Note: Descriptions are shown in the official language in which they were submitted.
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HUMAN TRANSFERASE MOLECULES
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human
transferase molecules
and to the use of these sequences in the diagnosis, treatment, and prevention
of cell proliferative
disorders and immune system disorders, and in the assessment of the effects of
exogenous compounds
on the expression of nucleic acid and amino acid sequences of human
transferase molecules.
BACKGROUND OF THE INVENTION
Transferases are enzymes that catalyze the transfer of molecular groups. The
reaction may
involve an oxidation, reduction, or cleavage of covalent bonds, and is often
specific to a substrate or
to particular sites on a type of substrate. Transferases participate in
reactions essential to such
functions as synthesis and degradation of cell components, regulation of cell
functions including cell
signaling, cell proliferation, infiamation, apoptosis, secretion and
excretion. Transferases are
involved in key steps in disease processes involving these functions.
Transferases are frequently
classified according to the type of group transferred. For example, methyl
transferases transfer one-
carbon methyl groups, amino transferases transfer nitrogenous amino groups,
and similarly
denominated enzymes transfer aldehyde or ketone, acyl, glycosyl, alkyl or
aryl, isoprenyl, saccharyl,
phosphorous-containing, sulfur-containing, or selenium-containing groups, as
well as small enzymatic
groups such as Coenzyme A.
Acyl transferases include peroxisomal carnitine octanoyl transferase, which is
involved in the
fatty acid beta-oxidation pathway, and mitochondria) carnitine palmitoyl
.transferases, involved in fatty
acid metabolism and transport. Choline O-acetyl transferase catalyzes the
biosynthesis of the
neurotransmitter acetylcholine.
Amino transferases play key roles in protein synthesis and degradation, and
they contribute to
other processes as well. For example, the amino transferase 5-aminolevulinic
acid synthase catalyzes
the addition of succinyl-CoA to glycine, the first step in heme biosynthesis.
Other amino transferases
participate in pathways important for neurological function and metabolism.
For example, glutamine-
phenylpyruvate amino transferase, also known as glutamine transaminase K
(GTK), catalyzes several
reactions with a pyridoxal phosphate cofactor. GTK catalyzes the reversible
conversion of L-glutamine
and phenylpyruvate to 2-oxoglutaramate and L-phenylalanine. Other amino acid
substrates for GTK
include L-methionine, L-histidine, and L-tyrosine. GTK also catalyzes the
conversion of kynurenine to
kynurenic acid, a tryptophan metabolite that is an antagonist of the N-methyl-
D-aspartate (NMDA)
receptor in the brain and may exert a neuromodulatory function. Alteration of
the kynurenine metabolic
pathway may be associated with several neurological disorders. GTK also plays
a role in the
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metabolism of halogenated xenobiotics conjugated to glutathione, leading to
nephrotoxicity in rats and
neurotoxicity in humans. GTK is expressed in kidney, liver, and brain. Both
human and rat GTKs
contain a putative pyridoxal phosphate binding site. (ExPASy ENZYME: EC
2.6.1.64; Perry, S.J. et
al. (1993) Mol. Pharmacol. 43:660-665; Perry, S. et al. (1995) FEBS Lett.
360:277-280; and Alberati-
Giani, D. et al. (1995) J. Neurochem. 64:1448-1455.) A second amino
transferase associated with this
pathway is kynurenine/a-aminoadipate amino transferase (AadAT). AadAT
catalyzes the reversible
conversion of a-aminoadipate and a-ketoglutarate to a-ketoadipate and L-
glutamate during lysine
metabolism. AadAT also catalyzes the transamination of kynurenine to kynurenic
acid. A cytosolic
AadAT is expressed in rat kidney, liver, and brain. (Nakatani, Y. et al.
(1970) Biochim. Biophys. Acta
198:219-228; Buchli, R. et al. (1995) J. Biol. Chem. 270:29330-29335).
Glycosyl transferases include the mammalian UDP-glucouronosyl transferases, a
family of
membrane-bound microsomal enzymes catalyzing the transfer of glucouronic acid
to lipophilic
substrates in reactions that play important roles in detoxification and
excretion of drugs, carcinogens,
and other foreign substances. Another mammalian glycosyl transferase,
mammalian UDP-galactose-
ceramide galactosyl transferase, catalyzes the transfer of galactose to
ceramide in the synthesis of
galactocerebrosides in myelin membranes of the nervous system. The UDP-
glycosyl transferases share
a conserved signature domain of about 50 amino acid residues (PROSITE:
PDOC00359,
http://expasy.hcuge. ch/sprobprosite.html).
Methyl transferases are involved in a variety of pharmacologically important
processes.
Nicotinamide N-methyl transferase catalyzes the N-methylation of nicotinamides
and other pyridines,
an important step in the cellular handling of drugs and other foreign
compounds. Phenylethanolamine
N-methyl transferase catalyzes the conversion of noradrenalin to adrenalin. 6-
O-methylguanine-DNA
methyl transferase reverses DNA methylation, an important step in
carcinogenesis. Uroporphyrin-III
C-methyl transferase, which catalyzes the transfer of two methyl groups from S-
adenosyl-L-methionine
to uroporphyrinogen III, is the first specific enzyme in the biosynthesis of
cobalamin, a dietary enzyme
whose uptake is deficient in pernicious anemia. Protein-arginine methyl
transferases catalyze the
posttranslational methylation of arginine residues in proteins, resulting in
the mono- and dimethylation
of arginine on the guanidino group. Substrates include histones, myelin basic
protein, and
heterogeneous nuclear ribonucleoproteins involved in mRNA processing,
splicing, and transport.
Protein-arginine methyl transferase interacts with proteins upregulated by
mitogens, with proteins
involved in chronic lymphocytic leukemia, and with interferon, suggesting an
important role for
methylation in cytokine receptor signaling (Lin, W.-J. et al. (1996) J. Biol.
Chem. 271:15034-15044;
Abramovich, C. et al. (1997) EMBO J. 16:260-266; and Scott, H.S. et al. (1998)
Genomics 48:330-
340.)
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Phospho-transferases catalyze the transfer of high-energy phosphate groups and
are important
in energy-requiring and -releasing reactions. The metabolic enzyme creatine
kinase catalyzes the
reversible phosphate transfer between creatine/creatine phosphate and ATP/ADP.
