular U.S. Ser. No. 60/144,992 and U.S. Ser. No. 60/168,858 are hereby
expressly incorporated
by reference.
EXAMPLES
I. Construction of cDNA Libraries
RNA was purchased from Clontech or isolated from tissues described in Table 4.
Scpe
tissues were homogenized and lysed in guanidinium isothiocyanate, while others
were homogenized
and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL
(Life Technologies), a
monophasic solution of phenol and guanidine isothiocyanate. The resulting
lysates were centrifuged
over CsCI cushions or extracted with chloroform. RNA was precipitated from the
lysates with either
isopropanol or sodium acetate and ethanol, or by other routine methods.
?5 Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A+) RNA was isolated
using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
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appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies),
pcDNA2.1 plasmid
(Invitrogen, Carlsbad CA), or pINCY plasmid (Incyte Genomics, Palo Alto CA).
Recombinant
plasmids were transformed into competent E. coli cells including XL1-Blue, XL1-
BIueMRF, or
SOLR from Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life
Technologies.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo
excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega); an
AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid,
QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
R.E.A.L. PREP 96
plasmid purification kit from QIAGEN. Following precipitation, plasmids were
resuspended in 0.1
ml of distilled water and stored, with or without lyophilization, at
4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically
using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence
scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation
such as the ABI CATALYST 800 (PE Biosystems) thermal cycler or the PTC-200
thermal cycler (MJ
Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or
the MICROLAB
2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were
prepared using reagents
provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such
as the ABI
PRISM BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems).
Electrophoretic
separation of cDNA sequencing reactions and detection of labeled
polynucleotides were carried out
using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI
PRISM 373
or 377 sequencing system (PE Biosystems) in conjunction with standard ABI
protocols and base
calling software; or other sequence analysis systems known in the art. Reading
frames within the
cDNA sequences were identified using standard methods (reviewed in Ausubel,
1997, supra, unit
7.7). Some of the cDNA sequences were selected for extension using the
techniques disclosed in
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Example VI.
The polynucleotide sequences derived from cDNA sequencing were assembled and
analyzed
using a combination of software programs which utilize algorithms well known
to those skilled in the
art. Table 5 summarizes the tools, programs, and algorithms used and provides
applicable
descriptions, references, and threshold parameters. The first column of Table
5 shows the tools,
programs, and algorithms used, the second column provides brief descriptions
thereof, the third
column presents appropriate references, all of which are incorporated by
reference herein in their
entirety, and the fourth column presents, where applicable, the scores,
probability values, and other
parameters used to evaluate the strength of a match between two sequences (the
higher the score, the
greater the homology between two sequences). Sequences were analyzed using
MACDNASIS PRO
software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE
software
(DNASTAR). Polynucleotide and polypeptide sequence alignments were generated
using the default
parameters specified by the clustal algorithm as incorporated into the
MEGALIGN multisequence
alignment program (DNASTAR), which also calculates the percent identity
between aligned
sequences.
The polynucleotide sequences were validated by removing vector, linker, and
polyA
sequences and by masking ambiguous bases, using algorithms and programs based
on BLAST,
dynamic programing, and dinucleotide nearest neighbor analysis. The sequences
were then queried
against a selection of public databases such as the GenBank primate, rodent,
mammalian, vertebrate,
and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM to acquire
annotation using programs based on BLAST, FASTA, and BLIMPS. The sequences
were assembled
into full length polynucleotide sequences using programs based on Phred,
Phrap, and Consed, and
were screened for open reading frames using programs based on GeneMark, BLAST,
and FASTA.
The full length polynucleotide sequences were translated to derive the
corresponding full length
amino acid sequences, and these full length sequences were subsequently
analyzed by querying
against databases such as the GenBank databases (described above), SwissProt,
BLOCKS, PRINTS,
DOMO, PRODOM, Prosite, and Hidden Markov Model (HMM)-based protein family
databases such
as PFAM. HMM is a probabilistic approach which analyzes consensus primary
structures of gene
families. (See, e.g., Eddy, S.R. (1996) Cutr. Opin. Struct. Biol. 6:361-365.)
The programs described above for the assembly and analysis of full length
polynucleotide
and amino acid sequences were also used to identify polynucleotide sequence
fragments from SEQ ID
N0:16-30. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
amplification technologies were described in The Invention section above.
IV. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
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from a particular cell type or tissue have been bound. (See, e.g., Sambrook,
sera, ch. 7; Ausubel,
1995, su ra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the
computer search can be modified to determine whether any particular match is
categorized as exact or
similar. The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity
5 x minimum { length(Seq. 1 ), length(Seq. 2) }
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. The product score is a normalized value between
0 and 100, and is
calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
product is divided by (5 times the length of the shorter of the two
sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by
gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate
the product score. The product score represents a balance between fractional
overlap and quality in a
BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the
entire length of the shorter of the two sequences being compared. A product
score of 70 is produced
either by 100% identity and 70% overlap at one end, or by 88% identity and
100% overlap at the
other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
The results of northern analyses are reported as a percentage distribution of
libraries in which
the transcript encoding SYNT occurred. Analysis involved the categorization of
cDNA libraries by
organ/tissue and disease. The organ/tissue categories included cardiovascular,
dermatologic,
developmental, endocrine, gastrointestinal, hematopoietic/immune,
musculoskeletal, nervous,
reproductive, and urologic. The disease/condition categories included cancer,
inflammation, trauma,
cell proliferation, neurological, and pooled. For each category, the number of
libraries expressing the
sequence of interest was counted and divided by the total number of libraries
across all categories.
Percentage values of tissue-specific and disease- or condition-specific
expression are reported in
Table 3.
V. Extension of SYNT Encoding Polynucleotides
The full length nucleic acid sequences of SEQ ID N0:16-30 were produced by
extension of
an appropriate fragment of the full length molecule using oligonucleotide
primers designed from this
fragment. One primer was synthesized to initiate 5' extension of the known
fragment, and the other
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primer, to initiate 3' extension of the known fragment. The initial primers
were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68°C to about 72°C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg'-+, (NHQ),S04,
and ~-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerase (Stratagene), with the following
parameters for primer
pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C,
2 nun; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5
min; Step 7: storage at 4°C. In the
alternative, the parameters for primer pair T7 and SK+ were as follows: Step
1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 p1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 Itl of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ~I to 10 ~l aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose mini-gel to determine which reactions were
successful in extending
the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to relegation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones
were relegated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18
vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in
restriction site
overhangs, and transfected into competent E. coli cells. Transformed cells
were selected on
antibiotic-containing media, and individual colonies were picked and cultured
overnight at 37 °C in
384-well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
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(Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the
following
parameters: Step I: 94°C, 3 min; Step 2: 94°C. 15 sec: Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step
7: storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted
with 20% dimethysulfoxide ( 1:2, v/v), and sequenced using DYENAMIC energy
transfer sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems).
In like manner, the polynucleotide sequences of SEQ ID N0:16-30 are used to
obtain 5'
regulatory sequences using the procedure above, along with oligonucleotides
designed for such
extension, and an appropriate genomic library.
V. Chromosomal Mapping of SNYT Encoding Polynucleotides
The cDNA sequences which were used to assemble SEQ ID N0:16-30 were compared
with
sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ ID N0:16-30 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 5). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for
Genome Research (WIGR), and Genethon were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ ID NO:, to that
map location.
The genetic map locations of SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:18, SEQ ID
N0:21, SEQ ID N0:24, SEQ ID N0:26, SEQ ID N0:27, SEQ ID N0:28, SEQ ID N0:29,
and SEQ
ID N0:30 are described in The Invention as ranges, or intervals, of human
chromosomes. More than
one map location is reported for SEQ ID N0:28 and SEQ ID N0:29, indicating
that previously
mapped sequences having similarity, but not complete identity, to SEQ ID N0:28
and SEQ ID N0:29
were assembled into their respective clusters. The map position of an
interval, in centiMorgans, is
measured relative to the terminus of the chromosome's p-arm. (The centiMorgan
(cM) is a unit of
measurement based on recombination frequencies between chromosomal markers. On
average, 1 cM
is roughly equivalent to I megabase (Mb) of DNA in humans, although this can
vary widely due to
hot and cold spots of recombination.) The cM distances are based on genetic
markers mapped by
Genethon which provide boundaries for radiation hybrid markers whose sequences
were included in
each of the clusters. Diseases associated with the public and Incyte sequences
located within the
indicated intervals are also reported in the Invention where applicable. Human
genome maps and
other resources available to the public, such as the NCBI "GeneMap'99" World
Wide Web site which
can be accessed at http://www.ncbi.nlm.nih.gov/genemap, can be employed to
determine if
CA 02380317 2002-O1-21
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previously identified disease genes map within or in proximity to the
intervals indicated above.
VI. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:16-30 are employed to screen
cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide
fragments. Oligonucleotides are designed using state-of-the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 ~Ci of
[y-''-P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-based
hybridization analysis of human genomic DNA digested with one of the following
endonucleases:
Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
VII. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink-jet printing, See, e.g.,
Baldeschweiler, su ra),
mechanical microspotting technologies, and derivatives thereof. The substrate
in each of the
aforementioned technologies should be uniform and solid with a non-porous
surface (Schena ( I 999),
supra). Suggested substrates include silicon, silica, glass slides, glass
chips, and silicon wafers.
Alternatively, a procedure analogous to a dot or slot blot may also be used to
arrange and link
elements to the surface of a substrate using thermal, I1V, chemical, or
mechanical bonding
procedures. A typical array may be produced using available methods and
machines well known to
those of ordinary skill in the art and may contain any appropriate number of
elements. (See, e.g.,
Schena, M. et al. ( 1995) Science 270:467-470; Shalom D. et al. ( 1996) Genome
Res. 6:639-645;
Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The
array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
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After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element
on the microarray may be assessed. In one embodiment, microarray preparation
and usage is
described in detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/pl oligo-(dT)
primer (2lmer), 1X
first strand buffer, 0.03 units/pl RNase inhibitor, 500 EtM dATP, 5001.~M
dGTP, 500 EtM dTTP, 40
l~M dCTP, 40 liM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)+ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)' RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37 °C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium
hydroxide and
incubated for 20 minutes at 85 °C to the stop the reaction and degrade
the RNA. Samples are purified
using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
using 1 ml of glycogen ( 1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is
then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
NY) and
resuspended in 14 p1 5X SSC/0.2% SDS.
Microarrav Preparation
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification
uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5
~tg. Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia
Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0. I % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water,
and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides
are cured in a
110°C oven.
Array elements are applied to the coated glass substrate using a procedure
described in US
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Patent No. 5,807,522, incorporated herein by reference. 1 p1 of the array
element DNA, at an average
concentration of 100 ng/ql, is loaded into the open capillary printing element
by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60
°C followed by washes in
0.2% SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 ~tl of sample mixture consisting of 0.2 pg
each of Cy3 and
Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65 °C for 5 minutes and is aliquoted onto the
microarray surface and covered
with an 1.8 cm- coverslip. The arrays are transferred to a waterproof chamber
having a cavity just
slightly larger than a microscope slide. The chamber is kept at 100% humidity
internally by the
addition of 140 p1 of SX SSC in a comer of the chamber. The chamber containing
the arrays is
incubated for about 6.5 hours at 60°C. The arrays are washed for 10 min
at 45 °C in a first wash
buffer ( 1X SSC, 0.1 % SDS), three times for 10 minutes each at 45 °C
in a second wash buffer (O.IX
SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that
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WO 01/07628 PCT/US00/19980
location to be correlated with a weight ratio of hybridizing species of
1:100,000. When two samples
from different sources (e.g., representing test and control cells), each
labeled with a different
fluorophore, are hybridized to a single array for the purpose of identifying
genes that are
differentially expressed, the calibration is done by labeling samples of the
calibrating cDNA with the
two fluorophores and adding identical amounts of each to the hybridization
mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping
emission spectra) between the fluorophores using each fluorophore's emission
spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each
spot is centered in each element of the grid. The fluorescence signal within
each element is then
integrated to obtain a numerical value corresponding to the average intensity
of the signal. The
software used for signal analysis is the GEMTOOLS gene expression analysis
program (Incyte).
VIII. Complementary Polynucleotides
Sequences complementary to the SYNT-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring SYNT. Although
use of
oligonucleotides comprising from about 15 to 30 base pairs is described,
essentially the same
procedure is used with smaller or with larger sequence fragments. Appropriate
oligonucleotides are
designed using OLIGO 4.06 software (National Biosciences) and the coding
sequence of SYNT. To
inhibit transcription, a complementary oligonucleotide is designed from the
most unique 5' sequence
and used to prevent promoter binding to the coding sequence. To inhibit
translation, a
complementary oligonucleotide is designed to prevent ribosomal binding to the
SYNT-encoding
transcript.
IX. Expression of SYNT
Expression and purification of SYNT is achieved using bacterial or virus-based
expression
systems. For expression of SYNT in bacteria, cDNA is subcloned into an
appropriate vector
containing an antibiotic resistance gene and an inducible promoter that
directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the
trp-lac (tac) hybrid
promoter and the TS or T7 bacteriophage promoter in conjunction with the !ac
operator regulatory
element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21(DE3).
Antibiotic resistant bacteria express SYNT upon induction with isopropyl beta-
D-
thiogalactopyranoside (IPTG). Expression of SYNT in eukaryotic cells is
achieved by infecting
insect or mammalian cell lines with recombinant Autographica californica
nuclear polyhedrosis virus
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(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding SYNT by either homologous recombination or
bacterial-mediated
transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect Spodoptera fru liperda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus. (See Engelhard, E.K.
et al. ( 1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (
1996) Hum. Gene Ther.
7:1937-1945.)
In most expression systems, SYNT is synthesized as a fusion protein with,
e.g., glutathione S-
transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting
rapid, single-step,
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-
kilodalton enzyme from Schistosoma japonicum, enables the purification of
fusion proteins on
immobilized glutathione under conditions that maintain protein activity and
antigenicity (Amersham
Pharmacia Biotech). Following purification, the GST moiety can be
proteolytically cleaved from
SYNT at specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity
purification using commercially available monoclonal and polyclonal anti-FLAG
antibodies (Eastman
Kodak). 6-His, a stretch of six consecutive histidine residues, enables
purification on metal-chelate
resins (QIAGEN). Methods for protein expression and purification are discussed
in Ausubel ( 1995,
supra, ch. 10 and 16). Purified SYNT obtained by these methods can be used
directly in the assays
shown in Examples X and XIV.
X. Demonstration of SYNT Activity
An SYNT activity assay measures aminoacylation of tRNA in the presence of a
radiolabeled
substrate. A cell-free extract depleted of endogenous aminoacyl-tRNA
synthetase is prepared from
Escherichia coli. SYNT, either biochemically purified or recombinantly
produced, is added to the
cell free extract. The cell-free extract is incubated with ['°C]-
labeled amino acid under conditions
favorable for translation. Incorporation of the ['4C]-labeled amino acid into
acid-precipitable
aminoacyl-tRNA is measured using a radioisotope counter. The amount of the
["C]-labeled amino
acid incorporated into aminoacyl tRNA is proportional to the amount of SYNT
activity. (See, for
example, Ibba, M. et al. ( 1997) Science 278:1 I 19-I 122).
Alternatively, SYNT activity may be assayed as follows. SYNT, or biologically
active
fragments thereof, are labeled with ''-5I Bolton-Hunter reagent. (See, e.g.,
Bolton et al. ( 1973)
Biochem. J. 133:529.) Candidate molecules previously arrayed in the wells of a
multi-well plate are
incubated with the labeled SYNT, washed, and any wells with labeled SYNT
complex are assayed.
Data obtained using different concentrations of SYNT are used to calculate
values for the number,
affinity, and association of SYNT with the candidate molecules.
XI. Functional Assays
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SYNT function is assessed by expressing the sequences encoding SYNT at
physiologically
elevated levels in mammalian cell culture systems. cDNA is subcloned into a
mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice
include pCMV SPORT plasmid (Life Technologies) and pCR3.1 plasmid
(Invitrogen), both of which
contain the cytomegalovirus promoter. 5-10 ~g of recombinant vector are
transiently transfected into
a human cell line, for example, an endothelial or hematopoietic cell line,
using either liposome
formulations or electroporation. 1-2 ,ug of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
Clontech), CD64, or a CD64.-GFP fusion protein. Flow cytometry (FCM), an
automated, laser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate
the apoptotic state of the cells and other cellular properties. FCM detects
and quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events
include changes in nuclear DNA content as measured by staining of DNA with
propidium iodide;
changes in cell size and granularity as measured by forward light scatter and
90 degree side light
scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as
measured by reactivity with
specific antibodies; and alterations in plasma membrane composition as
measured by the binding of
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M.G. ( 1994) Flow C ty ometry, Oxford, New York NY.
The influence of SYNT on gene expression can be assessed using highly purified
populations
of cells transfected with sequences encoding SYNT and either CD64 or CD64-GFP.
CD64 and
CD64-GFP are expressed on the surface of transfected cells and bind to
conserved regions of human
immunoglobulin G (IgG). Transfected cells are efficiently separated from
nontransfected cells using
magnetic beads coated with either human IgG or antibody against CD64 (DYNAL,
Lake Success
NY). mRNA can be purified from the cells using methods well known by those of
skill in the art.
Expression of mRNA encoding SYNT and other genes of interest can be analyzed
by northern
analysis or microarray techniques.
XII. Production of SYNT Specific Antibodies
SYNT substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
Harrington, M.G. ( 1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used to
immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the SYNT amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
66
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI431A
peptide synthesizer (PE Biosystems) using FMOC chemistry and coupled to KLH
(Sigma-Aldrich, St.
Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
to increase
immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with
the oligopeptide-
KLH complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide and anti-
SYNT activity by, for example, binding the peptide or SYNT to a substrate,
blocking with 1 ~Io BSA,
reacting with rabbit antisera, washing, and reacting with radio-iodinated goat
anti-rabbit IgG.
XIII. Purification of Naturally Occurring SYNT Using Specific Antibodies
Naturally occurring or recombinant SYNT is substantially purified by
immunoaffinity
chromatography using antibodies specific for SYNT. An immunoaffinity column is
constructed by
covalently coupling anti-SYNT antibody to an activated chromatographic resin,
such as
CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the
resin is
blocked and washed according to the manufacturer's instructions.
Media containing SYNT are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of SYNT (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/SYNT binding (e.g., a buffer of pH 2 to pH 3, or a high concentration
of a chaotrope, such
as urea or thiocyanate ion), and SYNT is collected.
XIV. Identification of Molecules Which Interact with SYNT
SYNT, or biologically active fragments thereof, are labeled with ''-5I Bolton-
Hunter reagent.
(See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.)
Candidate molecules
previously arrayed in the wells of a multi-well plate are incubated with the
labeled SYNT, washed,
and any wells with labeled SYNT complex are assayed. Data obtained using
different concentrations
of SYNT are used to calculate values for the number, affinity, and association
of SYNT with the
candidate molecules.
Alternatively, molecules interacting with SYNT are analyzed using the yeast
two-hybrid
system as described in Fields, S. and O. Song (1989, Nature 340:245-246), or
using commercially
available kits based on the two-hybrid system, such as the MATCHMAKER system
(Clontech).
SYNT may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S.
Patent No. 6,057,1 O1 ).
Various modifications and variations of the described methods and systems of
the invention
67
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
will be apparent to those skilled in the art without departing from the scope
and spirit of the
invention. Although the invention has been described in connection with
certain embodiments, it
should be understood that the invention as claimed should not be unduly
limited to such specific
embodiments. Indeed, various modifications of the described modes for carrying
out the invention
which are obvious to those skilled in molecular biology or related fields are
intended to be within the
scope of the following claims.
68
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
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CA 02380317 2002-O1-21
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CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
SEQUENCE LISTING
<110> INCYTE GENOMICS, INC.
TANG, Y. Tom
HILLMAN, Jennifer L.
BANDMAN, Olga
YUE, Henry
BAUGHN, Mariah R.
LAL, Preeti
LU, Dyung Aina M.
SHAH, Purvi
AZIMZAI, Yalda
<120> HUMAN SYNTHETASES
<130> PF-0721 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/144,992; 60,168,858
<151> 1999-07-22; 1999-12-02
<160> 30
<170> PERL Program
<210> 1
<211> 1176
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1806212CD1
<400> 1
Met Ala Glu Arg Lys Gly Thr Ala Lys Val Asp Phe Leu Lys Lys
1 5 10 15
Ile Glu Lys Glu Ile Gln Gln Lys Trp Asp Thr Glu Arg Val Phe
20 25 30
Glu Val Asn Ala Ser Asn Leu Glu Lys Gln Thr Ser Lys Gly Lys
35 40 45
Tyr Phe Val Thr Phe Pro Tyr Pro Tyr Met Asn Gly Arg Leu His
50 55 60
Leu Gly His Thr Phe Ser Leu Ser Lys Cys Glu Phe Ala Val Gly
65 70 75
Tyr Gln Arg Leu Lys Gly Lys Cys Cys Leu Phe Pro Phe Gly Leu
80 85 90
His Cys Thr Gly Met Pro Ile Lys Ala Cys Ala Asp Lys Leu Lys
95 100 105
Arg Glu Ile Glu Leu Tyr Gly Cys Pro Pro Asp Phe Pro Asp Glu
110 115 120
Glu Glu Glu Glu Glu Glu Thr Ser Val Lys Thr Glu Asp Ile Ile
125 130 135
Ile Lys Asp Lys Ala Lys Gly Lys Lys Ser Lys Ala Ala Ala Lys
140 145 150
Ala Gly Ser Ser Lys Tyr Gln Trp Gly Ile Met Lys Ser Leu Gly
155 160 165
Leu Ser Asp Glu Glu Ile Val Lys Phe Ser Glu Ala Glu His Trp
170 175 180
Leu Asp Tyr Phe Pro Pro Leu Ala Ile Gln Asp Leu Lys Arg Met
185 190 195
1/32
CA 02380317 2002-O1-21
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Gly Leu Lys Val Asp Trp Arg Arg Ser Phe Ile Thr Thr Asp Val
200 205 210
Asn Pro Tyr Tyr Asp Ser Phe Val Arg Trp Gln Phe Leu Thr Leu
215 220 225
Arg Glu Arg Asn Lys Ile Lys Phe Gly Lys Arg Tyr Thr Ile Tyr
230 235 240
Ser Pro Lys Asp Gly Gln Pro Cys Met Asp His Asp Arg Gln Thr
245 250 255
Gly Glu Gly Val Gly Pro Gln Glu Tyr Thr Leu Leu Lys Leu Lys
260 265 270
Val Leu G1u Pro Tyr Pro Ser Lys Leu Ser Gly Leu Lys Gly Lys
275 280 285
Asn Ile Phe Leu Val Ala Ala Thr Leu Arg Pro Glu Thr Met Phe
290 295 300
Gly Gln Thr Asn Cys Trp Val Arg Pro Asp Met Lys Tyr Ile Gly
305 310 315
Phe Glu Thr Val Asn Gly Asp Ile Phe Ile Cys Thr Gln Lys Ala
320 325 330
Ala Arg Asn Met Ser Tyr Gln Gly Phe Thr Lys Asp Asn Gly Val
335 340 345
Val Pro Val Val Lys Glu Leu Met Gly G1u Glu Ile Leu Gly Ala
350 355 360
Ser Leu Ser Ala Pro Leu Thr Ser Tyr Lys Val Ile Tyr Val Leu
365 370 375
Pro Met Leu Thr Ile Lys Glu Asp Lys Gly Thr Gly Val Val Thr
380 385 390
Ser Val Pro Ser Asp Ser Pro Asp Asp Ile Ala Ala Leu Arg Asp
395 400 405
Leu Lys Lys Lys Gln Ala Leu Arg Ala Lys Tyr Gly Ile Arg Asp
410 415 420
Asp Met Val Leu Pro Phe Glu Pro Val Pro Val Ile Glu Ile Pro
425 430 435
Gly Phe Gly Asn Leu Ser Ala Val Thr Ile Cys Asp Glu Leu Lys
440 445 450
Ile Gln Ser Gln Asn Asp Arg Glu Lys Leu Ala Glu Ala Lys Glu
455 460 465
Lys Ile Tyr Leu Lys Gly Phe Tyr Glu Gly Ile Met Leu Val Asp
470 475 480
G1y Phe Lys Gly Gln Lys Val Gln Asp Val Lys Lys Thr Ile Gln
485 490 495
Lys Lys Met Ile Asp Ala Gly Asp Ala Leu Ile Tyr Met Glu Pro
500 505 510
Glu Lys Gln Val Met Ser Arg Ser Ser Asp Glu Cys Val Val Ala
515 520 525
Leu Cys Asp Gln Trp Tyr Leu Asp Tyr Gly Glu Glu Asn Trp Lys
530 535 540
Lys Gln Thr Ser Gln Cys Leu Lys Asn Leu Glu Thr Phe Cys Glu
545 550 555
Glu Thr Arg Arg Asn Phe Glu Ala Thr Leu Gly Trp Leu Gln Glu
560 565 570
His Ala Cys Ser Arg Thr Tyr Gly Leu Gly Thr His Leu Pro Trp
575 580 585
Asp Glu Gln Trp Leu Ile Glu Ser Leu Ser Asp Ser Thr Ile Tyr
590 595 600
Met Ala Phe Tyr Thr Val Ala His Leu Leu Gln Gly Gly Asn Leu
605 610 615
His Gly Gln Ala Glu Ser Pro Leu Gly Ile Arg Pro Gln Gln Met
620 625 630
Thr Lys Glu Val Trp Asp Tyr Val Phe Phe Lys Glu Ala Pro Phe
635 640 645
Pro Lys Thr G1n Ile Ala Lys Glu Lys Leu Asp Gln Leu Lys Gln
650 655 660
Glu Phe Glu Phe Trp Tyr Pro Val Asp Leu Arg Val Ser Gly Lys
665 670 675
Asp Leu Val Pro Asn His Leu Ser Tyr Tyr Leu Tyr Asn His Val
2/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
680 685 690
Ala Met Trp Pro Glu Gln Ser Asp Lys Trp Pro Thr Ala Val Arg
695 700 705
Ala Asn Gly His Leu Leu Leu Asn Ser Glu Lys Met Ser Lys Ser
710 715 720
Thr Gly Asn Phe Leu Thr Leu Thr Gln Ala Ile Asp Lys Phe Ser
725 730 735
Ala Asp Gly Met Arg Leu Ala Leu Ala Asp Ala Gly Asp Thr Val
740 745 750
Glu Asp Ala Asn Phe Val Glu Ala Met Ala Asp Ala Gly Ile Leu
755 760 765
Arg Leu Tyr Thr Trp Val Glu Trp Val Lys Glu Met Val Ala Asn
770 775 780
Trp Asp Ser Leu Arg Ser Gly Pro Ala Ser Thr Phe Asn Asp Arg
785 790 795
Val Phe Ala Ser Glu Leu Asn Ala Gly Ile Ile Lys Thr Asp Gln
800 805 810
Asn Tyr Glu Lys Met Met Phe Lys Glu Ala Leu Lys Thr Gly Phe
815 820 825
Phe Glu Phe Gln Ala Ala Lys Asp Lys Tyr Arg Glu Leu Ala Val
830 835 840
Glu Gly Met His Arg Glu Leu Val Phe Arg Phe Ile Glu Val Gln
845 850 855
Thr Leu Leu Leu Ala Pro Phe Cys Pro His Leu Cys Glu His Ile
860 865 870
Trp Thr Leu Leu Gly Lys Pro Asp Ser Ile Met Asn Ala Ser Trp
875 880 885
Pro Val Ala Gly Pro Val Asn Glu Val Leu Ile His Ser Ser Gln
890 895 900
Tyr Leu Met Glu Val Thr His Asp Leu Arg Leu Arg Leu Lys Asn
905 910 915
Tyr Met Met Pro Ala Lys Gly Lys Lys Thr Asp Lys Gln Pro Leu
920 925 930
Gln Lys Pro Ser His Cys Thr Ile Tyr Val Ala Lys Asn Tyr Pro
935 940 945
Pro Trp Gln His Thr Thr Leu Ser Val Leu Arg Lys His Phe Glu
950 955 960
Ala Asn Asn Gly Lys Leu Pro Asp Asn Lys Val Ile Ala Ser Glu
965 970 975
Leu Gly Ser Met Pro Glu Leu Lys Lys Tyr Met Lys Lys Val Met
980 985 990
Pro Phe Val Ala Met Ile Lys Glu Asn Leu Giu Lys Met Gly Pro
995 1000 1005
Arg Ile Leu Asp Leu Gln Leu Glu Phe Asp Glu Lys Ala Val Leu
1010 1015 1020
Met Glu Asn Ile Val Tyr Leu Thr Asn Ser Leu Glu Leu Glu His
1025 1030 1035
Ile Glu Val Lys Phe Ala Ser Glu Ala Glu Asp Lys Ile Arg Glu
1040 1045 1050
Asp Cys Cys Pro Gly Lys Pro Leu Asn Val Phe Arg Ile Glu Pro
1055 1060 1065
Gly Val Ser Val Ser Leu Val Asn Pro Gln Pro Ser Asn Gly His
1070 1075 1080
Phe Ser Thr Lys Ile Glu Ile Arg Gln Gly Asp Asn Cys Asp Ser
1085 1090 1095
Ile Ile Arg Arg Leu Met Lys Met Asn Arg Gly Ile Lys Asp Leu
1100 1105 1110
Ser Lys Val Lys Leu Met Arg Phe Asp Asp Pro Leu Leu Gly Pro
1115 1120 1125
Arg Arg Val Pro Val Leu Gly Lys Glu Tyr Thr Glu Lys Thr Pro
1130 1135 1140
Ile Ser Glu His Ala Val Phe Asn Val Asp Leu Met Ser Lys Lys
1145 1150 1155
Ile His Leu Thr Glu Asn Gly Ile Arg Val Asp Ile Gly Asp Thr
1160 1165 1170
3/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
Ile Ile Tyr Leu Val His
1175
<210> 2
<211> 739
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2083883CD1
<400> 2
Met Asp Ala Leu Lys Pro Pro Cys Leu Trp Arg Asn His Glu Arg
1 5 10 15
Gly Lys Lys Asp Arg Asp Ser Cys Gly Arg Lys Asn Ser Glu Pro
20 25 30
Gly Ser Pro His Ser Leu Glu Ala Leu Arg Asp Ala Ala Pro Ser
35 40 45
Gln Gly Leu Asn Phe Leu Leu Leu Phe Thr Lys Met Leu Phe Ile
50 55 60
Phe Asn Phe Leu Phe Ser Pro Leu Pro Thr Pro Ala Leu Ile Cys
65 70 75
Ile Leu Thr Phe Gly Ala Ala Ile Phe Leu Trp Leu Ile Thr Arg
80 85 90
Pro Gln Pro Val Leu Pro Leu Leu Asp Leu Asn Asn Gln Ser Val
95 100 105
Gly Ile Glu Gly Gly Ala Arg Lys Gly Val Ser Gln Lys Asn Asn
110 115 120
Asp Leu Thr Ser Cys Cys Phe Ser Asp Ala Lys Thr Met Tyr Glu
125 130 135
Val Phe Gln Arg Gly Leu Ala Val Ser Asp Asn Gly Pro Cys Leu
140 145 150
Gly Tyr Arg Lys Pro Asn Gln Pro Tyr Arg Trp Leu Ser Tyr Lys
155 160 165
Gln Val Ser Asp Arg Ala Glu Tyr Leu Gly Ser Cys Leu Leu His
170 175 180
Lys Gly Tyr Lys Ser Ser Pro Asp Gln Phe Val Gly Ile Phe Ala
185 190 195
Gln Asn Arg Pro Glu Trp Ile Ile Ser Glu Leu Ala Cys Tyr Thr
200 205 210
Tyr Ser Met Val Ala Val Pro Leu Tyr Asp Thr Leu Gly Pro Glu
215 220 225
Ala Ile Val His Ile Val Asn Lys Ala Asp Ile Ala Val Val Ile
230 235 240
Cys Asp Thr Pro Gln Lys Ala Leu Val Leu Ile Gly Asn Val Glu
245 250 255
Lys Gly Phe Thr Pro Ser Leu Lys Val Ile Ile Leu Met Asp Pro
260 265 270
Phe Asp Asp Asp Leu Lys Gln Arg Gly Glu Lys Ser Gly Ile Glu
275 280 285
Ile Leu Ser Leu Tyr Asp Ala Glu Asn Leu Gly Lys Glu His Phe
290 295 300
Arg Lys Pro Val Pro Pro Ser Pro Glu Asp Leu Ser Val Ile Cys
305 310 315
Phe Thr Ser Gly Thr Thr Gly Asp Pro Lys Gly Ala Met Ile Thr
320 325 330
His Gln Asn Ile Val Ser Asn Ala Ala Ala Phe Leu Lys Cys Val
335 340 345
Glu His Ala Tyr Glu Pro Thr Pro Asp Asp Val Ala Ile Ser Tyr
350 355 360
Leu Pro Leu Ala His Met Phe Glu Arg Ile Val Gln Ala Val Val
365 370 375
Tyr Ser Cys Gly Ala Arg Val Gly Phe Phe Gln Gly Asp I1e Arg
380 385 390
Leu Leu Ala Asp Asp Met Lys Thr Leu Lys Pro Thr Leu Phe Pro
4/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
395 400 405
Ala Val Pro Arg Leu Leu Asn Arg Ile Tyr Asp Lys Val Gln Asn
410 415 420
Glu Ala Lys Thr Pro Leu Lys Lys Phe Leu Leu Lys Leu Ala Val
425 430 435
Ser Ser Lys Phe Lys Glu Leu Gln Lys Gly Ile Ile Arg His Asp
440 445 450
Ser Phe Trp Asp Lys Leu Ile Phe Ala Lys Ile Gln Asp Ser Leu
455 460 465
Gly Gly Arg Val Arg Val Ile Val Thr Gly Ala Ala Pro Met Ser
470 475 480
Thr Ser Val Met Thr Phe Phe Arg Ala Ala Met Gly Cys Gln Val
485 490 495
Tyr Glu Ala Tyr Gly Gln Thr Glu Cys Thr Gly Gly Cys Thr Phe
500 505 510
Thr Leu Pro Gly Asp Trp Thr Ser Gly His Val Gly Val Pro Leu
515 520 525
Ala Cys Asn Tyr Val Lys Leu Glu Asp Val Ala Asp Met Asn Tyr
530 535 540
Phe Thr Val Asn Asn Glu Gly Glu Val Cys Ile Lys Gly Thr Asn
545 550 555
Val Phe Lys Gly Tyr Leu Lys Asp Pro Glu Lys Thr Gln Glu Ala
560 565 570
Leu Asp Ser Asp Gly Trp Leu His Thr Gly Asp Ile Gly Arg Trp
575 580 585
Leu Pro Asn Gly Thr Leu Lys Ile Ile Asp Arg Lys Lys Asn Ile
590 595 600
Phe Lys Leu Ala Gln Gly Glu Tyr Ile Ala Pro Glu Lys Ile Glu
605 610 615
Asn Ile Tyr Asn Arg Ser Gln Pro Val Leu Gln Ile Phe Val His
620 625 630
Gly Glu Ser Leu Arg Ser Ser Leu Val Gly Val Val Val Pro Asp
635 640 645
Thr Asp Val Leu Pro Ser Phe Ala Ala Lys Leu Gly Val Lys Gly
650 655 660
Ser Phe Glu Glu Leu Cys Gln Asn Gln Val Val Arg Glu Ala Ile
665 670 675
Leu Glu Asp Leu Gln Lys Ile Gly Lys Glu Ser Gly Leu Lys Thr
680 685 690
Phe Glu Gln Val Lys Ala Ile Phe Leu His Pro Glu Pro Phe Ser
695 700 705
Ile Glu Asn Gly Leu Leu Thr Pro Thr Leu Lys Ala Lys Arg Gly
710 715 720
Glu Leu Ser Lys Tyr Phe Arg Thr Gln Ile Asp Ser Leu Tyr Glu
725 730 735
His Ile Gln Asp
<210> 3
<211> 589
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2454288CD1
<400> 3
Met Pro Thr Val Ser Val Lys Arg Asp Leu Leu Phe Gln Ala Leu
1 5 10 15
Gly Arg Thr Tyr Thr Asp Glu Glu Phe Asp Glu Leu Cys Phe Glu
20 25 30
Phe Gly Leu Glu Leu Asp Glu Ile Thr Ser Glu Lys Glu Ile Ile
35 40 45
Ser Lys Glu Gln Gly Asn Val Lys Ala Ala Gly Ala Ser Asp Val
50 55 60
5/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
Val Leu Tyr Lys Ile Asp Val Pro Ala Asn Arg Tyr Asp Leu Leu
65 70 75
Cys Leu Glu Gly Leu Val Arg G1y Leu Gln Val Phe Lys Glu Arg