Glycocyamine
kinase catalyzes phosphate transfer from ATP to guanidoacetate, and arginine
kinase catalyzes
phosphate transfer from ATP to argenine. A cysteine-containing active site is
conserved in this family
(PROSITE: PDOC00103).
Prenyl transferases are heterodimers, consisting of an alpha and a beta
subunit, that catalyze
the transfer of an isoprenyl group. An example of a prenyl transferase is the
mammalian protein
farnesyl transferase. The alpha subunit of farnesyl transferase consists of 5
repeats of 34 amino acids
each, with each repeat containing an invariant tryptophan (PROSITE:
PDOC00703).
Saccharyl transferases are glycating enzymes involved in a variety of
metabolic processes.
Oligosacchryl transferase-48, for example, is a receptor for advanced
glycation endproducts.
Accumulation of these endproducts is observed in vascular complications of
diabetes, macrovascular
disease, renal insufficiency, and Alzheimer's disease (Thornalley, P.J. (1998)
Cell Mol. Biol. (Noisy-
Le-Grand) 44:1013-1023).
Coenzyme A (CoA) transferase catalyzes the transfer of CoA between two
carboxylic acids.
Succinyl CoA:3-oxoacid CoA transferase, for example, transfers CoA from
succinyl-CoA to a recipient
such as acetoacetate. Acetoacetate is essential to the metabolism of ketone
bodies, which accumulate in
tissues affected by metabolic disorders such as diabetes (PROSITE: PDOC00980).
The discovery of new human transferase molecules 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 cell proliferative disorders and immune system disorders, and
in the assessment of the
effects of exogenous compounds on the expression of nucleic acid and amino
acid sequences of human
transferase molecules.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, human transferase molecules,
referred to
collectively as "HTFS" and individually as "HTFS-1," "HTFS-2," "HTFS-3," "HTFS-
4," "HTFS-5,"
"HTFS-6," "HTFS-7," "HTFS-8," "HTFS-9," "HTFS-10," "HTFS-11," "HTFS-12," "HTFS-
13,"
"HTFS-14," "HTFS-15," "HTFS-16," "HTFS-17," "HTFS-18," "HTFS-19," "HTFS-20,"
"HTFS-
21," "HTFS-22," "HTFS-23," "HTFS-24," "HTFS-25," "HTFS-26," "HTFS-27," "HTFS-
28,"
"HTFS-29," "HTFS-30," "HTFS-31," "HTFS-32," "HTFS-33," "HTFS-34," "HTFS-35,"
"HTFS-
36," "HTFS-37," "HTFS-38," "HTFS-39," "HTFS-40," "HTFS-41," and "HTFS-42." In
one aspect,
the invention provides an isolated polypeptide comprising an amino acid
sequence selected from the
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group consisting of a) an amino acid sequence selected from the group
consisting of SEQ ID NO:1-42,
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:1-42, c) a
biologically active fragment of
an amino acid sequence selected from the group consisting of SEQ ID NO:1-42,
and d) an
immunogenic fragment of an amino acid sequence selected from the group
consisting of SEQ ID NO:1-
42. In one alternative, the invention provides an isolated polypeptide
comprising the amino acid
sequence of SEQ ID NO:1-42. -
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:1-42, 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-
42, c) a biologically active fragment of an amino acid sequence selected from
the group consisting of
SEQ ID NO:1-42, and d) an immunogenic fragment of an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-42. In one alternative, the polynucleotide encodes a
polypeptide selected
from the group consisting of SEQ ID NO:1-42. In another alternative, the
polynucleotide is selected
from the group consisting of SEQ ID N0:43-84.
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:1-42, 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:1-42, c)
a biologically active fragment of an amino acid sequence selected from the
group consisting of SEQ ID
NO:1-42, and d) an immunogenic fragment of an amino acid sequence selected
from the group
consisting of SEQ ID N0:1-42. 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:1-42, 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:1-42, c)
a biologically active fragment of an amino acid sequence selected from the
group consisting of SEQ ID
NO:1-42, and d) an immunogenic fragment of an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-42. The method comprises a) culturing a cell under
conditions suitable for
expression of the polypeptide, wherein said cell is transformed with a
recombinant polynucleotide
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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:1-42, 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:1-42, c) a biologically active fragment of an amino
acid sequence selected
from the group consisting of SEQ ID N0:1-42, and d) an immunogenic fragment of
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-42.
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 N0:43-84, b) a naturally occurring polynucleetide sequence having at
least 90% sequence
identity to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:43-84, 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 N0:43-84, b) a naturally occurring polynucleotide sequence having at
least 90% sequence
identity to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:43-84, c) a
polynucle~tide 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 N0:43-84, b) a naturally occurring polynucleotide sequence having at
least 90% sequence
identity to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:43-84, c) a
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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 composition comprising an effective amount 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 N0:1-42, 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:1-42, c) a biologically active fragment of an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-42, and d) an immunogenic fragment of an amino acid
sequence selected
from the group consisting of SEQ ID N0:1-42, and a pharmaceutically acceptable
excipient. In one
embodiment, the composition comprises an amino acid sequence selected from the
group consisting of
SEQ ID NO:1-42. The invention additionally provides a method of treating a
disease or condition
associated with decreased expression of functional HTFS, comprising
administering to a patient in need
of such treatment the 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 ID NO:1-42, 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:1-42, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID N0:1-42, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-42. 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 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 HTFS, comprising administering to a patient in need of such
treatment the composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypepdde 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-
42, 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:1-42, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-42, and d) an
immunogenic fragment
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of an amino acid sequence selected from the group consisting of SEQ ID NO:1-
42. 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 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 HTFS, comprising administering to a patient in
need of such treatment
the composition.
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:1-42, 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:1-42, c) a biologically active fragment of an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-42, and d) an immunogenic fragment of
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-42. 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:1-42,
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:1-42, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-42, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-42. 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 N0:43-84, the method
comprising a)
exposing a sample comprising the target polynucleotide to a compound, and b)
detecting altered
expression of the target polynucleotide.
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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
N0:43-84, ii) a naturally occurring polynucleotide sequence having at least
90% sequence identity to
a polynucleotide sequence selected from the group consisting of SEQ ID N0:43-
84, iii) a
polynucleotide sequence complementary to i), iv) a polynucleotide sequence
complementary to ii),
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 i) a polynucleotide sequence selected from the group consisting
of SEQ ID N0:43-84,
ii) a naturally occurring polynucleotide sequence having at least 90% sequence
identity to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:43-84,
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
a polynucleotide sequence selected from the group consisting of i)-v) above;
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 HTFS.