80 85 90
I1e Lys Ala Pro Val Tyr Lys Arg Val Met Pro Asp Gly Lys Ile
95 100 105
Gln Lys Leu Ile Ile Thr Glu Glu Thr Ala Lys Ile Arg Pro Phe
110 115 120
Ala Val Ala Ala Val Leu Arg Asn Ile Lys Phe Thr Lys Asp Arg
125 130 135
Tyr Asp Ser Phe Ile Glu Leu Gln Glu Lys Leu His Gln Asn Ile
140 145 150
Cys Arg Lys Arg Ala Leu Val Ala Ile Gly Thr His Asp Leu Asp
155 160 165
Thr Leu Ser Gly Pro Phe Thr Tyr Thr Ala Lys Arg Pro Ser Asp
170 175 180
Ile Lys Phe Lys Pro Leu Asn Lys Thr Lys Glu Tyr Thr Ala Cys
185 190 195
Glu Leu Met Asn Ile Tyr Lys Thr Asp Asn His Leu Lys His Tyr
200 205 210
Leu His Ile Ile Glu Asn Lys Pro Leu Tyr Pro Val Ile Tyr Asp
215 220 225
Ser Asn Gly Val Val Leu Ser Met Pro Pro Ile Ile Asn Gly Asp
230 235 240
His Ser Arg Ile Thr Val Asn Thr Arg Asn Ile Phe Ile Glu Cys
245 250 255
Thr Gly Thr Asp Phe Thr Lys Ala Lys Ile Val Leu Asp Ile Ile
260 265 270
Val Thr Met Phe Ser Glu Tyr Cys Glu Asn Gln Phe Thr Val Glu
275 280 285
Ala Ala Glu Val Val Phe Pro Asn Gly Lys Ser His Thr Phe Pro
290 295 300
Glu Leu Ala Tyr Arg Lys Glu Met Val Arg Ala Asp Leu Ile Asn
305 310 315
Lys Lys Val Gly Ile Arg Glu Thr Pro Glu Asn Leu Ala Lys Leu
320 325 330
Leu Thr Arg Met Tyr Leu Lys Ser Glu Val Ile Gly Asp Gly Asn
335 340 345
Gln Ile Glu Ile Glu Ile Pro Pro Thr Arg Ala Asp Ile Ile His
350 355 360
Ala Cys Asp Ile Val Glu Asp Ala Ala Ile Ala Tyr Gly Tyr Asn
365 370 375
Asn Ile Gln Met Thr Leu Pro Lys Thr Tyr Thr Ile Ala Asn Gln
380 385 390
Phe Pro Leu Asn Lys Leu Thr Glu Leu Leu Arg His Asp Met Ala
395 400 405
Ala Ala Gly Phe Thr Glu Ala Leu Thr Phe Ala Leu Cys Ser Gln
410 415 420
Glu Asp Ile Ala Asp Lys Leu Gly Val Asp Ile Ser Ala Thr Lys
425 430 435
Ala Val His Ile Ser Asn Pro Lys Thr Ala Glu Phe Gln Val Ala
440 445 450
Arg Thr Thr Leu Leu Pro Gly Leu Leu Lys Thr Ile Ala Ala Asn
455 460 465
Arg Lys Met Pro Leu Pro Leu Lys Leu Phe Glu Ile Ser Asp Ile
470 475 480
Val Ile Lys Asp Ser Asn Thr Asp Val Gly Ala Lys Asn Tyr Arg
485 490 495
His Leu Cys Ala Val Tyr Tyr Asn Lys Asn Pro Gly Phe Glu Ile
500 505 510
Ile His Gly Leu Leu Asp Arg Ile Met Gln Leu Leu Asp Val Pro
515 520 525
Pro Gly G1u Asp Lys Gly Gly Tyr Val Ile Lys Ala Ser Glu Gly
530 535 540
Pro A1a Phe Phe Pro Gly Arg Cys Ala Glu Ile Phe Ala Arg Gly
6/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
545 550 555
Gln Ser Val Gly Lys Leu Gly Val Leu His Pro Asp Val Ile Thr
560 565 570
Lys Phe Glu Leu Thr Met Pro Cys Ser Ser Leu Glu Ile Asn Ile
575 580 585
Gly Pro Phe Leu
<210> 4
<211> 157
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1513539CD1
<400> 4
Met Lys Leu Lys Cys Ile Phe Gly Phe Ala Thr Lys Glu Thr Ser
1 5 10 15
Cys Tyr Asn Val Thr Asn Ile Gly Phe Lys Ser Pro Ser Asp Phe
20 25 30
Trp Gln Ser Val His Ser Thr Leu Pro Arg Glu Leu Ala Pro Cys
35 40 45
Leu Val Phe Asn Thr Ser Pro Asn Leu Ala Leu Phe Ser Ala Ala
50 55 60
Phe Ala Phe Ile Val Val Lys Asp Ser Ala Gly Asp Ser Asp Val
65 70 75
Val Val Gln Glu Leu Lys Ser Met Val Ala Thr Lys Ile Ala Lys
80 85 90
Tyr Ala Val Pro Asp Glu Ile Leu Val Val Lys Arg Leu Pro Lys
95 100 105
Thr Arg Ser Gly Lys Val Met Arg Arg Leu Leu Arg Lys Ile Ile
110 115 120
Thr Ser Glu Ala Gln Glu Leu Gly Asp Thr Thr Thr Leu Glu Asp
125 130 135
Pro Ser Ile Ile Ala Glu Ile Leu Ser Val Tyr Gln Lys Cys Lys
140 145 150
Asp Lys Gln Ala Ala Ala Lys
155
<210> 5
<211> 643
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2148623CD1
<400> 5
Met Cys Gly Ile Cys Cys Ser Val Asn Phe Ser Ala Glu His Phe
1 5 10 15
Ser Gln Asp Leu Lys Glu Asp Leu Leu Tyr Asn Leu Lys Gln Arg
20 25 30
Gly Pro Asn Ser Ser Lys Gln Leu Leu Lys Ser Asp Val Asn Tyr
35 40 45
Gln Cys Leu Phe Ser Ala His Val Leu His Leu Arg Gly Val Leu
50 55 60
Thr Thr Gln Pro Val Glu Asp Glu Arg Gly Asn Val Phe Leu Trp
65 70 75
Asn Gly Glu Ile Phe Ser Gly I1e Lys Va1 Glu Ala G1u Glu Asn
80 85 90
Asp Thr Gln Ile Leu Phe Asn Tyr Leu Ser Ser Cys Lys Asn Glu
95 100 105
Ser Glu Ile Leu Ser Leu Phe Ser Glu Val Gln Gly Pro Trp Ser
110 115 120
7/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
Phe Ile Tyr Tyr G1n Ala Ser Ser His Tyr Leu Trp Phe Gly Arg
125 130 135
Asp Phe Phe Gly Arg Arg Ser Leu Leu Trp His Phe Ser Asn Leu
140 145 150
Gly Lys Ser Phe Cys Leu Ser Ser Val Gly Thr Gln Thr Ser Gly
155 160 165
Leu Ala Asn Gln Trp Gln Glu Val Pro Ala Ser Gly Leu Phe Arg
170 175 180
Ile Asp Leu Lys Ser Thr Val Ile Ser Arg Cys Ile Ile Leu Gln
185 190 195
Leu Tyr Pro Trp Lys Tyr Ile Ser Arg Glu Asn Ile Ile G1u Glu
200 205 210
Asn Val Asn Ser Leu Ser Gln Ile Ser Ala Asp Leu Pro Ala Phe
215 220 225
Val Ser Val Val Ala Asn Glu Ala Lys Leu Tyr Leu Glu Lys Pro
230 235 240
Val Val Pro Leu Asn Met Met Leu Pro Gln Ala Ala Leu Glu Thr
245 250 255
His Cys Ser Asn Ile Ser Asn Val Pro Pro Thr Arg Glu Ile Leu
260 265 270
Gln Val Phe Leu Thr Asp Val His Met Lys Glu Val Ile Gln Gln
275 280 285
Phe Ile Asp Val Leu Ser Val Ala Val Lys Lys Arg Val Leu Cys
290 295 300
Leu Pro Arg Asp Glu Asn Leu Thr Ala Asn Glu Val Leu Lys Thr
305 310 315
Cys Asp Arg Lys Ala Asn Val Ala Ile Leu Phe Ser Gly Gly Ile
320 325 330
Asp Ser Met Val Ile Ala Thr Leu Ala Asp Arg His Ile Pro Leu
335 340 345
Asp Glu Pro Ile Asp Leu Leu Asn Val Ala Phe Ile Ala Glu Glu
350 355 360
Lys Thr Met Pro Thr Thr Phe Asn Arg Glu Gly Asn Lys Gln Lys
365 370 375
Asn Lys Cys Glu Ile Pro Ser Glu Glu Phe Ser Lys Asp Val Ala
380 385 390
Ala Ala Ala Ala Asp Ser Pro Asn Lys His Val Ser Val Pro Asp
395 400 405
Arg Ile Thr Gly Arg Ala Gly Leu Lys Glu Leu Gln Ala Val Ser
410 415 420
Pro Ser Arg Ile Trp Asn Phe Val Glu Ile Asn Val Ser Met Glu
425 430 435
Glu Leu Gln Lys Leu Arg Arg Thr Arg Ile Cys His Leu Ile Arg
440 445 450
Pro Leu Asp Thr Val Leu Asp Asp Ser Ile Gly Cys Ala Val Trp
455 460 465
Phe Ala Ser Arg Gly Ile Gly Trp Leu Val Ala Gln Glu Gly Val
470 475 480
Lys Ser Tyr Gln Ser Asn Ala Lys Val Val Leu Thr Gly Ile Gly
485 490 495
Ala Asp Glu Gln Leu Ala G1y Tyr Ser Arg His Arg Val Arg Phe
500 505 510
Gln Ser His Gly Leu Glu Gly Leu Asn Lys Glu Ile Met Met Glu
515 520 525
Leu Gly Arg Ile Ser Ser Arg Asn Leu Gly Arg Asp Asp Arg Val
530 535 540
Ile Gly Asp His Gly Lys Glu Ala Arg Phe Pro Phe Leu Asp Glu
545 550 555
Asn Val Val Ser Phe Leu Asn Ser Leu Pro Ile Trp Glu Lys Ala
560 565 570
Asn Leu Thr Leu Pro Arg Gly Ile Gly Glu Lys Leu Leu Leu Arg
575 580 585
Leu Ala Ala Val Glu Leu G1y Leu Thr Ala Ser Ala Leu Leu Pro
590 595 600
Lys Arg Ala Met Gln Phe Gly Ser Arg Ile Ala Lys Met Glu Lys
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
605 610 615
Ile Asn Glu Lys Ala Ser Asp Lys Cys Gly Arg Leu Gln Ile Met
620 625 630
Ser Leu Glu Asn Leu Ser Ile Glu Lys Glu Thr Lys Leu
635 640
<210> 6
<211> 660
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2579405CD1
<400> 6
Met Asp Ile Leu Val Ser Glu Cys Ser Ala Arg Leu Leu Gln Gln
1 5 10 15
Glu Glu Glu Ile Lys Ser Leu Thr Ala Glu Ile Asp Arg Leu Lys
20 25 30
Asn Cys Gly Cys Leu Gly Ala Ser Pro Asn Leu Glu Gln Leu Gln
35 40 45
Glu Glu Asn Leu Lys Leu Lys Tyr Arg Leu Asn Ile Leu Arg Lys
50 55 60
Ser Leu Gln Ala Glu Arg Asn Lys Pro Thr Lys Asn Met Ile Asn
65 70 75
Ile Ile Ser Arg Leu Gln Glu Val Phe Gly His Ala Ile Lys Ala
80 85 90
Ala Tyr Pro Asp Leu Glu Asn Pro Pro Leu Leu Val Thr Pro Ser
95 100 105
Gln Gln Ala Lys Phe Gly Asp Tyr Gln Cys Asn Ser Ala Met Gly
110 115 120
Ile Ser Gln Met Leu Lys Thr Lys Glu Gln Lys Val Asn Pro Arg
125 130 135
Glu Ile Ala Glu Asn Ile Thr Lys His Leu Pro Asp Asn Glu Cys
140 145 150
Ile Glu Lys Val Glu Ile Ala Gly Pro Gly Phe Ile Asn Val His
155 160 165
Leu Arg Lys Asp Phe Val Ser Glu Gln Leu Thr Ser Leu Leu Val
170 175 180
Asn Gly Val Gln Leu Pro Ala Leu Gly Glu Asn Lys Lys Val Ile
185 190 195
Val Asp Phe Ser Ser Pro Asn Ile Ala Lys Glu Met His Val Gly
200 205 210
His Leu Arg Ser Thr Ile Ile Gly Glu Ser Ile Ser Arg Leu Phe
215 220 225
Glu Phe Ala Gly Tyr Asp Val Leu Arg Leu Asn His Val Gly Asp
230 235 240
Trp Gly Thr Gln Phe Gly Met Leu Ile Ala His Leu Gln Asp Lys
245 250 255
Phe Pro Asp Tyr Leu Thr Val Ser Pro Pro Ile Gly Asp Leu Gln
260 265 270
Val Phe Tyr Lys Glu Ser Lys Lys Arg Phe Asp Thr Glu Glu Glu
275 280 285
Phe Lys Lys Arg Ala Tyr Gln Cys Val Val Leu Leu Gln Gly Lys
290 295 300
Asn Pro Asp Ile Thr Lys Ala Trp Lys Leu Ile Cys Asp Val Ser
305 310 315
Arg Gln Glu Leu Asn Lys Ile Tyr Asp Ala Leu Asp Val Ser Leu
320 325 330
Ile Glu Arg Gly Glu Ser Phe Tyr Gln Asp Arg Met Asn Asp Ile
335 340 345
Val Lys Glu Phe Glu Asp Arg Gly Phe Val Gln Val Asp Asp Gly
350 355 360
Arg Lys Ile Val Phe Val Pro Gly Cys Ser Ile Pro Leu Thr Ile
365 370 375
9/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
Val Lys Ser Asp G1y Gly Tyr Thr Tyr Asp Thr Ser Asp Leu Ala
380 385 390
Ala Ile Lys Gln Arg Leu Phe Glu Glu Lys Ala Asp Met Ile Ile
395 400 405
Tyr Val Val Asp Asn Gly Gln Ser Val His Phe Gln Thr Ile Phe
410 415 420
Ala Ala Ala Gln Met Ile Gly Trp Tyr Asp Pro Lys Val Thr Arg
425 430 435
Val Phe His Ala Gly Phe Gly Val Val Leu Gly Glu Asp Lys Lys
440 445 450
Lys Phe Lys Thr Arg Ser Gly Glu Thr Val Arg Leu Met Asp Leu
455 460 465
Leu Gly Glu Gly Leu Lys Arg Ser Met Asp Lys Leu Lys Glu Lys
470 475 480
Glu Arg Asp Lys Val Leu Thr Ala Glu Glu Leu Asn Ala Ala Gln
485 490 495
Thr Ser Val Ala Tyr Gly Cys Ile Lys Tyr Ala Asp Leu Ser His
500 505 510
Asn Arg Leu Asn Asp Tyr Ile Phe Ser Phe Asp Lys Met Leu Asp
515 520 525
Asp Arg Gly Asn Thr Ala Ala Tyr Leu Leu Tyr Ala Phe Thr Arg
530 535 540
Ile Arg Ser Ile Ala Arg Leu Ala Asn Ile Asp Glu Glu Met Leu
545 550 555
Gln Lys Ala Ala Arg Glu Thr Lys Ile Leu Leu Asp His Glu Lys
560 565 570
Glu Trp Lys Leu Gly Arg Cys Ile Leu Arg Phe Pro Glu Ile Leu
575 580 585
Gln Lys Ile Leu Asp Asp Leu Phe Leu His Thr Leu Cys Asp Tyr
590 595 600
Ile Tyr Glu Leu Ala Thr Ala Phe Thr Glu Phe Tyr Asp Ser Cys
605 610 615
Tyr Cys Val Glu Lys Asp Arg Gln Thr Gly Lys Ile Leu Lys Val
620 625 630
Asn Met Trp Arg Met Leu Leu Cys Glu Ala Val Ala Ala Val Met
635 640 645
Ala Lys Gly Phe Asp Ile Leu Gly Ile Lys Pro Val Gln Arg Met
650 655 660
<210> 7
<211> 725
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2662427CD1
<400> 7
Met Ala Ala Ala Ser Ala Val Ser Val Leu Leu Val Ala Ala Glu
1 5 10 15
Arg Asn Arg Trp His Arg Leu Pro Ser Leu Leu Leu Pro Pro Arg
20 25 30
Thr Trp Val Trp Arg Gln Arg Thr Met Lys Tyr Thr Thr Ala Thr
35 40 45
Gly Arg Asn Ile Thr Lys Val Leu Ile Ala Asn Arg Gly Glu Ile
50 55 60
Ala Cys Arg Val Met Arg Thr Ala Lys Lys Leu Gly Val Gln Thr
65 70 75
Val Ala Val Tyr Ser Glu Ala Asp Arg Asn Ser Met His Val Asp
80 85 90
Met Ala Asp Glu Ala Tyr Ser Ile Gly Pro Ala Pro Ser Gln Gln
95 100 105
Ser Tyr Leu Ser Met Glu Lys Ile Ile Gln Val Ala Lys Thr Ser
110 115 120
10/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/CTS00/19980
A1a A1a Gln Ala Ile His Pro Gly Cys Arg Phe Leu Ser Glu Asn
125 130 135
Met Glu Phe Ala Glu Leu Cys Lys Gln Glu Gly Ile Ile Phe Ile
140 145 150
Gly Pro Pro Pro Ser Ala Ile Arg Asp Met Gly Ile Lys Ser Thr
155 160 165
Ser Lys Ser Ile Met Ala Ala Ala Gly Val Pro Val Val Glu Gly
170 175 180
Tyr His Gly Glu Asp Gln Ser Asp Gln Cys Leu Lys Glu His Ala
185 190 195
Arg Arg Ile Gly Tyr Pro Val Met Ile Lys Ala Val Arg Gly Gly
200 205 210
Gly Gly Lys Gly Met Arg Ile Val Arg Ser Glu Gln Glu Phe Gln
215 220 225
Glu Gln Leu Glu Ser Ala Arg Arg Glu Ala Lys Lys Ser Phe Asn
230 235 240
Asp Asp Ala Met Leu Ile Glu Lys Phe Val Asp Thr Pro Arg His
245 250 255
Val Glu Val Gln Val Phe Gly Asp His His Gly Asn Ala Val Tyr
260 265 270
Leu Phe Glu Arg Asp Cys Ser Val Gln Arg Arg His Gln Lys Ile
275 280 285
Ile Glu Glu Ala Pro Ala Pro Gly Ile Lys Ser Glu Val Arg Lys
290 295 300
Lys Leu Gly Glu Ala Ala Val Arg Ala Ala Lys Ala Val Asn Tyr
305 310 315
Val Gly Ala Gly Thr Val Glu Phe Ile Met Asp Ser Lys His Asn
320 325 330
Phe Cys Phe Met Glu Met Asn Thr Arg Leu Gln Val Glu His Pro
335 340 345
Val Thr Glu Met Ile Thr Gly Thr Asp Leu Val Glu Trp Gln Leu
350 355 360
Arg Ile Ala Ala Gly Glu Lys Ile Pro Leu Ser Gln Glu Glu Ile
365 370 375
Thr Leu Gln Gly His Ala Phe Glu Ala Arg Ile Tyr Ala Glu Asp
380 385 390
Pro Ser Asn Asn Phe Met Pro Val Ala Gly Pro Leu Val His Leu
395 400 405
Ser Thr Pro Arg Ala Asp Pro Ser Thr Arg Ile Glu Thr Gly Val
410 415 420
Arg Gln Gly Asp Glu Val Ser Val His Tyr Asp Pro Met Ile Ala
425 430 435