Table 2 shows features of each polypeptide sequence, including potential
motifs, homologous
sequences, and methods, algorithms, and searchable databases used for analysis
of HTFS.
Table 3 shows 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 HTFS 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.
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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,"
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
"HTFS" refers to the amino acid sequences of substantially purified HTFS
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
HTFS. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of HTFS either by
directly interacting with
HTFS or by acting on components of the biological pathway in which HTFS
participates.
An "allelic variant" is an alternative form of the gene encoding HTFS. 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
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a given sequence.
"Altered" nucleic acid sequences encoding HTFS include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as HTFS or a
polypeptide with at least one functional characteristic of HTFS. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe of
the polynucleotide encoding HTFS, and improper or unexpected hybridization to
allelic variants, with a
locus other than the normal chromosomal locus for the polynucleotide sequence
encoding HTFS. 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 HTFS. Deliberate
amino acid substitutions may be made on the basis of similarity in polarity,
charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues,
as long as the biological
or immunological activity of HTFS 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
HTFS. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of HTFS either by
directly interacting with HTFS or by acting on components of the biological
pathway in which HTFS
participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments thereof,
such as Fab, F(ab')2, and Fv fragments, which are capable of binding an
epitopic determinant.
Antibodies that bind HTFS 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
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synthesized chemically, and can be conjugated to a carrier 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"
(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 HTFS, 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 HTFS or fragments of
HTFS 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
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deployed in an aqueous solution containing salts (e.g., NaCl), 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 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 Tle, 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.
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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
S 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 HTFS or the polynucleotide encoding HTFS
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,
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 N0:43-84 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:43-84, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:43-84 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID N0:43-84 from related polynucleotide sequences. The precise length of a
fragment of SEQ
ID N0:43-84 and the region of SEQ ID N0:43-84 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:1-42 is encoded by a fragment of SEQ ID N0:43-84. A
fragment
of SEQ ID NO:1-42 comprises a region of unique amino acid sequence that
specifically identifies
SEQ ID NO:1-42. For example, a fragment of SEQ ID NO:1-42 is useful as an
immunogenic peptide
for the development of antibodies that specifically recognize SEQ ID NO:1-42.
The precise length of
a fragment of SEQ ID N0:1-42 and the region of SEQ ID N0:1-42 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
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(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 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 (NCBn 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
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Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off. 50
Expect: 10
Word Size: 11
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.
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=1, 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: 1l and Extension Gap: 1 penalties
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Gap x drop-off. 50
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.
"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 ~g/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 (T"~ 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
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for nucleic acid hybridization are well known and can be found in Sambrook, J.
et al., 1989, Molecular
Cloning: A Laboratory Manual, 2"a 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 ~g/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 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 "immunogenic fragment" is a polypeptide or oligopeptide fragment of HTFS
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
HTFS 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.
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The term "modulate" refers to a change in the activity of HTFS. For example,
modulation may
cause an increase or a decrease in protein activity, binding characteristics,
or any other biological,
functional, or immunological properties of HTFS.
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 HTFS may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolykic cleavage, and other
modifications known in the
art. These processes may occur synthetically or biochemically. Biochemical
modifications will vary by
cell type depending on the enzymatic milieu of HTFS.
"Probe" refers to nucleic acid sequences encoding HTFS, 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.
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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"d
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (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 InstitutelMIT 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 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
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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 HTFS, or fragments thereof, or HTFS 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
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.
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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
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 transferring the DNA of the
present invention
into such organisms are widely known and provided in references such as
Sambrook, J. et al. (1989),
su ra.
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
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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 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%o, 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 transferase molecules
(HTFS), the
polynucleotides encoding HTFS, and the use of these compositions for the
diagnosis, treatment, or
prevention of cell proliferative disorders and immune system disorders.
Table 1 lists the Incyte clones used to assemble full length nucleotide
sequences encoding
HTFS. Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs) of
the polypeptide
and nucleotide sequences, respectively. Column 3 shows the clone IDs of the
Incyte clones in which
nucleic acids encoding each HTFS 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 HTFS 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 along with relevant
citations, all of
22
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WO 01/32888 PCT/US00/30485
which are expressly incorporated by reference herein in their entirety; 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 HTFS. The first column of Table
3 lists the nucleotide
SEQ ID NOs. Fragments of these nucleotide sequences are useful, for example,
in hybridization or
amplification technologies to identify SEQ ID N0:43-84 and to distinguish
between SEQ ID N0:43-
84 and related polynucleotide sequences. The polypeptides encoded by these
fragments are useful, for
example, as immunogenic peptides. Column 2 lists tissue categories which
express HTFS as a fraction
of total tissues expressing HTFS. Column 3 lists diseases, disorders, or
conditions associated with
those tissues expressing HTFS as a fraction of total tissues expressing HTFS.
Column 4 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 HTFS 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 N0:44 maps to chromosome 1 within the interval from 170.1 to 186.4
centiMorgans.
SEQ ID N0:46 maps to chromosome 11 within the interval from 58.2 to 59.5
centiMorgans. SEQ ID
N0:48 maps to chromosome 11 within the interval from 67.4 to 70.9
centiMorgans. SEQ ID N0:49
maps to chromosome 21 within the interval from 51.6 centiMorgans to the q-
terminus. SEQ ID
N0:52 maps to chromosome 3 within the interval from 63.3 to 77.4 centiMorgans.
SEQ ID N0:59
maps to chromosome 20 within the interval from 50.2 to 53.6 centiMorgans and
to chromosome 12
within the interval from 113.3 to 118.9 centiMorgans. SEQ ID N0:60 maps to
chromosome 12
within the interval from 62.7 to 70.6 centiMorgans. SEQ ID N0:62 maps to
chromosome 11 within
the interval from 62.5 to 70.9 centiMorgans. SEQ ID N0:68 maps to chromosome
11 within the
interval from 70.9 to 72.1 centiMorgans. SEQ ID N0:78 maps to chromosome 23
within the interval
from 94.4 to 97.4 centiMorgans and to chromosome 2 within the interval from
272.5 centiMorgans to
the q-terminus. SEQ ID N0:85 maps to chromosome 5 within the interval from 5.5
to 21.5
centiMorgans, to chromosome 17 within the interval from 53.9 to 62.9
centiMorgans, and to
chromosome 12 within the interval from 84.7 to 92.5 centiMorgans. SEQ ID N0:86
maps to
chromosome 6 within the interval from 42.0 to 45.4 centiMorgans, to chromosome
11 within the
interval from 58.2 to 59.5 centiMorgans, and to chromosome 16 within the
interval from 88.1 to 92.6
centiMorgans.
The invention also encompasses HTFS variants. A preferred HTFS variant is one
which has at
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WO 01/32888 PCT/US00/30485
least about 80%, or alternatively at least about 90%, or even at least about
95% amino acid sequence
identity to the HTFS amino acid sequence, and which contains at least one
functional or structural
characteristic of HTFS.
The invention also encompasses polynucleotides which encode HTFS. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected from
the group consisting of SEQ ID N0:43-84, which encodes HTFS. The
polynucleotide sequences of
SEQ ID N0:43-84, 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
HTFS. 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 HTFS. A particular aspect of the invention encompasses a variant of a
polynucleotide
sequence comprising a sequence selected from the group consisting of SEQ ID
N0:43-84 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 N0:43-84.
Any one of the polynucleotide variants described above can encode an amino
acid sequence which
contains at least one functional or structural characteristic of HTFS.
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 HTFS, 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
polynucleotide sequence of naturally
occurring HTFS, and all such variations are to be considered as being
specifically disclosed.
Although nucleotide sequences which encode HTFS and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally occurring HTFS under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding HTFS 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
HTFS 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
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WO 01/32888 PCT/US00/30485
from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode HTFS
and HTFS
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 HTFS 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
N0:43-84 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 polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise (PE
Biosystems,
Foster City CA), thermostable T7 polymerise (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerises 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 Biology, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A.
(1995) Molecular
Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853.)
The nucleic acid sequences encoding HTFS 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
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
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
legations may be used to insert an engineered double-stranded sequence into a
region 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 which may be
present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof which
encode HTFS may be cloned in recombinant DNA molecules that direct expression
of HTFS, 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 HTFS.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter HTFS-encoding sequences for a variety of
purposes including, but not
26
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
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 HTFS, 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 HTFS 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; Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-
232.) Alternatively,
HTFS 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 may be achieved
using the ABI 431 A peptide synthesizer (PE Biosystems). Additionally, the
amino acid sequence of
HTFS, 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.
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WO 01/32888 PCT/US00/30485
(See, e.g., Creighton, supra, pp. 28-53.)
In order to express a biologically active HTFS, the nucleotide sequences
encoding HTFS 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 HTFS. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
HTFS. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding HTFS 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 HTFS 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 Biolo~y> 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 HTFS. 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, or
tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or
animal cell systems. (See, e.g., Sambrook, su ra; Ausubel, supra; 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
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WO 01/32888 PCT/US00/30485
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 HTFS. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding HTFS 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 HTFS into the vector's
multiple cloning site
disrupts the IacZ 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 HTFS are needed, e.g. for the
production of antibodies,
vectors which direct high level expression of HTFS may be used. For example,
vectors containing the
strong, inducible TS or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of HTFS. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomvces cerevisiae or Pichia
nastoris. 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, supra; and Scorer, supra.)
Plant systems may also be used for expression of HTFS. Transcription of
sequences encoding
HTFS may be driven viral promoters, e.g., the 35S and 19S promoters of CaMV
used alone 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, su ra; Brogue, su ra; 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 Technolo~y (1992) McGraw Hill, New
York NY, pp.
191-196.)
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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 HTFS
may be ligated into an
adenovirus transcription/translation complex consisting of the late promoter
and tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses HTFS 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
HTFS in cell lines is preferred. For example, sequences encoding HTFS 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) 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),13
glucuronidase and its substrate B-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
CA 02387785 2002-04-17
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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 HTFS is inserted within a marker gene sequence, transformed
cells containing
sequences encoding HTFS can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding HTFS 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 HTFS
and that express
HTFS 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 HTFS 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 HTFS 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 HTFS
include oligolabeling,
nick translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the
sequences encoding HTFS, 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 polymerise
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,
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cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with nucleotide sequences encoding HTFS 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 HTFS may be designed to contain signal sequences
which direct secretion
of HTFS 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 HTFS 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
HTFS protein containing a
heterologous moiety that can be recognized by a commercially available
antibody may facilitate the
screening of peptide libraries for inhibitors of HTFS 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 HTFS encoding sequence and the heterologous protein
sequence, so that HTFS 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). 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 HTFS may
be achieved in
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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.
HTFS of the present invention or fragments thereof may be used to screen for
compounds that
specifically bind to HTFS. At least one and up to a plurality of test
compounds may be screened for
specific binding to HTFS. 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
HTFS, e.g., a ligand or fragment thereof, a natural substrate, a structural or
functional mimetic, or a
natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current
Protocols in ImmunoloQV 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which HTFS
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 HTFS,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or E.
coli. Cells expressing HTFS or cell membrane fractions which contain HTFS are
then contacted with
a test compound and binding, stimulation, or inhibition of activity of either
HTFS 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
HTFS, either in
solution or affixed to a solid support, and detecting the binding of HTFS 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.
HTFS of the present invention or fragments thereof may be used to screen for
compounds that
modulate the activity of HTFS. Such compounds may include agonists,
antagonists, or partial or
inverse agonists. In one embodiment, an assay is performed under conditions
permissive for HTFS
activity, wherein HTFS is combined with at least one test compound, and the
activity of HTFS in the
presence of a test compound is compared with the activity of HTFS in the
absence of the test
compound. A change in the activity of HTFS in the presence of the test
compound is indicative of a
compound that modulates the activity of HTFS. Alternatively, a test compound
is combined with an
in vitro or cell-free system comprising HTFS under conditions suitable for
HTFS activity, and the
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assay is performed. In either of these assays, a test compound which modulates
the activity of HTFS
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 HTFS 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-loxP 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 HTFS 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 HTFS 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 HTFS 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 HTFS, e.g., by secreting HTFS 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
between regions of HTFS and human transferase molecules. In addition, the
expression of HTFS is
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closely associated with proliferating tissues and inflammation. Therefore,
HTFS appears to play a
role in cell proliferative disorders and immune system disorders. In the
treatment of disorders
associated with increased HTFS expression or activity, it is desirable to
decrease the expression or
activity of HTFS. In the treatment of disorders associated with decreased HTFS
expression or
activity, it is desirable to increase the expression or activity of HTFS.