Lys Leu Val Val Trp Ala Ala Asp Arg Gln Ala Ala Leu Thr Lys
440 445 450
Leu Arg Tyr Ser Leu Arg Gln Tyr Asn Ile Val Gly Leu Pro Thr
455 460 465
Asn Ile Asp Phe Leu Leu Asn Leu Ser Gly His Pro Glu Phe Glu
470 475 480
Ala Gly Asn Val His Thr Asp Phe Ile Pro Gln His His Lys Gln
485 490 495
Leu Leu Leu Ser Arg Lys Ala Ala Ala Lys Glu Ser Leu Cys Gln
500 505 510
Ala Ala Leu Gly Leu Ile Leu Lys Glu Lys Ala Met Thr Asp Thr
515 520 525
Phe Thr Leu Gln Ala His Asp Gln Phe Ser Pro Phe Ser Ser Ser
530 535 540
Ser Gly Arg Arg Leu Asn Ile Ser Tyr Thr Arg Asn Met Thr Leu
545 550 555
Lys Asp Gly Lys Asn Asn Val Ala Ile Ala Val Thr Tyr Asn His
560 565 570
Asp Gly Ser Tyr Ser Met Gln Ile Glu Asp Lys Thr Phe Gln Va1
575 580 585
Leu Gly Asn Leu Tyr Ser Glu Gly Asp Cys Thr Tyr Leu Lys Cys
590 595 600
Ser Val Asn Gly Val A1a Ser Lys Ala Lys Leu I1e Ile Leu Glu
11/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
605 610 615
Asn Thr Ile Tyr Leu Phe Ser Lys Glu Gly Ser Ile Glu Ile Asp
620 625 630
Ile Pro Val Pro Lys Tyr Leu Ser Ser Val Ser Ser Gln Glu Thr
635 640 645
Gln Gly Gly Pro Leu Ala Pro Met Thr Gly Thr Ile Glu Lys Val
650 655 660
Phe Val Lys Ala Gly Asp Lys Val Lys Ala Gly Asp Ser Leu Met
665 670 675
Val Met Ile Ala Met Lys Met Glu His Thr Ile Lys Ser Pro Lys
680 685 690
Asp Gly Thr Val Lys Lys Val Phe Tyr Arg Glu Gly Ala Gln Ala
695 700 705
Asn Arg His Thr Pro Leu Val Glu Phe Glu Glu Glu Glu Ser Asp
710 715 720
Lys Arg Glu Ser Glu
725
<210> 8
<211> 644
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2844928CD1
<400> 8
Met Ala Val Tyr Val Gly Met Leu Arg Leu Gly Arg Leu Cys Ala
1 5 10 15
Gly Ser Ser Gly Val Leu Gly Ala Arg Ala Ala Leu Ser Arg Ser
20 25 30
Trp Gln Glu Ala Arg Leu Gln Gly Val Arg Phe Leu Ser Ser Arg
35 40 45
Glu Val Asp Arg Met Val Ser Thr Pro Ile Gly Gly Leu Ser Tyr
50 55 60
Val.Gln Gly Cys Thr Lys Lys His Leu Asn Ser Lys Thr Val Gly
65 70 75
Gln Cys Leu Glu Thr Thr Ala Gln Arg Val Pro Glu Arg Glu Ala
80 85 90
Leu Val Val Leu His Glu Asp Val Arg Leu Thr Phe Ala Gln Leu
95 100 105
Lys Glu Glu Val Asp Lys Ala Ala Ser Gly Leu Leu Ser Ile Gly
110 115 120
Leu Cys Lys Gly Asp Arg Leu Gly Met Trp Gly Pro Asn Ser Tyr
125 130 135
Ala Trp Val Leu Met Gln Leu Ala Thr Ala Gln Ala Gly Ile Ile
140 145 150
Leu Val Ser Val Asn Pro Ala Tyr Gln Ala Met Glu Leu Glu Tyr
155 160 165
Val Leu Lys Lys Val Gly Cys Lys Ala Leu Val Phe Pro Lys Gln
170 175 180
Phe Lys Thr Gln Gln Tyr Tyr Asn Val Leu Lys Gln Ile Cys Pro
185 190 195
Glu Val Glu Asn Ala Gln Pro Gly Ala Leu Lys Ser Gln Arg Leu
200 205 210
Pro Asp Leu Thr Thr Val Ile Ser Val Asp Ala Pro Leu Pro Gly
215 220 225
Thr Leu Leu Leu Asp Glu Val Val Ala Ala Gly Ser Thr Arg Gln
230 235 240
His Leu Asp Gln Leu Gln Tyr Asn Gln Gln Phe Leu Ser Cys His
245 250 255
Asp Pro Ile Asn Ile Gln Phe Thr Ser Gly Thr Thr Gly Ser Pro
260 265 270
Lys Gly Ala Thr Leu Ser His Tyr Asn Ile Val Asn Asn Ser Asn
275 280 285
12/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
Ile Leu Gly Glu Arg Leu Lys Leu His Glu Lys Thr Pro Glu Gln
290 295 300
Leu Arg Met Ile Leu Pro Asn Pro Leu Tyr His Cys Leu Gly Ser
305 310 315
Val Ala Gly Thr Met Met Cys Leu Met Tyr Gly Ala Thr Leu Ile
320 325 330
Leu Ala Ser Pro Ile Phe Asn Gly Lys Lys Ala Leu Glu Ala Ile
335 340 345
Ser Arg Glu Arg Gly Thr Phe Leu Tyr Gly Thr Pro Thr Met Phe
350 355 360
Val Asp I1e Leu Asn Gln Pro Asp Phe Ser Ser Tyr Asp Ile Ser
365 370 375
Thr Met Cys Gly Gly Val Ile Ala Gly Ser Pro Ala Pro Pro Glu
380 385 390
Leu Ile Arg Ala Ile Ile Asn Lys Ile Asn Met Lys Asp Leu Val
395 400 405
Val Ala Tyr Gly Thr Thr Glu Asn Ser Pro Val Thr Phe Ala His
410 415 420
Phe Pro Glu Asp Thr Val Glu Gln Lys Ala Glu Ser Val Gly Arg
425 430 435
Ile Met Pro His Thr Glu Ala Arg Ile Met Asn Met Glu Ala Gly
440 445 450
Thr Leu Ala Lys Leu Asn Thr Pro Gly Glu Leu Cys Ile Arg Gly
455 460 465
Tyr Cys Val Met Leu Gly Tyr Trp Gly Glu Pro Gln Lys Thr Glu
470 475 480
Glu Ala Val Asp Gln Asp Lys Trp Tyr Trp Thr Gly Asp Val Ala
485 490 495
Thr Met Asn Glu Gln Gly Phe Cys Lys Ile Val Gly Arg Ser Lys
500 505 510
Asp Met Ile Ile Arg Gly Gly Glu Asn Ile Tyr Pro Ala Glu Leu
515 520 525
Glu Asp Phe Phe His Thr His Pro Lys Val Gln Glu Val Gln Val
530 535 540
Arg His Leu Ala Gln Val Ser Pro G1n Lys Gln Glu Thr His Met
545 550 555
Asn Thr Val Met Ser Asp Ile Phe Leu Trp Pro Trp Asn Val Val
560 565 570
Gly Val Lys Asp Asp Arg Met Gly Glu Glu Ile Cys Ala Cys Ile
575 580 585
Arg Leu Lys Asp Gly Glu Glu Thr Thr Val Glu Glu Ile Lys Ala
590 595 600
Phe Cys Lys Gly Lys Ile Ser His Phe Lys Ile Pro Lys Tyr Ile
605 610 615
Val Phe Val Thr Asn Tyr Pro Leu Thr Ile Ser Gly Lys Ile Gln
620 625 630
Lys Phe Lys Leu Arg Glu Gln Met Glu Arg His Leu Asn Leu
635 640
<210> 9
<211> 504
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3231586CD1
<400> 9
Met Phe Pro Arg Glu Lys Thr Trp Asn I1e Ser Phe Ala Gly Cys
1 5 10 15
Gly Phe Leu Gly Val Tyr Tyr Val Gly Val Ala Ser Cys Leu Arg
20 25 30
Glu His Ala Pro Phe Leu Val Ala Asn Ala Thr His Ile Tyr Gly
35 40 45
Ala Ser Ala Gly Ala Leu Thr Ala Thr Ala Leu Val Thr Gly Val
13/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
50 55 60
Cys Leu Gly G1u Ala Gly Ala Lys Phe Ile Glu Val Ser Lys Glu
65 70 75
Ala Arg Lys Arg Phe Leu Gly Pro Leu His Pro Ser Phe Asn Leu
80 85 90
Val Lys Ile Ile Arg Ser Phe Leu Leu Lys Val Leu Pro Ala Asp
95 100 105
Ser His Glu His Ala Ser Gly Arg Leu Gly Ile Ser Leu Thr Arg
110 115 120
Val Ser Asp Gly Glu Asn Val Ile Ile Ser His Phe Asn Ser Lys
125 130 135
Asp Glu Leu I1e Gln Ala Asn Val Cys Ser Gly Phe Ile Pro Val
140 145 150
Tyr Cys Gly Leu Ile Pro Pro Ser Leu Gln Gly Val Arg Tyr Val
155 160 165
Asp Gly Gly Ile Ser Asp Asn Leu Pro Leu Tyr Glu Leu Lys Asn
170 175 180
Thr Ile Thr Val Ser Pro Phe Ser Gly Glu Ser Asp Ile Cys Pro
185 190 195
Gln Asp Ser Ser Thr Asn Ile His Glu Leu Arg Val Thr Asn Thr
200 205 210
Ser Ile Gln Phe Asn Leu Arg Asn Leu Tyr Arg Leu Ser Lys Ala
215 220 225
Leu Phe Pro Pro Glu Pro Leu Val Leu Arg Glu Met Cys Lys Gln
230 235 240
Gly Tyr Arg Asp Gly Leu Arg Phe Leu Gln Arg Asn Gly Leu Leu
245 250 255
Asn Arg Pro Asn Pro Leu Leu Ala Leu Pro Pro Ala Arg Pro His
260 265 270
Gly Pro Glu Asp Lys Asp Gln Ala Val Glu Ser Ala Gln Ala Glu
275 280 285
Asp Tyr Ser Gln Leu Pro Gly Glu Asp His Ile Leu Glu His Leu
290 295 300
Pro Ala Arg Leu Asn Glu Ala Leu Leu Glu Ala Cys Val Glu Pro
305 310 315
Thr Asp Leu Leu Thr Thr Leu Ser Asn Met Leu Pro Val Arg Leu
320 325 330
Ala Thr Ala Met Met Val Pro Tyr Thr Leu Pro Leu Glu Ser Ala
335 340 345
Leu Ser Phe Thr Ile Arg Leu Leu Glu Trp Leu Pro Asp Val Pro
350 355 360
Glu Asp Ile Arg Trp Met Lys Glu Gln Thr Gly Ser Ile Cys Gln
365 370 375
Tyr Leu Va1 Met Arg Ala Lys Arg Lys Leu Gly Arg His Leu Pro
380 385 390
Ser Arg Leu Pro Glu Gln Val Glu Leu Arg Arg Val Gln Ser Leu
395 400 405
Pro Ser Val Pro Leu Ser Cys Ala Ala Tyr Arg Glu Ala Leu Pro
410 415 420
Gly Trp Met Arg Asn Asn Leu Ser Leu Gly Asp Ala Leu Ala Lys
425 430 435
Trp Glu Glu Cys Gln Arg Gln Leu Leu Leu Gly Leu Phe Cys Thr
440 445 450
Asn Val Ala Phe Pro Pro Glu Ala Leu Arg Met Arg Ala Pro Ala
455 460 465
Asp Pro Ala Pro Ala Pro Ala Asp Pro Ala Ser Pro Gln His Gln
470 475 480
Leu Ala Gly Pro Ala Pro Leu Leu Ser Thr Pro Ala Pro Glu Ala
485 490 495
Arg Pro Va1 Ile Gly Ala Leu Gly Leu
500
<210> 10
<211> 489
<212> PRT
<213> Homo sapiens
14/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
<220>
<221> misc_feature
<223> Incyte ID No: 3580770CD1
<400> 10
Met Lys Tyr Ile Leu Val Thr Gly Gly Val Ile Ser Gly Ile Gly
1 5 10 15
Lys Gly Ile Ile Ala Ser Ser Ile Gly Thr Ile Leu Lys Ser Cys
20 25 30
Gly Leu Arg Val Thr Ala Ile Lys Ile Asp Pro Tyr Ile Asn Ile
35 40 45
Asp Ala Gly Thr Phe Ser Pro Tyr Glu His Gly Glu Val Phe Val
50 55 60
Leu Asn Asp Gly Gly Glu Val Asp Leu Asp Leu Gly Asp Tyr Glu
65 70 75
Arg Phe Leu Asp Ile Asn Leu Tyr Lys Asp Thr Ile Val Thr Thr
80 85 90
Gly Lys Ile Tyr Gln His Val Ile Asn Lys Glu Arg Arg Gly Asp
95 100 105
Tyr Leu Gly Lys Thr Val Gln Val Val Pro His Ile Thr Asp Ala
110 115 120
Val Gln Glu Trp Val Met Asn Gln Ala Lys Val Pro Val Asp Gly
125 130 135
Asn Lys Glu G1u Pro Gln Ile Cys Val Ile Glu Leu Gly Gly Thr
140 145 150
Ile Gly Asp Ile Glu Gly Met Pro Phe Val Glu Ala Phe Arg Gln
155 160 165
Phe Gln Phe Lys Ala Lys Arg Glu Asn Phe Cys Asn Ile His Val
170 175 180
Ser Leu Val Pro Gln Leu Ser Ala Thr Gly Glu Gln Lys Thr Lys
185 190 195
Pro Thr Gln Asn Ser Val Arg Ala Leu Arg Gly Leu Gly Leu Ser
200 205 210
Pro Asp Leu Ile Val Cys Arg Ser Ser Thr Pro Ile Glu Met Ala
215 220 225
Val Lys Glu Lys Ile Ser Met Phe Cys His Val Asn Pro Glu Gln
230 235 240
Val Ile Cys Ile His Asp Val Ser Ser Thr Tyr Arg Val Pro Val
245 250 255
Leu Leu Glu Glu Gln Ser Ile Val Lys Tyr Phe Lys Glu Arg Leu
260 265 270
His Leu Pro Ile Gly Asp Ser Ala Ser Asn Leu Leu Phe Lys Trp
275 280 285
Arg Asn Met Ala Asp Arg Tyr Glu Arg Leu Gln Lys Ile Cys Ser
290 295 300
Ile Ala Leu Val Gly Lys Tyr Thr Lys Leu Arg Asp Cys Tyr Ala
305 310 315
Ser Val Phe Lys Ala Leu Glu His Ser Ala Leu Ala Ile Asn His
320 325 330
Lys Leu Asn Leu Met Val Ile Asp Met Pro Glu His Asn Pro Gly
335 340 345
Asn Leu Gly Gly Thr Met Arg Leu Gly Ile Arg Arg Thr Val Phe
350 355 360
Lys Thr Glu Asn Ser Ile Leu Arg Lys Leu Tyr Gly Asp Val Pro
365 370 375
Phe Ile Glu Glu Arg His Arg His Arg Phe Glu Val Asn Pro Asn
380 385 390
Leu Ile Lys Gln Phe Glu Gln Asn Asp Leu Ser Phe Val Gly Gln
395 400 405
Asp Val Asp Gly Asp Arg Met Glu Ile Ile Glu Leu Ala Asn His
410 415 420
Pro Tyr Phe Val Gly Val Gln Phe His Pro Glu Phe Ser Ser Arg
425 430 435
Pro Met Lys Pro Ser Pro Pro Tyr Leu Gly Leu Leu Leu Ala Ala
440 445 450
15/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
Thr Gly Leu Ala Tyr Leu Gln Gly Cys Lys Leu
Asn Asn Gln Ser
455 460 465
Ser Ser Arg Ser Asp Ala Ser Asp Ser Phe Ser
Asp Tyr Asp Glu
470 475 480
Pro Arg Ala Leu Glu Ile Ser
Ile Glu
485
<210>
11
<211>
258
<212>
PRT
<213> Sapiens
Homo
<220>
<221> misc_feature
<223> Incyte ID No: 3778612CD1
<400> 11
Met Glu Arg Gln Lys Arg Lys Ala Asp Ile Glu Lys Gly Leu Gln
1 5 10 15
Phe Ile Gln Ser Thr Leu Pro Leu Lys Gln Glu Glu Tyr Glu Ala
20 25 30
Phe Leu Leu Lys Leu Val Gln Asn Leu Phe Ala Glu Gly Asn Asp
35 40 45
Leu Phe Arg Glu Lys Asp Tyr Lys Gln Ala Leu Val Gln Tyr Met
50 55 60
Glu Gly Leu Asn Val Ala Asp Tyr Ala Ala Ser Asp Gln Val Ala
65 70 75
Leu Pro Arg Glu Leu Leu Cys Lys Leu His Val Asn Arg Ala Ala
80 85 90
Cys Tyr Phe Thr Met Gly Leu Tyr Glu Lys Ala Leu Glu Asp Ser
95 100 105
Glu Lys Ala Leu Gly Pro Asp Ser Glu Ser Ile Arg Ala Leu Phe
110 115 120
Arg Lys Ala Arg Ala Leu Asn Glu Leu Gly Arg His Lys Glu Ala
125 130 135
Tyr Glu Cys Ser Ser Arg Cys Ser Leu Ala Leu Pro His Asp Glu
140 145 150
Ser Val Thr Gln Leu Gly Gln Gly Pro Leu Gly Ser Gly Ala Ser
155 160 165
Trp Pro Gly Gln Ser Trp Ser Pro His Arg Val Arg Lys Arg Glu
170 175 180
Trp Glu Ala Glu Cys Asp Gly Glu Glu Gly Gln Glu Asp Pro Phe
185 190 195
Asn Asp Glu Gly Asn Tyr Phe Ser Cys Glu Pro Ser Arg Ala Pro
200 205 210
Gly Trp Glu Ala Gln Arg Thr Glu Ser Gly Thr Cys Val Pro Pro
215 220 225
Gly Arg Gln Gly Gln Asp Gly Met Ala Ser Met Gly Ala Gly Trp
230 235 240
Val Gly Arg Asp Ala Ala Phe Leu Ser Trp Ala Val Ile Asn Leu
245 250 255
Met Val Leu
<210> 12
<211> 555
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4574912CD1
<220>
<221> unsure
<222> 63-65
<223> unknown or other
16/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
<400> 12
Met Glu Gly Ala Val Leu Glu Ala Gly Gly Ala Arg Cys Phe Cys
1 5 10 15
Arg Phe Gly Cys Glu Leu Ser Lys Tyr Glu Asn Pro Gly Tyr Ser
20 25 30
Ser Pro Arg Ser Asp Tyr Phe Lys Asn Tyr Met Ile Ile Ile Thr
35 40 45
Gln Asn Arg Met Ser Phe Leu Ala Asn Met Phe His Thr Met Asp
50 55 60
Cys Val Xaa Xaa Xaa Arg Tyr Ser Cys Gly Pro Thr Val Tyr Asp
65 70 75
His Ala His Leu Gly His Ala Cys Ser Tyr Val Arg Phe Asp Ile
80 85 90
Ile Arg Arg Ile Leu Thr Lys Val Phe Gly Cys Ser Ile Val Met
95 100 105
Val Met Gly Ile Thr Asp Val Asp Asp Lys Ile Ile Lys Arg Ala
110 115 120
Asn Glu Met Asn Ile Ser Pro Ala Ser Leu Ala Ser Leu Tyr Glu
125 130 135
Glu Asp Phe Lys Gln Asp Met Ala Ala Leu Lys Val Leu Pro Pro
140 145 150
Thr Val Tyr Leu Arg Val Thr Glu Asn Ile Pro Gln Ile Ile Ser
155 160 165