Therefore, in one embodiment, HTFS 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 HTFS. Examples of such disorders include, but are not limited to,
a cell proliferative
disorder such as actinic keratosis, arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis, mixed
connective tissue disease (MCTD), myelolibrosis, paroxysmal nocturnal
hemoglobinuria,
polycythemia vera, psoriasis, primary thrombocythemia, and cancers including
adenocarcinoma,
leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in
particular, cancers 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; and an
immune system 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, and hematopoietic cancer including
lymphoma, leukemia, and
myeloma.
In another embodiment, a vector capable of expressing HTFS 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 HTFS including, but not limited to, those described
above.
In a further embodiment, a composition comprising a substantially purified
HTFS in
conjunction with a suitable pharmaceutical carrier may be administered to a
subject to treat or prevent a
CA 02387785 2002-04-17
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disorder associated with decreased expression or activity of HTFS including,
but not limited to, those
provided above.
In still another embodiment, an agonist which modulates the activity of HTFS
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or activity
of HTFS including, but not limited to, those listed above.
In a further embodiment, an antagonist of HTFS may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of HTFS.
Examples of such
disorders include, but are not limited to, those cell proliferative disorders
and immune system disorders
described above. In one aspect, an antibody which specifically binds HTFS 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 HTFS.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding HTFS may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of HTFS 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 HTFS may be produced using methods which are generally known
in the art.
In particular, purified HTFS may be used to produce antibodies or to screen
libraries of pharmaceutical
agents to identify those which specifically bind HTFS. Antibodies to HTFS 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 HTFS or with any fragment or
oligopeptide thereof
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
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(bacilli Calmette-Guerin) and Corynebacterium parvum are especially
preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to HTFS
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 HTFS 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 HTFS 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
HTFS-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) Froc. Natl. Acad. Sci.
USA 86:3833-3837; Winter,
G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for HTFS may also be
generated. For
example, such fragments include, but are not limited to, F(ab')2 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.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
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polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
HTFS and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal
antibodies reactive to two
non-interfering HTFS epitopes is generally used, but a competitive binding
assay may also be employed
(Pound, su ra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay techniques
may be used to assess the affinity of antibodies for HTFS. Affinity is
expressed as an association
constant, Ka, which is defined as the molar concentration of HTFS-antibody
complex divided by the
molar concentrations of free antigen and free antibody under equilibrium
conditions. The Ka determined
for a preparation of polyclonal antibodies, which are heterogeneous in their
affinities for multiple HTFS
epitopes, represents the average affinity, or avidity, of the antibodies for
HTFS. The Ka determined for
a preparation of monoclonal antibodies, which are monospecific for a
particular HTFS epitope,
represents a true measure of affinity. High-affinity antibody preparations
with Ka ranging from about
109 to 10'2 L/mole are preferred for use in immunoassays in which the HTFS-
antibody complex must
withstand rigorous manipulations. Low-affinity antibody preparations with Ka
ranging from about 106
to 10' L/mole are preferred for use in immunopurification and similar
procedures which ultimately
require dissociation of HTFS, 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 1-2 mg specific
antibody/ml, preferably 5-10 mg
specific antibody/ml, is generally employed in procedures requiring
precipitation of HTFS-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, su ra, and
Coligan et al., supra.)
In another embodiment of the invention, the polynucleotides encoding HTFS, 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 HTFS.
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 HTFS. (See,
e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press Inc.,
Totawa NJ.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
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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 Moms, M.C. et al. (1997)
Nucleic Acids Res.
25(14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding HTFS 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 N. Somia (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 (HIS
(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 HTFS expression or regulation causes
disease, the expression of
HTFS 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 HTFS
are treated by constructing mammalian expression vectors encoding HTFS and
introducing these
vectors by mechanical means into HTFS-deficient cells. Mechanical transfer
technologies for use with
cells in vivo or ex vitro include (i) direct DNA microinjection into
individual cells, (ii) ballistic gold
particle delivery, (iii) liposome-mediated transfection, (iv) receptor-
mediated gene transfer, and (v) the
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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. R6cipon (1998) Curr.
Opin. Biotechnol. 9:445-
450).
Expression vectors that may be effective for the expression of HTFS 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). HTFS may be
expressed
using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV),
Rous sarcoma virus
(RSV), SV40 virus, thymidine kinase (TK), or (3-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 HTFS from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KIT, 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 HTFS expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding HTFS 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 VS Vg
(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
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A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R.
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 HTFS to cells which have one or more genetic
abnormalities with respect to
the expression of HTFS. 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 HTFS to target cells which have one or more genetic
abnormalities with
respect to the expression of HTFS. The use of herpes simplex virus (HSV)-based
vectors may be
especially valuable for introducing HTFS 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
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sequences, the generation of recombinant virus following the transfection of
multiple plasmids
containing different segments of the large herpesvirus genomes, the growth and
propagation 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 HTFS 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. Biotechnol. 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 HTFS
into the alphavirus
genome in place of the capsid-coding region results in the production of a
large number of HTFS-
coding RNAs and the synthesis of high levels of HTFS 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 HTFS 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 Immunolo~ic Annroaches, 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,
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engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding HTFS.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
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 HTFS. 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 HTFS. 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 HTFS
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding HTFS may be therapeutically useful, and in the treament of disorders
associated with
decreased HTFS expression or activity, a compound which specifically promotes
expression of the
polynucleotide encoding HTFS may be therapeutically useful.
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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
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 HTFS 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
HTFS 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 HTFS. The amount of hybridization may be quantified,
thus forming the
basis for a comparison of the expression of the polynucleodde 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.
Biotechno1.15:462-466.)
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
composition which
generally comprises an active ingredient formulated with a pharmaceutically
acceptable excipient.
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Excipients may include, for example, sugars, starches, celluloses, gums, and
proteins. Various
formulations are commonly known and are thoroughly discussed in the latest
edition of ReminQton's
Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may
consist of HTFS,
antibodies to HTFS, and mimetics, agonists, antagonists, or inhibitors of
HTFS.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, infra-
arterial, intramedullary, intrathecal,
intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal,
intranasal, enteral, topical,
sublingual, or rectal means.
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.
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 compositions may be prepared for direct intracellular
delivery of
macromolecules comprising HTFS or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of the
macromolecule. Alternatively, HTFS or a fragment thereof may be joined to a
short cationic N-
terminal portion from the HIV Tat-1 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 HTFS
or fragments thereof, antibodies of HTFS, and agonists, antagonists or
inhibitors of HTFS, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by
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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
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. 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 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 ~cg to 100,000 ~cg, 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 HTFS may be used for
the diagnosis
of disorders characterized by expression of HTFS, or in assays to monitor
patients being treated with
HTFS or agonists, antagonists, or inhibitors of HTFS. Antibodies useful for
diagnostic purposes may
be prepared in the same manner as described above for therapeutics. Diagnostic
assays for HTFS
include methods which utilize the antibody and a label to detect HTFS 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 HTFS, including ELISAs, RIAs, and FACS,
are known in
the art and provide a basis for diagnosing altered or abnormal levels of HTFS
expression. Normal or
standard values for HTFS expression are established by combining body fluids
or cell extracts taken
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from normal mammalian subjects, for example, human subjects, with antibody to
HTFS under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of HTFS
expressed in 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 HTFS 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 HTFS may
be correlated with
disease. The diagnostic assay may be used to determine absence, presence, and
excess expression of
HTFS, and to monitor regulation of HTFS levels during therapeutic
intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding HTFS or closely related
molecules may be used to
identify nucleic acid sequences which encode HTFS. 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 HTFS, 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 HTFS encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:43-84 or from
genomic sequences including promoters, enhancers, and introns of the HTFS
gene.
Means for producing specific hybridization probes for DNAs encoding HTFS
include the
cloning of polynucleotide sequences encoding HTFS or HTFS 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 32P or 355, or by
enzymatic labels, such as
alkaline phosphatase coupled to the probe via avidin/biotin coupling systems,
and the like.
Polynucleotide sequences encoding HTFS may be used for the diagnosis of
disorders associated
with expression of HTFS. Examples of such disorders include, but are not
limited to, 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
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particular, cancers 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; and an immune
system 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, and hematopoietic cancer including
lymphoma, leukemia, and
myeloma. The polynucleotide sequences encoding HTFS 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 HTFS
expression. Such qualitative or quantitative methods are well known in the
art.
In a particular aspect, the nucleotide sequences encoding HTFS may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding HTFS 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 a control
sample then the presence of altered levels of nucleotide sequences encoding
HTFS 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 HTFS,
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 HTFS, under conditions suitable for hybridization
or amplification.
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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 HTFS
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 HTFS,
or a fragment of a polynucleotide complementary to the polynucleotide encoding
HTFS, 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 HTFS 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,
oligonucleotide primers
derived from the polynucleotide sequences encoding HTFS are used to amplify
DNA using the
polymerise 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
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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 HTFS 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 HTFS, or HTFS 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.
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
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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://www.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 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
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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, supra). 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 HTFS
to quantify the
levels of HTFS 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 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
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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 HTFS
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. For 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
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.,
Larder, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83:7353-7357.)
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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
HTFS on a physical map
and a specific disorder, or a predisposition to a specific disorder, may help
def ne 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 11q22-23, 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, HTFS, 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 HTFS 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 HTFS, or
fragments thereof, and
washed. Bound HTFS is then detected by methods well known in the art. Purified
HTFS 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.
In another embodiment, one may use competitive drug screening assays in which
neutralizing
antibodies capable of binding HTFS specifically compete with a test compound
for binding HTFS. In
this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with HTFS.
In additional embodiments, the nucleotide sequences which encode HTFS may be
used in any
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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. Serial No. 60/163,595, 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.
Some 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 CsCl
cushions or extracted with chloroform. RNA was precipitated from the lysates
with either isopropanol
or sodium acetate and ethanol, or by other routine methods.
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, su ra, 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
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-1000
bp) using SEPHACRYL S1000, 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
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(Invitrogen, Carlsbad CA), or pINCY plasmid (Incyte Genomics, Palo Alto CA).
Recombinant
plasmids were transformed into competent E. coli cells including XL1-Blue, XL1-
BlueMRF, or SOLR
from Stratagene or DHSa, DH10B, or ElectroMAX DHlOB 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 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,
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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
Conned, 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), Swissl?rot, 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) Curr. 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
N0:43-84. 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 from a
particular cell type or tissue have been bound. (See, e.g., Sambrook, supra,
ch. 7; Ausubel, 1995,
supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
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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 Identitv
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 HTFS 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. Chromosomal Mapping of HTFS Encoding Polynucleotides
The cDNA sequences which were used to assemble SEQ ID N0:43-84 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 N0:43-84 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
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Genome Research (WIGR), and G~n6thon 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 N0:44, 46, 48, 49, 52, 59, 60, 62, 68, 78,
85, and 86
are described in The Invention as ranges, or intervals, of human chromosomes.
More than one map
location is reported for SEQ ID N0:59, 78, 85, and 86, indicating that
previously mapped sequences
having similarity, but not complete identity, to SEQ ID N0:59, 78, 85, and 86
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 1 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
G6n6thon which provide
boundaries for radiation hybrid markers whose sequences were included in each
of the clusters.
Human genome maps and other resources available to the public, such as the
NCBI "GeneMap'99"
World Wide Web site (http:llwv:w.nc:bi.nlm.nih.~ov; eenemapll. can be employed
to determine if
previously identified disease genes map within or in proximity to the
intervals indicated above.
VI. Extension of HTFS Encoding Polynucleotides
The full length nucleic acid sequences of SEQ ID N0:43-84 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
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,
Ine.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+, (NH4)ZS04,
and (3-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 min; 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:
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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 ~1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 ~1 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 ~1 to 10 ~1 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 religation 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
religated 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
(Amersham
Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following
parameters: Step 1:
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 N0:43-84 are used to
obtain 5'
regulatory sequences using the procedure above, along with oligonucleotides
designed for such
extension, and an appropriate genomic library.
VII. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:43-84 are employed to screen
cDNAs, genomic
DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about
20 base pairs, is
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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 ~cCi of
[~y-32P] 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.
VIII. 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, supra), 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
(1999), 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, UV, 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.