Phe I1e Glu Gly Ile Ile Ala Arg Gly Asn Ala Tyr Ser Thr Ala
170 175 180
Lys Gly Asn Val Tyr Phe Asp Leu Lys Ser Arg Gly Asp Lys Tyr
185 190 195
Gly Lys Leu Val Gly Val Val Pro Gly Pro Val Gly Glu Pro Ala
200 205 210
Asp Ser Asp Lys Arg His Ala Ser Asp Phe Ala Leu Trp Lys Ala
215 220 225
Ala Lys Pro Gln Glu Val Phe Trp Ala Ser Pro Trp Gly Pro Gly
230 235 240
Arg Pro Gly Trp His Ile Glu Cys Ser Ala Ile Ala Ser Met Val
245 250 255
Phe Gly Ser Gln Leu Asp Ile His Ser Gly Gly Ile Asp Leu Ala
260 265 270
Phe Pro His His Glu Asn Glu Ile Ala Gln Cys Glu Val Phe His
275 280 285
Gln Cys Glu Gln Trp Gly Asn Tyr Phe Leu His Ser Gly His Leu
290 295 300
His Ala Lys Gly Lys Glu Glu Lys Met Ser Lys Ser Leu Lys Asn
305 310 315
Tyr Ile Thr Ile Lys Asp Phe Leu Lys Thr Phe Ser Pro Asp Val
320 325 330
Phe Arg Phe Phe Cys Leu Arg Ser Ser Tyr Arg Ser Ala Ile Asp
335 340 345
Tyr Ser Asp Ser Ala Met Leu Gln Ala Gln Gln Leu Leu Leu Gly
350 355 360
Leu Gly Ser Phe Leu Glu Asp Ala Arg Ala Tyr Met Lys Gly Gln
365 370 375
Leu Ala Cys Gly Ser Val Arg Glu Ala Met Leu Trp Glu Arg Leu
380 385 390
Ser Ser Thr Lys Arg Ala Val Lys Ala Ala Leu Ala Asp Asp Phe
395 400 405
Asp Thr Pro Arg Val Val Asp Ala Ile Leu Gly Leu Ala His His
410 415 420
Gly Asn Gly Gln Leu Arg Ala Ser Leu Lys Glu Pro Glu Gly Pro
425 430 435
Arg Ser Pro Ala Val Phe Gly Ala Ile Ile Ser Tyr Phe Glu Gln
440 445 450
Phe Phe Glu Thr Val Gly Ile Ser Leu Ala Asn Gln Gln Tyr Val
455 460 465
Ser Gly Asp Gly Ser Glu Ala Thr Leu His Gly Val Val Asp Glu
470 475 480
17/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
Leu Val Arg Phe Arg Gln Lys Val Arg Gln Phe Ala Leu Ala Met
485 490 495
Pro Glu Ala Thr Gly Asp Ala Arg Arg Gln Gln Leu Leu Glu Arg
500 505 510
Gln Pro Leu Leu Glu Ala Cys Asp Thr Leu Arg Arg Gly Leu Thr
515 520 525
Ala His G1y Ile Asn Ile Lys Asp Arg Ser Ser Thr Thr Ser Thr
530 535 540
Trp Glu Leu Leu Asp Gln Arg Thr Lys Asp Gln Lys Ser Ala G1y
545 550 555
<210> 13
<211> 463
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5630806CD1
<400> 13
Met Ala Ala Ser Met Phe Tyr Gly Arg Leu Val Ala Val Ala Thr
1 5 10 15
Leu Arg Asn His Arg Pro Arg Thr Ala Gln Arg Ala Ala Ala Gln
20 25 30
Val Leu G1y Ser Ser Gly Leu Phe Asn Asn His Gly Leu Gln Val
35 40 45
Gln Gln Gln Gln Gln Arg Asn Leu Ser Leu His Glu Tyr Met Ser
50 55 60
Met Glu Leu Leu Gln Glu Ala Gly Val Ser Val Pro Lys Gly Tyr
65 70 75
Val Ala Lys Ser Pro Asp Glu Ala Tyr Ala Ile Ala Lys Lys Leu
80 85 90
Gly Ser Lys Asp Val Val I1e Lys Ala Gln Val Leu Ala Gly Gly
95 100 105
Arg Gly Lys Gly Thr Phe Glu Ser Gly Leu Lys Gly Gly Val Lys
110 115 120
Ile Val Phe Ser Pro Glu Glu Ala Lys Ala Val Ser Ser Gln Met
125 130 135
Ile Gly Lys Lys Leu Phe Thr Lys Gln Thr Gly Glu Lys Gly Arg
140 145 150
Ile Cys Asn Gln Val Leu Val Cys Glu Arg Lys Tyr Pro Arg Arg
155 160 165
Glu Tyr Tyr Phe Ala Ile Thr Met Glu Arg Ser Phe Gln Gly Pro
170 175 180
Val Leu Ile Gly Ser Ser His Gly Gly Val Asn Ile Glu Asp Val
185 190 195
Ala Ala Glu Thr Pro Glu Ala Ile Ile Lys Glu Pro Ile Asp Ile
200 205 210
Glu Glu Gly Ile Lys Lys Glu Gln Ala Leu Gln Leu Ala Gln Lys
215 220 225
Met Gly Phe Pro Pro Asn Ile Val Glu Ser Ala Ala Glu Asn Met
230 235 240
Val Lys Leu Tyr Ser Leu Phe Leu Lys Tyr Asp Ala Thr Met Ile
245 250 255
Glu Ile Asn Pro Met Val Glu Asp Ser Asp Gly Ala Val Leu Cys
260 265 270
Met Asp Ala Lys Ile Asn Phe Asp Ser Asn Ser Ala Tyr Arg Gln
275 280 285
Lys Lys Ile Phe Asp Leu Gln Asp Trp Thr Gln Glu Asp Glu Arg
290 295 300
Asp Lys Asp Ala Ala Lys Ala Asn Leu Asn Tyr Ile Gly Leu Asp
305 310 315
Gly Asn Ile Gly Cys Leu Val Asn Gly Ala Gly Leu Ala Met Ala
320 325 330
18/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
Thr Met Asp Ile Ile Lys Leu His Gly Gly Thr Pro Ala Asn Phe
335 340 345
Leu Asp Val Gly Gly Gly Ala Thr Val His Gln Val Thr Glu Ala
350 355 360
Phe Lys Leu Ile Thr Ser Asp Lys Lys Val Leu Ala Ile Leu Val
365 370 375
Asn Ile Phe Gly Gly Ile Met Arg Cys Asp Val Ile Ala Gln Gly
380 385 390
Ile Val Met Ala Val Lys Asp Leu Glu Ile Lys Ile Pro Val Val
395 400 405
Val Arg Leu Gln Gly Thr Arg Val Asp Asp Ala Lys Ala Leu Ile
410 415 420
Ala Asp Ser Gly Leu Lys Ile Leu Ala Cys Asp Asp Leu Asp Glu
425 430 435
Ala Ala Arg Met Val Val Lys Leu Ser Glu Ile Val Thr Leu Ala
440 445 450
Lys Gln Ala His Val Asp Val Lys Phe Gln Leu Pro Ile
455 460
<210> 14
<211> 399
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5854855CD1
<400> 14
Met Tyr Phe Gly Ala Val Ser Trp Leu Cys Gly Pro Arg Ala Pro
1 5 10 15
Asp Glu Val Ser Arg Arg Pro Asp Pro Arg Lys Gly Gln Leu Gly
20 25 30
Val Ala Phe Val Leu Leu Pro Pro His Ser Glu Gly Ala Arg Val
35 40 45
Phe Gly Ala Leu Gly Pro Ile Gly Pro Ser Ser Pro Gly Leu Thr
50 55 60
Leu Gly Gly Leu Ala Val Ser Glu His Arg Leu Ser Asn Lys Leu
65 70 75
Leu Ala Trp Ser Gly Val Leu Glu Trp Gln Glu Lys Arg Arg Pro
80 85 90
Tyr Ser Asp Ser Thr Ala Lys Leu Lys Arg Thr Leu Pro Cys Gln
95 100 105
Ala Tyr Val Asn Gln Gly Glu Asn Leu Glu Thr Asp Gln Trp Pro
110 115 120
Gln Lys Leu Ile Met Gln Leu Ile Pro Gln Gln Leu Leu Thr Thr
125 130 135
Leu Gly Pro Leu Phe Arg Asn Ser Gln Leu Ala Gln Phe His Phe
140 145 150
Thr Asn Arg Asp Cys Asp Ser Leu Lys Gly Leu Cys Arg Ile Met
155 160 165
Gly Asn Gly Phe Ala Gly Cys Met Leu Phe Pro His Ile Ser Pro
170 175 180
Cys Glu Val Arg Val Leu Met Leu Leu Tyr Ser Ser Lys Lys Lys
185 190 195
Ile Phe Met Gly Leu Ile Pro Tyr Asp Gln Ser Gly Phe Val Ser
200 205 210
Ala Ile Arg Gln Val Ile Thr Thr Arg Lys Gln Ala Val Gly Pro
215 220 225
Gly Gly Val Asn Ser Gly Pro Val Gln Ile Val Asn Asn Lys Phe
230 235 240
Leu Ala Trp Ser Gly Val Met Glu Trp Gln Glu Pro Arg Pro Glu
245 250 255
Pro Asn Ser Arg Ser Lys Arg Trp Leu Pro Ser His Val Tyr Val
260 265 270
Asn Gln Gly Glu Ile Leu Arg Thr Glu Gln Trp Pro Arg Lys Leu
19/3?
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
275 280 285
Tyr Met Gln Leu Ile Pro Gln Gln Leu Leu Thr Thr Leu Val Pro
290 295 300
Leu Phe Arg Asn Ser Arg Leu Val Gln Phe His Phe Thr Lys Asp
305 310 315
Leu Glu Thr Leu Lys Ser Leu Cys Arg Ile Met Asp Asn Gly Phe
320 325 330
Ala Gly Cys Val His Phe Ser Tyr Lys Ala Ser Cys Glu Ile Arg
335 340 345
Val Leu Met Leu Leu Tyr Ser Ser Glu Lys Lys Ile Phe Ile Gly
350 355 360
Leu Ile Pro His Asp Gln Gly Asn Phe Val Asn Gly Ile Arg Arg
365 370 375
Val Ile Ala Asn Gln Gln Gln Val Leu Gln Arg Asn Leu Glu Gln
380 385 390
Glu Gln Gln Gln Arg Gly Met Gly Gly
395
<210> 15
<211> 339
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5993973CD1
<400> 15
Met Val Ser Gly Cys Gln Thr Arg Ser Ile Leu Glu Tyr Leu Arg
1 5 10 15
Val Gly Gly Arg Gly Gly Gly Lys Gly Lys Gly Arg Ala Glu Gly
20 25 30
Ser Glu Lys Glu Glu Ser Arg Arg Lys Arg Arg Glu Arg Lys Gln
35 40 45
Arg Arg Glu Gly Gly Asp Gly Glu Glu Gln Asp Val Gly Asp Ala
50 55 60
Gly Arg Leu Leu Leu Arg Val Leu His Val Ser Glu Asn Pro Val
65 70 75
Pro Leu Thr Val Arg Val Ser Pro Glu Val Arg Asp Val Arg Pro
80 85 90
Tyr Ile Val Gly Ala Val Val Arg Gly Met Asp Leu Gln Pro Gly
95 100 105
Asn Ala Leu Lys Arg Phe Leu Thr Ser Gln Thr Lys Leu His Glu
110 115 120
Asp Leu Cys Glu Lys Arg Thr Ala Ala Thr Leu Ala Thr His Glu
125 130 135
Leu Arg Ala Val Lys Gly Pro Leu Leu Tyr Cys Ala Arg Pro Pro
140 145 150
Gln Asp Leu Lys Ile Val Pro Leu Gly Arg Lys Glu Ala Lys Ala
155 160 165
Lys Glu Leu Val Arg Gln Leu Gln Leu Glu Ala Glu Glu Gln Arg
170 175 180
Lys Gln Lys Lys Arg Gln Ser Val Ser Gly Leu His Arg Tyr Leu
185 190 195
His Leu Leu Asp Gly Asn Glu Asn Tyr Pro Cys Leu Val Asp Ala
200 205 210
Asp Gly Asp Val Ile Ser Phe Pro Pro Ile Thr Asn Ser Glu Lys
215 220 225
Thr Lys Val Lys Lys Thr Thr Ser Asp Leu Phe Leu Glu Val Thr
230 235 240
Ser Ala Thr Ser Leu Gln Ile Cys Lys Asp Val Met Asp Ala Leu
245 250 255
Ile Leu Lys Met Ala Glu Met Lys Lys Tyr Thr Leu Glu Asn Lys
260 265 270
Glu Glu Gly Ser Leu Ser Asp Thr Glu Ala Asp Ala Val Ser Gly
275 280 285
20/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
Gln Leu Pro Asp Pro Thr Thr Asn Pro Ser Ala Gly Lys Asp Gly
290 295 300
Pro Ser Leu Leu Val Val Glu Gln Val Arg Val Val Asp Leu Glu
305 310 315
Gly Ser Leu Lys Val Val Tyr Pro Ser Lys Ala Asp Leu Ala Thr
320 325 330
Ala Pro Pro His Val Thr Val Val Arg
335
<210> 16
<211> 3902
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1806212CB1
<400> 16
ggatgattgc ctcagcaggt gtgaagcgtg tgctttagtt tcgtgggagg cctggcatcc 60
ccgagaggga ggggaaaggt aaccactcct ttgtggaggt cgccagggtc attgtcgtgg 120
atttgcacag tcggctgggc ggtgcaatgg cggaaagaaa aggaacagcc aaagtggact 180
ttttgaagaa gattgagaaa gaaatccaac agaaatggga tactgagaga gtgtttgagg 240
tcaatgcatc taatttagag aaacagacca gcaagggcaa gtattttgta accttcccat 300
acccatatat gaatggacgc cttcatttgg gacacacgtt ttctttatcc aaatgtgagt 360
ttgctgtagg gtaccagcga ttgaaaggaa aatgttgtct gtttcccttt ggcctgcact 420
gtactggaat gcctattaag gcatgtgctg ataagttgaa aagagaaata gagctgtatg 480
gttgcccccc tgattttcca gatgaagaag aggaagagga agaaaccagt gttaaaacag 540
aagatataat aattaaggat aaagctaaag gaaaaaagag taaagctgct gctaaagctg 600
gatcttctaa ataccagtgg ggcattatga aatcccttgg cctgtctgat gaagagatag 660
taaaattttc tgaagcagaa cattggcttg attatttccc gccactggct attcaggatt 720
taaaaagaat gggtttgaag gtagactggc gtcgttcctt catcaccact gatgttaatc 780
cttactatga ttcatttgtc agatggcaat ttttaacatt aagagaaaga aacaaaatta 840
aatttgggaa gcggtataca atttactctc cgaaagatgg acagccttgc atggatcatg 900
atagacaaac tggagagggt gttggacctc aggaatatac tttactcaaa ttgaaggtgc 960
ttgagccata cccatctaaa ttaagtggcc tgaaaggtaa aaatattttc ttggtggctg 1020
ctactctcag acctgagacc atgtttgggc agacaaattg ttgggttcgt cctgatatga 1080
agtacattgg atttgagacg gtgaatggtg atatattcat ctgtacccaa aaagcagcca 1140
ggaatatgtc ataccagggc tttaccaaag acaatggcgt ggtgcctgtt gttaaggaat 1200
taatggggga ggaaattctt ggtgcatcac tttctgcacc tttaacatca tacaaggtga 1260
tctatgttct cccaatgcta actattaagg aggataaagg cactggtgtg gttacaagtg 1320
ttccttccga ctcccctgat gatattgctg ccctcagaga cttgaagaaa aagcaagcct 1380
tacgagcaaa atatggaatt agagatgaca tggtcttgcc atttgagccg gtgccagtca 1440
ttgaaatccc aggttttgga aatctttctg ctgtaaccat ttgtgatgag ttgaaaattc 1500
agagccagaa tgaccgggaa aaacttgcag aagcaaagga gaagatatat ctaaaaggat 1560
tttatgaggg tatcatgttg gtggatggat ttaaaggaca gaaggttcaa gatgtaaaga 1620
agactattca gaaaaagatg attgacgctg gagatgcact tatttacatg gaaccagaga 1680
aacaagtgat gtccaggtcg tcagatgaat gtgttgtggc tctgtgtgac cagtggtact 1740
tggattatgg agaagagaat tggaagaaac agacatctca gtgcttgaag aacctggaaa 1800
cattctgtga ggagaccagg aggaattttg aagccacctt aggttggcta caagaacatg 1860
cttgctcaag aacttatggt ctaggcactc acctgccttg ggatgagcag tggctgattg 1920
aatcactttc tgactccact atttacatgg cattttacac agttgcacac ctattgcagg 1980
ggggtaactt gcatggacag gcagagtctc cgctgggcat tagaccgcaa cagatgacca 2040
aggaagtttg ggattatgtt ttcttcaagg aggctccatt tcctaagact cagattgcaa 2100
aggaaaaatt agatcagtta aagcaggagt ttgaattctg gtatcctgtt gatcttcgcg 2160
tctctggcaa ggatcttgtt ccaaatcatc tttcatatta cctttataat catgtggcta 2220
tgtggccgga acaaagtgac aaatggccta cagctgtgag agcaaatgga catctcctcc 2280
tgaactctga gaagatgtca aaatccacag gcaacttcct cactttgacc caagctattg 2340
acaaattttc agcagatgga atgcgtttgg ctctggctga tgctggtgac actgtagaag 2400
atgccaactt tgtggaagcc atggcagatg caggtattct ccgtctgtac acctgggtag 2460
agtgggtgaa agaaatggtt gccaactggg acagcctaag aagtggtcct gccagcactt 2520
tcaatgatag agtttttgcc agtgaattga atgcaggaat tataaaaaca gatcaaaact 2580
atgaaaagat gatgtttaaa gaagctttga aaacagggtt ttttgagttt caggccgcaa 2640
aagataagta ccgtgaattg gctgtggaag ggatgcacag agaacttgtg ttccggttta 2700
ttgaagttca gacacttctc ctcgctccat tctgtccaca tttgtgtgag cacatctgga 2760
cactcctggg aaagcctgac tcaattatga atgcttcatg gcctgtggca ggtcctgtta 2820
21/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
atgaagtttt aatacactcc tcacagtatc ttatggaagt aacacatgac cttagactac 2880
gactcaagaa ctatatgatg ccagctaaag ggaagaagac tgacaaacaa cccctgcaga 2940
agccctcaca ttgcaccatc tatgtggcaa agaactatcc accttggcaa cataccaccc 3000
tgtctgttct acgtaaacac tttgaggcca ataacggaaa actgcctgac aacaaagtca 3060
ttgctagtga actaggcagt atgccagaac tgaagaaata catgaagaaa gtcatgccat 3120
ttgttgccat gattaaggaa aatctggaga agatggggcc tcgtattctg gatttgcaat 3180
tagaatttga tgaaaaggct gtgcttatgg agaatatagt ctatctgact aattcgcttg 3240
agctagaaca catagaagtc aagtttgcct ccgaagcaga agataaaatc agggaagact 3300