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
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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/~.~1 oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 units/Nl RNase inhibitor, 500 ECM dATP, 500 ~tM dGTP, 500
~M dTTP, 40 ~M
dCTP, 40 NM 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 O.SM 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 Ed SX SSC/0.2% SDS.
Microarray 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
fig. 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 0.1 % 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
Patent No. 5,807,522, incorporated herein by reference. 1 Erl of the array
element DNA, at an average
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concentration of 100 ng/Nl, 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 Nl of sample mixture consisting of 0.2 ~g
each of Cy3 and
Cy5 labeled cDNA synthesis products in SX 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 cm2 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 ~.Q of SX SSC in a corner 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 (0.1X 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
location to be correlated with a weight ratio of hybridizing species of
1:100,000. When two samples
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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).
IX. Complementary Polynucleotides
Sequences complementary to the HTFS-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring HTFS. 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 HTFS. 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 HTFS-encoding transcript.
X. Expression of HTFS
Expression and purification of HTFS is achieved using bacterial or virus-based
expression
systems. For expression of HTFS 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 lac operator
regulatory element.
Recombinant vectors are transformed into suitable bacterial hosts, e.g.,
BL21(DE3). Antibiotic
resistant bacteria express HTFS upon induction with isopropyl beta-D-
thiogalactopyranoside (IPTG).
Expression of HTFS in eukaryotic cells is achieved by infecting insect or
mammalian cell lines with
recombinant Auto, raphica californica nuclear polyhedrosis virus (AcMNP~,
commonly known as
baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with
cDNA encoding HTFS
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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~perda (SP9) 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, HTFS 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 iaponicum, 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 HTFS 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 HTFS obtained by these methods can be used directly
in the assays shown in
Examples XI and XV.
XI. Demonstration of HTFS Activity
Galactosyltransferase activity is determined by measuring the transfer of
galactose from UDP-
galactose to a GlcNAc-terminated oligosaccharide chain in a radioactive assay.
(Kolbinger, F. et al.
(1998) J. Biol. Chem 273:58-65.) The HTFS sample is incubated with 14 p1 of
assay stock solution
(180 mM sodium cacodylate, pH 6.5, 1 mg_Jml bovine serum albumin, 0.26 mM UDP-
galactose, 2 ~1 of
UDP-['H]galactose), 1 ~tl of MnCl2 (500 mM), and 2.5 ~1 of GlcNAc(30-(CH2)8
COZMe (37 mg/ml in
dimethyl sulfoxide) for 60 minutes at 37 ° C. The reaction is quenched
by the addition of 1 ml of water
and loaded on a C 18 Sep-Pak cartridge (Waters), and the column is washed
twice with 5 ml of water to
remove unreacted UDP-['H]galactose. The ['H]galactosylated GlcNAc(30-(CHZ)e
COZMe remains
bound to the column during the water washes and is eluted with 5 ml of
methanol. Radioactivity in the
eluted material is measured by liquid scintillation counting and is
proportional to galactosyltransferase
activity in the starting sample.
Alternatively, methyltransferase activity is determined using a method that
measures transfer of
radiolabeled methyl groups from a donor substrate to an acceptor substrate
(Bokar, J.A. et al. (supra)).
Reaction mixtures (50 ~1 final volume) contain 15 mM HEPES, pH 7.9, 1.5 mM
MgCl2, 10 mM
dithiothreitol, 3% polyvinylalcohol, 1.5 ~Ci [methyl-3H]AdoMet (0.375 ~M
AdoMet) (DuPont-NEN),
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0.6 pg HTFS, and acceptor substrate (0.4 ~g [35S]RNA or 6-mercaptopurine (6-
MP) to 1 mM final
concentration). Reaction mixtures are incubated at 30°C for 30 minutes,
then 65 °C for 5 minutes.
Analysis of [methyl-3H]RNA is as follows: 1) 50 ~1 of 2 x loading buffer (20
mM tris-HCl, pH
7.6, 1 M LiCl, 1 mM EDTA, 1% sodium dodecyl sulphate (SDS)) and SO ~1 oligo
d(T)-cellulose (10
mg/ml in 1 x loading buffer) are added to the reaction mixture, and incubated
at ambient temperature
with shaking for 30 minutes. 2) Reaction mixtures are transferred to a 96-well
filtration plate attached
to a vacuum apparatus. 3) Each sample is washed sequentially with three 2.4 ml
aliquots of 1 x oligo
d(T) loading buffer containing 0.5 % SDS, 0.1 % SDS, or no SDS. and 4) RNA is
eluted with 300 p1 of
water into a 96-well collection plate, transferred to scintillation vials
containing liquid scintillant, and
radioactivity determined.
Analysis of [methyl-3H]6-MP is as follows: 1) 500 X10.5 M borate buffer, pH
10.0, and then
2.5 ml of 20% (v/v) isoamyl alcohol in toluene are added to the reaction
mixtures. 2) The samples
mixed by vigorous vortexing for ten seconds. 3) After centrifugation at 700g
for 10 minutes, 1.5 ml of
the organic phase is transferred to scintillation vials containing 0.5 ml
absolute ethanol and liquid
scintillant, and radioactivity determined. and 4) Results are corrected for
the extraction of 6-MP into
the organic phase (approximately 41 %).
XII. Functional Assays
HTFS function is assessed by expressing the sequences encoding HTFS 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 ~g 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
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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 Cvtometry, Oxford, New York NY.
The influence of HTFS on gene expression can be assessed using highly purified
populations of
cells transfected with sequences encoding HTFS 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 HTFS and other genes of interest can be analyzed by northern
analysis or
microarray techniques.
XIII. Production of HTFS Specific Antibodies
HTFS 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 HTFS amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of hiigh immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophiilic regions are well
described in the art. (See, e.g., Ausubel, 1995, su ra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
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, suQra.) Rabbits are immunized with
the oligopeptide-KLH
complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide and and-HTFS
activity by, for example, binding the peptide or HTFS to a substrate, blocking
with 1 % BSA, reacting
with rabbit antisera, washing, and reacting with radio-iodinated goat anti-
rabbit IgG.
XIV. Purification of Naturally Occurring HTFS Using Specific Antibodies
Naturally occurring or recombinant HTFS is substantially purified by
immunoaffinity
chromatography using antibodies specific for HTFS. An immunoaffinity column is
constructed by
covalently coupling anti-HTFS antibody to an activated chromatographiic resin,
such as CNBr-activated
SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is
blocked and washed
according to the manufacturer's instructions.