gctgtcctgg gaaaccactt aatgttttta gaatagaacc tggtgtgtcc gtttctctgg 3360
tgaatcccca gccatccaat ggccacttct caaccaaaat tgaaatcagg caaggagata 3420
actgtgattc cataatcagg cgtttaatga aaatgaatcg aggaattaaa gacctttcca 3480
aagtgaaact gatgagattt gatgatccac tgttggggcc tcgacgagtt cctgtcctgg 3540
gaaaggagta caccgagaag acccccattt ctgagcatgc tgttttcaat gtggacctca 3600
tgagcaagaa aattcatctg actgagaatg ggataagggt ggatattggc gatacaataa 3660
tctatctggt tcattaaact catgcacatt ggagatttat cctggtttct taggaatact 3720
actactctga ttgtgtctac tgattggcta tcagaacctt aggctggacc taaatagatt 3780
gatttcattt ctaaccatcc aattctgcat gtattcataa ttctatcaag tcatctttga 3840
ttcctggacc taataaattt tttttccctg ttctttgggt gtccaagaaa aaaaaaaaaa 3900
as 3902
<210> 17
<211> 3317
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2083883CB1
<400> 17
catggcctga ctcgggacag ctcagagcag ggcagaactg gggacactct gggccggcct 60
tctgcctgca tggacgctct gaagccaccc tgtctctgga ggaaccacga gcgagggaag 120
aaggacaggg actcgtgtgg caggaagaac tcagagccgg gaagccccca ttcactagaa 180
gcactgagag atgcggcccc ctcgcagggt ctgaatttcc tgctgctgtt cacaaagatg 240
ctttttatct ttaacttttt gttttcccca cttccgaccc cggcgttgat ctgcatcctg 300
acatttggag ctgccatctt cttgtggctg atcaccagac ctcaacccgt cttacctctt 360
cttgacctga acaatcagtc tgtgggaatt gagggaggag cacggaaggg ggtttcccag 420
aagaacaatg acctaacaag ttgctgcttc tcagatgcca agactatgta tgaggttttc 480
caaagaggac tcgctgtgtc tgacaatggg ccctgcttgg gatatagaaa accaaaccag 540
ccctacagat ggctatctta caaacaggtg tctgatagag cagagtacct gggttcctgt 600
ctcttgcata aaggttataa atcatcacca gaccagtttg tcggcatctt tgctcagaat 660
aggccagagt ggatcatctc cgaattggct tgttacacgt actctatggt agctgtacct 720
ctgtatgaca ccttgggacc agaagccatc gtacatattg tcaacaaggc tgatatcgcc 780
gtggtgatct gtgacacacc ccaaaaggca ttggtgctga tagggaatgt agagaaaggc 840
ttcaccccga gcctgaaggt gatcatcctt atggacccct ttgatgatga cctgaagcaa 900
agaggggaga agagtggaat tgagatctta tccctatatg atgctgagaa cctaggcaaa 960
gagcacttca gaaaacctgt gcctcctagc ccagaagacc tgagcgtcat ctgcttcacc 1020
agtgggacca caggtgaccc caaaggagcc atgataaccc atcaaaatat tgtttcaaat 1080
gctgctgcct ttctcaaatg tgtggagcat gcttatgagc ccactcctga tgatgtggcc 1140
atatcctacc tccctctggc tcatatgttt gagaggattg tacaggctgt tgtgtacagc 1200
tgtggagcca gagttggatt cttccaaggg gatattcggt tgctggctga cgacatgaag 1260
actttgaagc ccacattgtt tcccgcggtg cctcgactcc ttaacaggat ctacgataag 1320
gtacaaaatg aggccaagac acccttgaag aagttcttgt tgaagctggc tgtttccagt 1380
aaattcaaag agcttcaaaa gggtatcatc aggcatgata gtttctggga caagctcatc 1440
tttgcaaaga tccaggacag cctgggcgga agggttcgtg taattgtcac tggagctgcc 1500
cccatgtcca cttcagtcat gacattcttc cgggcagcaa tgggatgtca ggtgtatgaa 1560
gcttatggtc aaacagaatg cacaggtggc tgtacattta cattacctgg ggactggaca 1620
tcaggtcacg ttggggtgcc cctggcttgc aattacgtga agctggaaga tgtggctgac 1680
atgaactact ttacagtgaa taatgaagga gaggtctgca tcaagggtac aaacgtgttc 1740
aaaggatacc tgaaggaccc tgagaagaca caggaagccc tggacagtga tggctggctt 1800
cacacaggag acattggtcg ctggctcccg aatggaactc tgaagatcat cgaccgtaaa 1860
aagaacattt tcaagctggc ccaaggagaa tacattgcac cagagaagat agaaaatatc 1920
tacaacagga gtcaaccagt gttacaaatt tttgtacacg gggagagctt acggtcatcc 1980
ttagtaggag tggtggttcc tgacacagat gtacttccct catttgcagc caagcttggg 2040
gtgaagggct cctttgagga actgtgccaa aaccaagttg taagggaagc cattttagaa 2100
22/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
gacttgcaga aaattgggaa agaaagtggc cttaaaactt ttgaacaggt caaagccatt 2160
tttcttcatc cagagccatt ttccattgaa aatgggctct tgacaccaac attgaaagca 2220
aagcgaggag agctttccaa atactttcgg acccaaattg acagcctgta tgagcacatc 2280
caggattagg ataaggtact taagtacctg ccggcccact gtgcactgct tgtgagaaaa 2340
tggattaaaa actattctta catttgtttt gcctttcctc ctattttttt ttaacctgtt 2400
aaactctaaa gccatagctt ttgttttata ttgagacata taatgtgtaa acttagttcc 2460
caaataaatc aatcctgtct ttcccatctt cgatgttgct aatattaagg cttcagggct 2520
acttttatca acatgcctgt cttcaagatc ccagtttatg ttctgtgtcc ttcctcatga 2580
tttccaacct taatactatt agtaaccaca agttcaaggg tcaaagggac cctctgtgcc 2640
ttcttctttg ttttgtgata aacataactt gccaacagtc tctatgctta tttacatctt 2700
ctactgttca aactaagaga tttttaaatt ctgaaaaact gcttacaatt catgttttct 2760
agccactcca caaaccacta aaattttagt tttagcctat cactcatgtc aatcatatct 2820
atgagacaaa tgtctccgat gctcttctgc gtaaattaaa ttgtgtactg aagggaaaag 2880
tttgatcata ccaaacattt cctaaactct ctagttagat atctgacttg ggagtattaa 2940
aaattgggtc tatgacatac tgtccaaaag gaatgctgtt cttaaagcat tatttacagt 3000
aggaactggg gagtaaatct gttccctaca gtttgctgct gagctggaag ctgtggggga 3060
aggagttgac aggtgggccc agtgaacttt tccagtaaat gaagcaagca ctgaataaaa 3120
acctcctgaa ctgggaacaa agatctacag gcaagcaaga tgcccacaca acaggcttat 3180
tttctgtgaa ggaaccaact gatctccccc acccttggat tagagttcct gctctacctt 3240
acccacagat aacacatgtt gtttctactt gtaaatgtaa agtctttaaa ataaactatt 3300
acagataaaa aaaaaaa 3317
<210> 18
<211> 1928
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2454288CB1
<400> 18
cccacgcgtc cgcgacacac catgccgact gtcagcgtga agcgtgatct gctcttccaa 60
gccctgggcc gcacctacac tgacgaagaa tttgatgaac tatgttttga atttggtctg 120
gagcttgatg aaattacatc tgagaaggaa ataataagta aagaacaagg taatgtaaag 180
gcagcaggag cctctgatgt tgttctttac aaaattgacg tccctgccaa tagatatgat 240
ctcctgtgtc tggaaggatt ggttcgagga cttcaggtct tcaaagaaag gataaaggct 300
ccagtgtata aacgggtaat gcctgatgga aaaatccaga aattgattat cacagaagag 360
acagctaaga tacgtccttt tgcggtagca gcagttctcc gtaatataaa gtttactaaa 420
gatcgatatg acagcttcat tgaacttcag gagaaattac atcagaatat ttgcaggaaa 480
agagcactgg ttgccattgg tacccatgat ttggacactt tgtcgggccc atttacttat 540
actgcaaagc gtccttcaga tatcaaattc aagcctctaa ataagaccaa ggagtataca 600
gcctgtgaac tgatgaacat atacaagact gacaatcacc tgaaacatta tttacatatc 660
attgaaaaca aacccctgta tccagttatc tatgatagca atggtgtcgt cctttcaatg 720
cctcccatca tcaatgggga tcattccaga ataacagtaa atactagaaa tatttttatt 780
gaatgcacgg gaactgactt tactaaggca aaaatagttc ttgatattat tgtcaccatg 840
ttcagtgaat attgtgagaa tcaatttacg gtcgaagctg ctgaagtggt ttttcctaat 900
ggaaaatcac atacctttcc agaattagct taccgaaagg agatggtgag agctgaccta 960
attaacaaaa aagttggaat cagagaaact ccagaaaatc ttgccaaact tctgaccagg 1020
atgtatttaa aatcagaagt cataggtgat gggaatcaga ttgagattga aatccctcca 1080
accagagctg acattatcca tgcatgtgat attgtagaag atgcagctat tgcttatgga 1140
tataacaaca ttcagatgac tctcccgaaa acttacacca tagctaatca atttcctctt 1200
aataagctca ctgaacttct ccgacatgac atggcagccg ctggcttcac tgaagcactt 1260
acctttgccc tgtgctccca agaagatatt gctgataaac taggtgtgga tatctctgca 1320
acaaaggcag tccacataag taatcctaaa acagctgaat ttcaggtggc acgcactacc 1380
cttcttcctg gcctcctgaa gaccatagca gcaaatcgta agatgcccct tccactgaaa 1440
ctgtttgaaa tctctgacat tgtaataaaa gattctaata cagatgtagg tgcaaaaaac 1500
tacagacatc tctgtgctgt ttattacaac aagaatcctg ggtttgagat cattcatggg 1560
ctgctggaca gaattatgca gttgctcgat gtgcctcctg gtgaagacaa ggggggatat 1620
gtgatcaaag catcagaagg gcctgctttc ttccccgggc gatgtgcaga gatctttgcc 1680
aggggtcaaa gcgtcgggaa gcttggggtc cttcatcctg acgttatcac caaatttgag 1740
ctgaccatgc cctgctcctc cctagaaatc aatattggac cctttttgtg aagattggtc 1800
tctgtggtgt gattctcttc ccaggtgtcc ctttctcctc ccctagtgtc cttaagtcct 1860
cctccacagg gaacatctat ttgggctttg atgtttaata aagtagaaag cactgtcaaa 1920
aaaaaaaa 1928
23/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
<210> 19
<211> 2122
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1513539CB1
<400> 19
caggtcacac gcagccagtc agcattagag ctgagatgcc aagctctcag ccacacatgt 60
gcactcacct ggaacatctg tggagctgtt agaatcgctg ccacttttca tgaaactgaa 120
gtgcatcttt gggtttgcaa ctaaagaaac cagctgctac aatgtcacta acataggatt 180
caaaagccct tcggatttct ggcagtctgt gcatagcacc ctgcctcggg agttggctcc 240
ttgtctagta tttaatacat ccccaaactt ggctttattt tcagctgcct ttgccttcat 300
tgtggtgaaa gatagtgcgg gtgactcaga tgtggtggtg caggagctca agtccatggt 360
ggccaccaag atcgccaaat atgctgtgcc tgatgagatc ctggtggtga aacgtcttcc 420
aaaaaccagg tctgggaagg tcatgcggcg gctcctgagg aagatcatca ctagtgaggc 480
ccaggagctg ggagacacta ccaccttgga ggaccccagc atcatcgcag agatcctgag 540
tgtctaccag aagtgcaagg acaagcaggc tgctgctaag tgagctggca ccttgtgggg 600
ctcttgggat gggcgggcac ccaagccctg gcttgtcctt cccagaaggt acccctgagg 660
ttggcgtctt cctacgtccc agaagcagcc cccaccccac acatgaccca caccgccctc 720
acgtgaagct gggctgagag ccctttctcc catccattgg aggtcccagg agtgtcaccc 780
atggagaggc tatgcgacat ggctagggct ggttctgcca tctgagtttg gtttcctgga 840
atgaaaaggc attgccatct ccattcctct gccctcttga gccagcacag gaaggtgagg 900
ccctgggata gcgcgcctgc tcagataaca cagagctagt tagctagtag caaccgtgtt 960
ttctccagat ctgtctagat acaaaggtca gaaatcttat ttttatactt ttatattgtg 1020
gaagaacagc atgcaacact cacatgtagt gtgtggattt acttgaacat gttcttttta 1080
acatgtagtt atgaaaatct ccttttttgc ctctactggt gaggaaacat gaggatcaga 1140
ggccacattt ttaattattg ttagtgtatt tggaagtctg aattggagat gtttgtacct 1200
ctgtctaaac agttcccttg agaacttcca agcctccggc atcttttcct ggtgagtgtt 1260
tctcctgtgc ttggttgtgt ataatggagc taactcctaa gcggtggggt gaatgtggcc 1320
gccttagttc tgaagctact ccagttatgt tctgtttctt caagctgtga tccagaaaga 1380
tttttgtgcc cccagatgcc tcttgatagg agaggcaaca tactccaaat agttgggttc 1440
ttcagggaag ctattagaaa ctcaggtgac ttgttagagc actaacttgg tcagagccaa 1500
atcctggcaa acgctgcctg accttcactc tgtggttggg gcagtgagaa ccactgaggt 1560
ccaatgatga gacttggagg tctggatcca gtctctcttt gttttaatgt gacttaggtg 1620
ctgtcaacat tagcaagata atggaaatca cgacgccagt gggtgcttac ctccctgcta 1680
ggcatgcagg ggctggcggt tggcagggga aggaggccca gtgagccggg tcccttaggg 1740
gagggagagt ttgtcctctt tgccccacag tctacccttc agggccttgt ggcagtgcca 1800
gtgttcgggg ggtgtctggg ccactgagta cccactcggt cgtggttgtg ctggcctctt 1860
gggtgagtga acctgtgaag cccaggaggt ggtgttggct gcagggtaca caaatactga 1920
gtggtggtct tttgttacag gcttagcaac aaagctgtgc cctgggcatg gggggctgta 1980
gtgtagctac agttgtgcgt ttgtgaaatg gcttagcttt ccatgttgct gagaggaacc 2040
tggacatggt cccgggcatc tgaatgatct gtaggggagg gagttcaaat aaagctttat 2100
tttgttcatt ttcaaaaaaa as 2122
<210> 20
<211> 2357
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2148623CB1
<400> 20
gctctttcag ggctgagcca tcctgcgtgt cttgcgctcg gtggaaatgc ccagccgagg 60
gacgcgacca gaggacagct ctgtgctgat ccccaccgac aattcgaccc cacacaagga 120
ggatctaagc agcaagatta aagaacaaaa aattgtggtg gatgaacttt ctaaccttaa 180
gaagaatagg accgaacaga aatgctgtct gagagcaaga atatattgga tgaactgaaa 240
aaagaatacc aagaaataga aaacttagac aagaccaaaa tcaagaaata gtcaacctga 300
tttcacataa caatgtgtgg catttgttgt tctgtaaact tttctgctga gcatttcagt 360
caagatttaa aagaggactt actatataat cttaaacagc ggggacccaa tagtagtaaa 420
caattgttaa agtctgatgt taactaccag tgtttatttt ctgctcacgt cctacacttg 480
24/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/LJS00/19980
aggggtgttt tgactaccca gcctgtggaa gatgaaagag gcaatgtgtt tctatggaat 540
ggagaaattt ttagtggaat aaaggttgaa gctgaagaga atgacactca aattttgttt 600
aattatcttt cctcctgtaa gaatgaatct gagattttgt cactcttctc agaagtacaa 660
ggtccctggt catttatata ttatcaagca tctagtcatt atttatggtt tggtagggat 720
ttttttggtc gccgtagctt gctttggcat tttagtaatt tgggcaagag tttctgcctc 780
tcttcagttg gcacccaaac atctggattg gcaaatcagt ggcaagaagt tccagcatct 840
ggacttttca gaattgatct taagtctact gtcatttcca gatgcattat tttacaactg 900
tatccttgga aatatatttc tagggagaat attattgaag aaaatgttaa tagcctgagt 960
caaatttcag cagacttacc agcatttgta tcagtggtag caaatgaagc caaactgtat 1020
cttgaaaaac ctgttgttcc tttaaatatg atgttgccac aagctgcatt ggagactcat 1080
tgcagtaata tttccaatgt gccacctaca agagagatac ttcaagtctt tcttactgat 1140
gtacacatga aggaagtaat tcagcagttc attgatgtcc tgagtgtagc agtcaagaaa 1200
cgtgtcttgt gtttacctag ggatgaaaac ctgacagcaa atgaagtttt gaaaacgtgt 1260
gataggaaag caaatgttgc aatcctgttt tctgggggca ttgattccat ggttattgca 1320
acccttgctg accgtcatat tcctttagat gaaccaattg atcttcttaa tgtagctttc 1380
atagctgaag aaaagaccat gccaactacc tttaacagag aagggaataa acagaaaaat 1440
aaatgtgaaa taccttcaga agaattctct aaagatgttg ctgctgctgc tgctgacagt 1500
cctaataaac atgtcagtgt accagatcga atcacaggaa gggcgggact aaaggaacta 1560
caagctgtta gcccttcccg aatttggaat tttgttgaaa ttaatgtttc tatggaagaa 1620
ctgcagaaat taagaagaac tcgaatatgt cacttaattc ggccattgga tacagttttg 1680
gatgatagca ttggctgtgc agtctggttt gcttctagag gaattggttg gttagtggcc 1740
caggaaggag tgaaatccta tcagagcaat gcaaaggtag ttctcactgg aattggtgca 1800
gatgagcaac ttgcaggtta ttctcgtcat cgtgtccgct ttcagtcgca tgggctggaa 1860
ggattgaata aggaaataat gatggaactg ggtcgaattt cttctagaaa tcttggtcgt 1920
gatgacagag ttattggtga tcatggaaaa gaagcaagat ttcctttcct ggatgaaaat 1980
gttgtctcct ttctaaattc tctgccgatt tgggaaaaag caaacttgac tttaccccga 2040
ggaattggtg aaaaattact tttacgcctt gcagctgtgg aacttggtct tacagcctct 2100
gctcttctgc ccaaacgggc catgcagttt ggatcaagaa ttgcaaaaat ggaaaaaatt 2160
aatgaaaagg catctgataa atgtggacgg ctccaaatca tgtccttaga aaatctttct 2220
attgaaaagg agactaaatt gtaatgtgat tcacaatgta acaatataaa aataagtttt 2280
tatataatta tataaaagta agatactctg ctgctttact attgtataat atagtagttt 2340
taaagttcaa aaaaaaa 2357
<210> 21
<211> 2136
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2579405CB1
<400> 21
ccgtccactt ggcgagtgag acgctgatgg gaggatggac atactggtgt ctgagtgctc 60
cgcgcggctg ctgcagcagg aagaagagat taaatctctg actgctgaaa ttgaccggtt 120
gaaaaactgt ggctgtttag gagcttctcc aaatttggag cagttacaag aagaaaattt 180
aaaattaaag tatcgactga atattcttcg aaagagtctt caggcagaaa ggaacaaacc 240
aactaaaaat atgattaaca ttattagccg cctacaagag gtctttggtc atgcaattaa 300
ggctgcatat ccagatttgg aaaatcctcc tctgctagtg acaccaagtc agcaggccaa 360
gtttggggac tatcagtgta atagtgctat gggtatttct cagatgctca aaaccaagga 420
acagaaagtt aatccaagag aaattgctga aaacattacc aaacacctcc cagacaatga 480
atgtattgaa aaagttgaaa ttgctggtcc tggttttatt aatgtccact taagaaagga 540
ttttgtatca gaacaattga ccagtcttct agtgaatgga gttcaactac ctgctctggg 600
agagaataaa aaggttatag ttgacttttc ctcccctaat atagctaaag agatgcatgt 660
aggccacctg aggtcaacta tcataggaga gagtataagc cgcctctttg aatttgcagg 720
gtatgacgtg ctcaggttaa atcatgtagg agactggggg acccagtttg gcatgctcat 780
cgctcacctg caagacaaat ttccagatta tctaacagtt tcacctccta ttggggatct 840
tcaggtcttt tataaggaat ctaagaagag gtttgatact gaggaggaat ttaagaagcg 900
agcatatcag tgtgtagttc tgctccaggg taaaaaccca gatattacaa aagcttggaa 960
gcttatctgt gatgtctccc gccaagagtt aaataaaatc tatgatgcat tggacgtctc 1020
tttaatagag agaggggaat ccttctatca agataggatg aatgatattg taaaggaatt 1080
tgaagataga ggatttgtgc aggtggatga tggcagaaag attgtatttg tcccagggtg 1140
ttccatacca ttaaccatag taaaatcaga tggaggttat acctatgata catctgacct 1200
ggctgctatt aaacaaagac tatttgagga aaaagcagat atgattatct atgttgtgga 1260
caatggacaa tctgtgcact tccagacaat atttgctgct gctcaaatga ttggttggta 1320
25132
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
tgaccctaaa gtaactcgag tcttccatgc tggatttggt gtggtgctag gggaagacaa 1380
gaaaaagttt aaaacacgtt cgggtgaaac agtgcgcctc atggatcttc tgggagaagg 1440
actaaaacga tccatggaca agttgaagga aaaagaaaga gacaaggtct taactgcaga 1500
ggaattgaat gctgctcaga catccgttgc atatggctgc atcaaatatg ctgacctttc 1560
ccataaccgg ttgaatgact acatcttctc ctttgacaaa atgctagatg acagaggaaa 1620
tacagctgct tacttgttgt atgccttcac tagaatcagg tctattgcac gtctggccaa 1680
tattgatgaa gaaatgctcc aaaaagctgc tcgagaaacc aagattcttt tggatcatga 1740
gaaggaatgg aaactaggcc ggtgcatttt acggttccct gagattctgc aaaagatttt 1800
agatgactta tttctccaca ctctctgtga ttatatatat gagctggcaa ctgctttcac 1860
agagttctat gatagctgct actgtgtgga gaaagataga cagactggaa aaatattgaa 1920
ggtgaacatg tggcgtatgc tgctatgtga agcagtagct gctgtcatgg ccaaggggtt 1980
tgatatcctg ggaataaaac ctgtccaaag gatgtaatcc ttcataggtt tgaacactgt 2040
gtgtttttac caaagtggcc attggcactg tttgcttttt tacaatcatg tggacacaag 2100
cataagtaaa gaaaatttgt caaccaaaaa aaaaaa 2136
<210> 22
<211> 2480
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2662427CB1
<400> 22
ggaacgggtt tggcgtgtgg gacgcagctg cctctgtact ggggagtcac ggagtggccg 60
ggctccaggg acatggcggc ggcctctgcg gtgtcggtgc tgctggtggc ggcggagagg 120
aaccggtggc atcgtctccc gagcctgctc ctgccgccga ggacatgggt gtggaggcaa 180
agaaccatga agtacacaac agccacagga agaaacatta ccaaggtcct cattgcaaac 240
agaggagaaa ttgcctgcag ggtgatgcgc acagccaaaa aactgggtgt acagactgtg 300
gcggtttata gtgaggctga cagaaattcc atgcatgtag atatggcaga tgaagcatat 360
tccatcggcc ccgctccctc ccagcagagc tacctatcta tggagaaaat cattcaagtg 420
gccaagacct ctgctgcaca ggctatccat ccaggatgcc gttttctttc agaaaacatg 480
gaatttgctg aactttgtaa gcaagaagga attattttta taggccctcc tccatctgca 540
attagagaca tgggtataaa gagcacatcc aaatccataa tggctgctgc tggagtacct 600
gttgtggagg gttatcatgg tgaggaccaa tcagaccagt gcctgaagga acacgccagg 660
agaattggct atcctgtcat gattaaagcc gtccggggtg gaggaggaaa aggaatgagg 720
attgttagat cagaacaaga atttcaagaa cagttagagt cagcacggag agaagctaag 780
aagtctttca atgatgatgc tatgctgatc gagaagtttg tagacacacc gaggcatgta 840
gaagtccagg tgtttggtga tcaccatggc aatgctgtgt acttgtttga aagagactgt 900
agtgtgcaga ggcgacatca gaagatcatt gaggaggccc cagcgcctgg tattaaatct 960
gaagtaagaa aaaagctggg agaagctgca gtcagagctg ctaaagctgt aaattatgtt 1020
ggagcaggga ctgtggagtt tattatggac tcaaaacata atttctgttt catggagatg 1080
aatacaaggc tgcaagtgga acatcctgtt actgagatga tcacaggaac tgacttggtg 1140
gagtggcagc ttagaattgc agcaggagag aagattcctt tgagccagga agaaataact 1200
ctgcagggcc atgccttcga agctagaata tatgcagaag atcctagcaa taacttcatg 1260
cctgtggcag gcccattagt gcacctctct actcctcgag cagacccttc caccaggatt 1320
gaaactggag tacggcaagg agacgaagtt tccgtgcatt atgaccccat gattgcgaag 1380
ctggtcgtgt gggcagcaga tcgccaggcg gcattgacaa aactgaggta cagccttcgt 1440
cagtacaata ttgttggact gcccaccaac attgacttct tactcaacct gtctggccac 1500
ccagagtttg aagctgggaa cgtgcacact gatttcatcc ctcaacacca caaacagttg 1560
ttgctcagtc ggaaggctgc agccaaagag tctttatgcc aggcagccct gggtctcatc 1620
ctcaaggaga aagccatgac cgacactttc actcttcagg cacatgatca attctctcca 1680
ttttcgtcta gcagtggaag aagactgaat atctcgtata ccagaaacat gactcttaaa 1740
gatggtaaaa acaatgtagc catagctgta acgtataacc atgatgggtc ttatagcatg 1800
cagattgaag ataaaacttt ccaagtcctt ggtaatcttt acagcgaggg agactgcact 1860
tacctgaaat gttctgttaa tggagttgct agtaaagcga agctgattat cctggaaaac 1920
actatttacc tattttccaa ggaaggaagt attgagattg acattccagt ccccaaatac 1980
ttatcttctg tgagctcaca agaaactcag ggcggcccct tagctcctat gactggaacc 2040
attgaaaagg tgtttgtcaa agctggagac aaagtgaaag cgggagattc cctcatggtt 2100
atgatcgcca tgaagatgga gcataccata aagtctccaa aggatggcac agtaaagaaa 2160
gtgttctaca gagaaggtgc tcaggccaac agacacactc ctttagtcga gtttgaggag 2220
gaagaatcag acaaaaggga atcggaataa actccagcaa ggaaatggcc agttaagtag 2280
tgtcttctct ctccaccaaa aagaggaagt gcctccagct tttctggggg tctcataaag 2340
agcagtttta ctaaatgatt gtatgcttat gctgaacacc tttcatattg gagaatcatg 2400
26/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
catttgggtc actaattatc tcaaaatatt tcatactaat aaagttgaat tattttttat 2460
tggaagccaa aaaaaaaaaa 2480
<210> 23
<211> 2254
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2844928CB1
<400> 23
ggccgggacg cagggcaaag cgagccatgg ctgtctacgt cgggatgctg cgcctgggga 60
ggctgtgcgc cgggagctcg ggggtgctgg gggcccgggc cgccctctct cggagttggc 120
aggaagccag gttgcagggt gtccgcttcc tcagttccag agaggtggat cgcatggtct 180
ccacgcccat cggaggcctc agctacgttc aggggtgcac caaaaagcat cttaacagca 240
agactgtggg ccagtgcctg gagaccacag cacagagggt cccagaacga gaggccttgg 300
tcgtcctcca tgaagacgtc aggttgacct ttgcccaact caaggaggag gtggacaaag 360
ctgcttctgg cctcctgagc attggcctct gcaaaggtga ccggctgggc atgtggggac 420
ctaactccta tgcatgggtg ctcatgcagt tggccaccgc ccaggcgggc atcattctgg 480
tgtctgtgaa cccagcctac caggctatgg aactggagta tgtcctcaag aaggtgggct 540
gcaaggccct tgtgttcccc aagcaattca agacccagca atactacaac gtcctgaagc 600
agatctgtcc agaagtggag aatgcccagc caggggcctt gaagagtcag aggctcccag 660
atctgaccac agtcatctcg gtggatgccc ctttgccggg gaccctgctc ctggatgaag 720
tggtggcggc tggcagcaca cggcagcatc tggaccagct ccaatacaac cagcagttcc 780
tgtcctgcca tgaccccatc aacatccagt tcacctcggg gacaacaggc agccccaagg 840
gggccaccct ctcccactac aacattgtca acaactccaa cattttagga gagcgcctga 900
aactgcatga gaagacacca gagcagttgc ggatgatcct gcccaacccc ctgtaccatt 960
gcctgggttc cgtggcaggc acaatgatgt gtctgatgta cggtgccacc ctcatcctgg 1020
cctctcccat cttcaatggc aagaaggcac tggaggccat cagcagagag agaggcacct 1080
tcctgtatgg tacccccacg atgttcgtgg acattctgaa ccagccagac ttctccagtt 1140
atgacatctc gaccatgtgt ggaggtgtca ttgctgggtc ccctgcacct ccagagttga 1200
tccgagccat catcaacaag ataaatatga aggacctggt ggttgcttat ggaaccacag 1260
agaacagtcc cgtgacattc gcgcacttcc ctgaggacac tgtggagcag aaggcagaaa 1320
gcgtgggcag aattatgcct cacacggagg cccggatcat gaacatggag gcagggacgc 1380
tggcaaagct gaacacgccc ggggagctgt gcatccgagg gtactgcgtc atgctgggct 1440
actggggtga gcctcagaag acagaggaag cagtggatca ggacaagtgg tattggacag 1500
gagatgtcgc cacaatgaat gagcagggct tctgcaagat cgtgggccgc tctaaggata 1560
tgatcatccg gggtggtgag aacatctacc ccgcagagct cgaggacttc tttcacacac 1620
acccgaaggt gcaggaagtg caggtgaggc acttggctca ggtgagcccc cagaaacaag 1680
aaacacacat gaacacggtg atgtctgata tttttctttg gccctggaac gtggtgggag 1740
tgaaggacga tcggatgggg gaagagattt gtgcctgcat tcggctgaag gacggggagg 1800
agaccacggt ggaggagata aaagctttct gcaaagggaa gatctctcac ttcaagattc 1860
cgaagtacat cgtgtttgtc acaaactacc ccctcaccat ttcaggaaag atccagaaat 1920
tcaaacttcg agagcagatg gaacgacatc taaatctgtg aataaagcag caggcctgtc 1980
ctggccggtt ggcttgactc tctcctgtca gaatgcaacc tggctttatg cacctagatg 2040
tccccagcac ccagttctga gccaggcaca tcaaatgtca aggaattgac tgaacgaact 2100
aagagctcct ggatgggtcc gggaactcgc ctgggcacaa ggtgccaaaa ggcaggcagc 2160
ctgcccaggc cctccctcct gtccatcccc cacattcccc tgtctgtcct tgtgatttgg 2220
cataaagagc ttctgttttc aaaaaaaaaa aaaa 2254
<210> 24
<211> 1954
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3231586CB1
<400> 24
atccggtacc acggccagtg caagctaaaa ttaaccctca ctaaagggaa taagcttggc 60
ccccgccgcg atgtttcccc gcgagaagac gtggaacatc tcgttcgcgg gctgcggctt 120
cctcggcgtc tactacgtcg gcgtggcctc ctgcctccgc gagcacgcgc ccttcctggt 180
27/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
ggccaacgcc acgcacatct acggcgcctc ggccggggcg ctcacggcca cggcgctggt 240
caccggggtc tgcctgggtg aggctggtgc caagttcatt gaggtatcta aagaggcccg 300
gaagcggttc ctgggccccc tgcacccctc cttcaacctg gtaaagatca tccgcagttt 360
cctgctgaag gtcctgcctg ctgatagcca tgagcatgcc agtgggcgcc tgggcatctc 420
cctgacccgc gtgtcagacg gcgagaatgt cattatatcc cacttcaact ccaaggacga 480
gctcatccag gccaatgtct gcagcggttt catccccgtg tactgtgggc tcatccctcc 540
ctccctccag ggggtgcgct acgtggatgg tggcatttca gacaacctgc cactctatga 600
gcttaagaac accatcacag tgtccccctt ctcgggcgag agtgacatct gtccgcagga 660
cagctccacc aacatccacg agctgcgggt caccaacacc agcatccagt tcaacctgcg 720
caacctctac cgcctctcca aggccctctt cccgccggag cccctggtgc tgcgagagat 780
gtgcaagcag ggataccggg atggcctgcg ctttctgcag cggaacggcc tcctgaaccg 840
gcccaacccc ttgctggcgt tgccccccgc ccgcccccac ggcccagagg acaaggacca 900
ggcagtggag agcgcccaag cggaggatta ctcgcagctg ccgggagaag atcacatcct 960
ggagcacctg cccgcccggc tcaatgaggc cctgctggag gcctgcgtgg agcccacgga 1020
cctgctgacc accctctcca acatgctgcc tgtgcgtctg gccacggcca tgatggtgcc 1080
ctacacgctg ccgctggaga gcgctctgtc cttcaccatc cgcttgctgg agtggctgcc 1140
cgacgttccc gaggacatcc ggtggatgaa ggagcagacg ggcagcatct gccagtacct 1200
ggtgatgcgc gccaagagga agctgggcag gcacctgccc tccaggctgc cggagcaggt 1260
ggagctgcgc cgcgtccagt cgctgccgtc cgtgccgctg tcctgcgccg cctacagaga 1320
ggcactgccc ggctggatgc gcaacaacct ctcgctgggg gacgcgctgg ccaagtggga 1380
ggagtgccag cgccagctgc tgctcggcct cttctgcacc aacgtggcct tcccgcccga 1440
agctctgcgc atgcgcgcac ccgccgaccc ggctcccgcc cccgcggacc cagcatcccc 1500
gcagcaccag ctggccgggc ctgccccttt gctgagcacc cctgctcccg aggcccggcc 1560
cgtgatcggg gccctggggc tgtgagaccc cgaccctctc gaggaaccct gcctgagacg 1620
cctccattac cactgcgcag tgagatgagg ggactcacag ttgccaagag gggtctttgc 1680
cgtgggcccc ctcgccagcc actcaccagc tgcatgcact gagaggggag gtttccacac 1740
ccctcccctg ggccgctgag gccccgcgca cctgtgcctt aatcttccct cccctgtgct 1800
gcccgagcac ctcccccgcc cctttactcc tgggaacttt gcagctgccc ttccctcccc 1860
gtttttcatg gcctgctgaa atatgtgtgt gaagaattat ttattttcgc caaagcacat 1920
gtaataaatg ctgcagccca aaaaaaaaaa aaaa 1954
<210> 25
<211> 1937
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3580770CB1
<400> 25
gcagttcctg gggcgtagta ggggatccac aagcgtttgt gaccagtgaa gttctttaca 60
agggtgagat ctgcacggga ggacccgagc gagggtctcg gcttgccagg aagccggggt 120
tccccgggaa gcgtggagtt cacccgcgca ctcgaagtgc ctttgcaaaa ttatatctgg 180
gtgttggcac ccagccacta ttctgccaat gaagtacatc ctggtcacgg gtggggtcat 240
ctcaggcatt ggtaaaggga tcattgccag cagcattgga acgattctaa aatcatgtgg 300
actccgagtt actgccataa aaatcgaccc ctatattaac atcgatgctg gcactttttc 360