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Media containing HTFS are passed over the immunoaffinity column, and the
column is washed
under conditions that allow the preferential absorbance of HTFS (e.g., high
ionic strength buffers in the
presence of detergent). The column is eluted under conditions that disrupt
antibody/HTFS 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 HTFS is collected.
XV. Identification of Molecules Which Interact with HTFS
HTFS, or biologically active fragments thereof, are labeled with'ZSI 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 HTFS, washed, and
any wells with labeled HTFS complex are assayed. Data obtained using different
concentrations of
HTFS are used to calculate values for the number, affinity, and association of
HTFS with the candidate
molecules.
Alternatively, molecules interacting with HTFS 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).
HTFS 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. 6,057,101 ).
Various modifications and variations of the described methods and systems of
the invention 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
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~ c a °' ~ '~ ~c d A.
v C w z on ~ '° a~
r y a Y U a ...,
U CC ' w Q9 b ~ y 'D
c~O ~ L. U ~ '~ U ~ U ~ O
~ vi ~ ~ ~ ~ C v ~ O C~
Ov. ~ cd Dp
w v is N p., ~ O N b0 O O C
r~ U t L T ~ _b0 v~ _C p., N 'O
~ .o '° .b 3 ~ c ~ G
~ a y '~ E ~ ~ C ~' >,
U O" -y, '~ ~ C > cW .~. U
ii ~" y bTp O C O. c~
iaC.~ ooo.m QO~o w c~a~ ~O
'~ y~ 'b ~, ~e s
'~'0.~~ O 'c'~aO 'fib
.C O ~ ~~
O r G. G .c~ L N U ''' .t U ~ m ~ ,t
'i.~~ UN ~re~~U ~U c~
b ,~ ~ b4 C b4
~ w ~ ~ y ~ a> ~ ca a> O
v c o ~= ~ v~ a. o ~ a. on .3 Q~ a. ~
~1 d ~ =o d ~ d U o ~ d d ~ d y
C
U
~.
W U cO ~ U ._.w-.
O ~ O
P~.~ ~ 0.~ 0.. U v~
97
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
SEQUENCE LISTING
<110> INCYTE GENOMICS, INC.
TANG, Y. Tom
YUE, Henry
HILLMAN, Jennifer L.
LAL, Preeti
BANDMAN, Olga
PATTERSON, Chandra
SHIH, Leo
AZIMZAI, Yalda
LU, Dyung Aina M.
BAUGHN, Mariah R.
<120> HUMAN TRANSFERASE MOLECULES
<130> PF-0753 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/163,595
(151> 1999-11-04
<160> 84
<170> PERL Program
<210> 1
<211> 261
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 016233CD1
<400> 1
Met Ala Gly Asp Ser Glu Gln Thr Leu Gln Asn His Gln Gln Pro
1 5 10 15
Asn Gly Gly Glu Pro Phe Leu Ile Gly Val Ser Gly Gly Thr Ala
20 25 30
Ser Gly Lys Ser Ser Val Cys Ala Lys Ile Val Gln Leu Leu Gly
35 40 45
Gln Asn Glu Val Asp Tyr Arg Gln Lys Gln Val Val Ile Leu Ser
50 55 60
Gln Asp Ser Phe Tyr Arg Val Leu Thr Ser Glu Gln Lys Ala Lys
65 70 75
Ala Leu Lys Gly Gln Phe Asn Phe Asp His Pro Asp Ala Phe Asp
80 85 90
Asn Glu Leu Ile Leu Lys Thr Leu Lys Glu Ile Thr Glu Gly Lys
95 100 105
Thr Val Gln Ile Pro Val Tyr Asp Phe Val Ser His Ser Arg Lys
110 115 120
Glu Glu Thr Val Thr Val Tyr Pro Ala Asp Val Val Leu Phe Glu
125 130 135
Gly Ile Leu Ala Phe Tyr Ser Gln Glu Val Arg Asp Leu Phe Gln
140 145 150
Met Lys Leu Phe Val Asp Thr Asp Ala Asp Thr Arg Leu Ser Arg
155 160 165
Arg Val Leu Arg Asp Ile Ser Glu Arg Gly Arg Asp Leu Glu Gln
170 175 180
Ile Leu Ser Gln Tyr Ile Thr Phe Val Lys Pro Ala Phe Glu Glu
185 190 195
Phe Cys Leu Pro Thr Lys Lys Tyr Ala Asp Val Ile Ile Pro Arg
200 205 210
Gly Ala Asp Asn Leu Val Ala Ile Asn Leu Ile Val Gln His Ile
1/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
215 220 225
Gln Asp Ile Leu Asn Gly Gly Pro Ser Lys Arg Gln Thr Asn Gly
230 235 240
Cys Leu Asn Gly Tyr Thr Pro Ser Arg Lys Arg Gln Ala Ser Glu
245 250 255
Ser Ser Ser Arg Pro His
260
<210> 2
<211> 197
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 078336CD1
<400> 2
Met Phe Tyr Leu Val Val Phe Leu Asn Ser Gly Asp Ile Gln Glu
1 5 10 15
Leu Tyr Asp Thr Thr Leu Ala Leu Gly His Ala Ala Ala Phe Ser
20 25 30
Asp Asp Cys Asp Leu Pro Ser Ala Gln Asp Ile Asn Arg Leu Val
35 40 45
Gly Leu Gln Asn Thr Tyr Met Gly Tyr Leu Asp Tyr Arg Lys Lys
50 55 60
Ala Ile Lys Asp Leu Gly Ile Ser Pro Ser Thr Cys Ser Phe Asn
65 70 75
Pro Gly Val Ile Val Ala Asn Met Thr Glu Trp Lys His Gln Arg
80 85 90
Ile Thr Lys Gln Leu Glu Lys Trp Met Gln Lys Asn Val Glu Glu
95 100 105
Asn Leu Tyr Ser Ser Ser Leu Gly Gly Gly Val Ala Thr Ser Pro
110 115 120
Met Leu Ile Val Phe His Gly Lys Tyr Ser Thr Ile Asn Pro Leu
125 130 135
Trp His Ile Arg His Leu Gly Trp Asn Pro Asp Ala Arg Tyr