accttatgaa cacggtgaag tcttcgtctt aaatgatggt ggagaagttg atttagacct 420
tggagattat gaaagatttt tggatattaa tctttataaa gacacaatag tcaccacggg 480
gaagatatat cagcatgtga tcaataaaga gaggcgtggt gattacctgg ggaaaacagt 540
gcaagttgtc cctcacatta ctgatgctgt ccaggagtgg gttatgaatc aagccaaggt 600
gccggtggat ggtaataagg aagagcccca aatatgcgtt attgagctgg gaggcaccat 660
tggagacatc gaaggaatgc cgtttgtgga ggcgtttaga caattccagt ttaaggcgaa 720
aagagagaat ttctgtaata tccacgttag ccttgtccca cagctcagtg ctaccggaga 780
acaaaaaacc aaacccaccc aaaacagcgt ccgcgcactg aggggtttag gcctgtctcc 840
agatctgatt gtctgccgaa gttcaacgcc cattgagatg gccgtgaagg agaagatttc 900
tatgttttgt cacgtgaacc ctgaacaggt catatgtatc catgatgttt cttccacata 960
ccgagttcct gtgcttttag aggaacaaag cattgtgaaa tattttaagg agagattgca 1020
cctgcccatc ggtgattctg caagtaattt gctttttaag tggagaaata tggctgacag 1080
gtatgaaagg ttacagaaaa tatgctccat agccctggtt ggcaaataca ccaagctcag 1140
agactgctac gcctctgtgt tcaaagccct ggaacactca gccctggcca tcaaccacaa 1200
gttgaatctg atggtgattg atatgcccga gcacaaccct ggcaatttgg gaggaacaat 1260
gagactggga ataagaagaa ctgttttcaa aactgaaaat tcaatattaa ggaaacttta 1320
tggtgatgtt ccttttatag aagaaagaca cagacatcgg ttcgaggtaa accctaacct 1380
gatcaaacaa tttgagcaga atgacttaag ttttgtaggt caggatgttg atggagacag 1440
28/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
gatggaaatc attgaactgg caaatcatcc ttattttgtt ggtgtccagt tccatcctga 1500
gttttcttct aggccgatga agccttcccc tccgtatctg gggctgttac ttgcagcaac 1560
tgggaacctg aatgcctact tgcaacaggg ttgcaaactg tcttccagtg atagatacag 1620
tgatgccagt gatgacagct tttcagagcc aaggatagct gagttggaaa taagctgaaa 1680
tgaatacatg actgggaata atggggactg cctgtgaggc ctctgaaata attgaaggca 1740
agatgaagga actatctgaa gaaatcacta cactcttaga gaatccctct gttctccagc 1800
aaacatggga tgtaaagcct cacagggaat ctgataatac atacttctgt caaccagaac 1860
cagaggggta gttttctttt ccctccagag gcaacctttg atacttaaaa tatctgtagc 1920
tgattaaatt tcgccca 1937
<210> 26
<211> 970
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3778612CB1
<400> 26
taataataat aacgtaatca tacctctagt catagcatac catttatcgg gctcggcgca 60
ggcccgcggg gagcgcagcc cggcggagag actgatggag aggcagaaac ggaaggcgga 120
catcgagaaa gggctgcagt tcattcagtc gacactaccc ctaaagcaag aagaatatga 180
ggcctttctg ctcaagctgg tgcagaatct gtttgctgag ggcaatgatc tgttccggga 240
gaaggactat aagcaggctc tggtgcagta catggaaggg ctgaacgtgg ccgactacgc 300
tgcctctgac caggtggccc tgccccggga gctgctgtgc aagctgcatg tcaatagggc 360
cgcctgctac ttcaccatgg gcctgtatga gaaggcgctg gaggacagcg agaaggcgct 420
gggccctgac agtgagagta tccgggcgtt gttccgcaaa gcacgcgctc tcaatgaact 480
gggacgccac aaggaggcct acgagtgcag cagccggtgt tccctcgccc tgccccacga 540
tgaaagcgtg actcagcttg gtcagggacc cttgggatct ggggcttcct ggcctggcca 600
gagctggagc ccccacaggg taaggaagag agagtgggag gcagagtgtg atggggagga 660
gggacaggaa gaccctttta atgatgaggg taactatttc agttgtgagc cttctagggc 720
cccaggctgg gaggctcaga ggactgaatc tgggacctgt gttccccccg gcaggcaggg 780
acaagatggc atggcaagca tgggggcggg gtgggtgggg agggatgctg catttctcag 840
ctgggcagta atcaatttaa tggtccttta aaatgtctgt gtattaaaaa tttaagaata 900
ccacacttta atattaaata ttcataaggt ctagtatctt gataataatg tagatgtttt 960
aataacaatt 970
<210> 27
<211> 1810
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4574912CB1
<220>
<221> unsure
<222> 193, 196-198
<223> a, t, c, g, or other
<400> 27
gctgcatgga gggcgccgtt ctagaagctg ggggtgcgcg gtgcttttgc aggtttgggt 60
gtgaattgag caagtatgaa aaccccggct attcatcccc cagaagtgat tactttaaaa 120
attatatgat cattataact cagaatcgaa tgtcctttct tgcaaatatg ttccacacga 180
tggactgtgt tgntgnnncc aggtatagct gtggaccaac tgtatatgat catgcgcacc 240
ttggccatgc ttgctcatat gttagatttg atatcattcg aaggatccta accaaggttt 300
ttggatgcag catagtcatg gtgatgggta ttacagatgt agatgataaa atcatcaaaa 360
gagccaatga gatgaatatt tcccccgctt ccctcgccag tctttatgag gaagacttca 420
agcaggacat ggcagccctg aaggttctcc cacccacggt gtacctgagg gtaaccgaaa 480
atattcctca gataatttct ttcattgaag gaatcattgc tcgtgggaac gcttattcaa 540
cggcaaaagg caatgtctac ttcgatctga agtctagagg agacaagtat ggcaaattgg 600
tcggcgtggt ccctggtcca gtcggagagc cagcggactc tgacaagcgt catgccagtg 660
acttcgccct gtggaaggcg gccaaacccc aggaggtgtt ctgggcctct ccctggggac 720
29/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
ccgggaggcc gggctggcac atcgagtgct ctgccatcgc tagtatggta tttggaagtc 780
aactggatat ccattcaggt gggatagatt tagcttttcc acatcatgag aacgaaattg 840
cacagtgcga agtctttcat cagtgcgagc agtggggaaa ttattttctg cattctgggc 900
atttgcacgc caaaggcaaa gaagaaaaaa tgtccaaatc attaaagaac tacattacta 960
ttaaggactt tctgaagacc ttttcccccg atgtcttccg gttcttctgc ctgcggagca 1020
gctaccgctc agccatcgac tacagtgaca gcgccatgct ccaagctcag cagctgctcc 1080
tggggctggg ctctttcctg gaggacgcac gtgcctacat gaaggggcag ctggcctgcg 1140
gctccgtcag ggaagcgatg ctgtgggaga ggctctccag caccaagagg gccgtgaagg 1200
cggccttggc agatgacttt gacacaccca gggtggttga tgccatcctg ggccttgcac 1260
accacgggaa tggacagctc agggcgtccc tgaaggaacc tgaagggccg agaagtcctg 1320
ctgtgtttgg tgccatcatc tcttactttg aacagttttt tgaaactgtt ggaatttctc 1380
tggcaaatca acagtacgtt tcaggagacg gcagcgaggc taccttgcat ggtgtggtgg 1440
acgagctggt gcggttccgg cagaaggtcc ggcagtttgc gctggccatg cccgaggcca 1500
cgggggacgc ccggcggcag cagctcctag aaaggcagcc cctgctggaa gcatgcgaca 1560
ccctgcgccg gggcctgact gcccacggca tcaacatcaa ggacagaagc agtacaacat 1620
ccacgtggga actgctggat caaaggacaa aagaccaaaa atcagcgggc tgaggatgga 1680
gcacagccat gaacctgctc acgacaagac gcacccatgc ttctcagggt caaggcttta 1740
tgttaaagct tcctgtcggg gctgctaggt cagcattaaa gtaaggcaac caacagtgaa 1800
aaaaaaaaaa 1810
<210> 28
<211> 2162
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5630806CB1
<400> 28
gaccgtgcgc gcggcacgag gggacggggt ccgactcaga aatggcggcc tccatgttct 60
acggcaggct agtggccgtg gccacccttc ggaaccaccg gcctcggacg gcccagcggg 120
ctgctgctca ggttctggga agttctggat tgtttaataa ccatggactc caagtacagc 180
agcaacagca aaggaatctc tcactacatg aatacatgag tatggaatta ttgcaagaag 240
ctggtgtctc cgttcccaaa ggatatgtgg caaagtcacc agatgaagct tatgcaattg 300
ccaaaaaatt aggttcaaaa gatgtcgtga taaaggcaca ggttttagct ggtggtagag 360
gaaaaggaac atttgaaagt ggcctcaaag gaggagtgaa gatagttttc tctccagaag 420
aagcaaaagc tgtttcttca caaatgattg ggaaaaaatt gtttaccaag caaacgggag 480
aaaagggcag aatatgcaat caagtattgg tctgtgagcg aaaatatccc aggagagaat 540
actactttgc aataacaatg gaaaggtcat ttcaaggtcc tgtattaata ggaagttcac 600
atggtggtgt caacattgaa gatgttgctg ctgagactcc tgaagcaata attaaagaac 660
ctattgatat tgaagaaggc atcaaaaagg aacaagctct ccagcttgca cagaagatgg 720
gatttccacc taatattgtg gaatcagcag cagaaaacat ggtcaagctt tacagccttt 780
ttctgaaata cgatgcaacc atgatagaaa taaatccaat ggtggaagat tcagatggag 840
ctgtattgtg tatggatgca aagatcaatt ttgactctaa ttcagcctat cgccaaaaga 900
aaatctttga tctacaggac tggacccagg aagatgaaag ggacaaagat gctgctaagg 960
caaatctcaa ctacattggc ctcgatggaa atataggctg cctagtaaat ggtgctggtt 1020
tggctatggc cacaatggat ataataaaac ttcatggagg gactccagcc aacttccttg 1080
atgttggtgg tggtgctaca gtccatcaag taacagaagc atttaagctt atcacttcag 1140
ataaaaaggt actggctatt ctggtcaaca tttttggagg aatcatgcgc tgtgatgtta 1200
ttgcacaggg tatagtcatg gcagtaaaag acttggaaat taaaatacct gttgtggtac 1260
ggttacaagg tacacgagtc gatgatgcta aggcactgat agcggacagt ggacttaaaa 1320
tacttgcttg tgatgacttg gatgaagctg ctagaatggt tgtaaagctc tctgaaatag 1380
tgaccttagc gaagcaagca catgtggatg tgaaatttca gttgccaata tgatctgaaa 1440
acccagtgga tggctgaagg tgttaaatgt gctataatca ttaagaatac tgtgttctgt 1500
gttattgttc tttttctttt tagtgtgtgg agattgtaat tgccatctag gcacacaaac 1560
atttaaaagg atttggactg catttaattg taccattcag aatggactgt ttgtacgaag 1620
catgtataat gcagttatct tctttctttc gtcgcagcca gtcttttttg cttctcctac 1680
aaaacgtaac ttgcaatttg ccagtttatt attgttggat acaaagttct tcattgataa 1740
gagtcctata aataagataa atacgaagat aaagctttat tctttagtgt taaaatacag 1800
tatatctaat aactagcctc attagtagag cagtatatta aaacaatgtt ttatgtaaaa 1860
agtgtttatc ttcagcacca aatacatgat aaatgtatca atcactattt ataaacagag 1920
ctttcaaaca ctcctcagaa tattcttcta agtattttga tgaagtaact ttgtaattat 1980
ttgaacattg ttttaatcat taggaaacac tgattaactg caagtcttca tgattctgtc 2040
atattaagaa acacctgtag gtttgcttca aataaaggca tatataccaa ggacttacag 2100
30/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
acaaaattaa gaatgtcaat ttaagttaat aaaaatctcc caatatgaaa aaaaaaaaaa 2160
as 2162
<210> 29
<211> 1477
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5854855CB1
<400> 29
gcaatccgta ccctcagtgg gttccctttc agtgggttcc tttgtcccca ggcccattat 60
tccgtcctcc cctcttccct gatgtatttt ggcgcggtct cctggctctg cgggcccagg 120
gctccggatg aggtctcccg ccgtcccgac ccccgcaagg gccagcttgg tgtcgccttc 180
gttcttctgc caccccattc ggaaggtgct cgggtcttcg gggcactggg tcccatcggt 240
ccctcctcac ctgggctcac cctcgggggt ctggccgtga gcgagcaccg gctcagcaac 300
aagctgctgg cttggagcgg cgtcctcgag tggcaggaga agcgcagacc ctactctgac 360
tccactgcaa agctgaagcg gaccctgccc tgccaagcct acgtgaacca aggcgagaac 420
ctggagaccg accagtggcc gcagaagctg atcatgcagc tgatcccgca gcagctgctg 480
accaccctgg gccccctgtt ccggaactcc cagttggcac agttccactt caccaacaga 540
gactgcgact cgctcaaggg gctctgccgc atcatgggca acggcttcgc gggctgcatg 600
ctgttccccc acatctcccc ctgtgaggtg cgcgtgctca tgctcctgta ctcgtccaag 660
aagaagatct tcatgggcct catcccctac gaccagagcg gcttcgtcag tgccatccgg 720
caggtcatca ccacccgcaa gcaggcagtg ggacctggtg gtgtcaactc aggcccagtc 780
cagatcgtca acaacaagtt tctggcatgg agtggtgtca tggagtggca ggagcccagg 840
cctgagccca acagtcggtc caagaggtgg ctgccatccc acgtctacgt gaaccagggg 900
gagatcctga ggaccgagca gtggccaagg aagctgtaca tgcagctcat cccgcagcag 960
ctgctgacca ccctagtgcc gctgttccgg aactcgcgcc tggtccagtt ccacttcacc 1020
aaggacctgg agacactgaa gagcctgtgc cggatcatgg acaatggctt cgccggctgc 1080
gtgcactttt cctacaaagc atcgtgtgag atccgcgtgc ttatgctcct gtactcttca 1140
gagaagaaaa tcttcattgg cctcatcccc catgaccagg gcaactttgt caacggcatc 1200
cggcgtgtca ttgccaacca gcagcaggtc ctgcagcgga acctggagca ggagcaacag 1260
caacgaggga tgggggggta gtggttaccc cgggctgggc ccctccagga gtcacagatg 1320
aggcccccgc agagactggt gacacgcttc tgagcagggg cccctgggga cttcaactgc 1380
ccagcaacat ggaggatggt gtcctgaggc ctccaaggac ggtccccacc cctctacgtt 1440
tccccaataa agccttttaa aaacctgaaa aaaaaaa 1477
<210> 30
<211> 1660
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5993973CB1
<400> 30
gggccttccg cagctgcaga gcctcaacct cagcggcaac cggctgcgcg agccgccagc 60
cgacctggcg cgctgcgccc cgcgcctgca gagcctcaac ctcaccggca attgcctaga 120
ctcctttccc gccgagctct ttcgccccgg cgcgctgccc ctgctcagtg aactggcggc 180
tgctgacaac tgcctccgag aactcagccc cgacatcgcc cacctggcct cgctcaagac 240
gttggacctc tcgaacaacc agctgagcga gatccctgca gagcttgcgg actgccccaa 300
gctcaaggag atcaatttcc gtgggaacaa gctgagggac aagcgcctgg agaagatggt 360
cagcggctgc cagaccagat ccatcctgga gtacctgcgc gtcggaggcc gtggtggcgg 420
gaagggcaag ggccgtgccg agggctcgga gaaggaagag agccggagga agaggaggga 480
gaggaagcag aggcgggaag gtggtgatgg ggaggagcag gacgtgggag atgccggccg 540
gctgctgctc agggtcctgc acgtctctga aaaccccgta cctctgacag tcagagtgag 600
ccccgaggtc cgggatgtgc ggccctacat tgtgggggcc gtggtgcgag gcatggacct 660
gcagccaggg aatgcactca agcgcttcct cacctcgcag accaagctcc acgaagatct 720
ctgtgagaag aggacggctg ccacccttgc cacccacgag ctccgtgccg tcaaagggcc 780
cctgctgtac tgcgcccggc ccccacagga cctcaagatt gtccccttgg ggcggaaaga 840
agccaaggcc aaggagctgg tgcggcagct gcagctggag gccgaggagc agaggaagca 900
gaagaagcgg cagagtgtgt cgggcctgca cagatacctt cacttgctgg atggaaatga 960
31/32
CA 02380317 2002-O1-21
WO 01/07628 PCT/US00/19980
aaattacccg tgtcttgtgg atgcagacgg tgatgtgatt tccttcccac caataaccaa 1020
cagtgagaag acaaaggtta agaaaacgac ttctgatttg tttttggaag taacaagtgc 1080
caccagtctg cagatttgca aggatgtcat ggatgccctc attctgaaaa tggcagaaat 1140
gaaaaagtac actttagaaa ataaagagga aggatcactc tcagatactg aagccgatgc 1200
agtctctgga caacttccag atcccacaac gaatcccagt gctggaaagg acgggccctc 1260
ccttctggtg gtggagcagg tccgggtggt ggatctggaa gggagcctga aggtggtgta 1320
cccgtccaag gccgacctgg ccactgcccc tccccacgtg actgtcgtgc gctgacgcca 1380
gggccgcctg tccgcgtttg tttggccggt tttgcggagg tttctatgcg gcaatgctga 1440
attatccgtt agattttcac cccagttttt ttgttggttt tttttttttg agatggagtc 1500
tcgctctgtc gccaggctgg agtgcagtgg cgtgatctcg agtcactgca gcctgtgtct 1560
cctgggttca agcgattctc ctgcctcagc ctcccaagta gctgggacta caggtgtgtg 1620
ccactaagct cagctaattt ttgtattttt agtagagacg 1660
32/32
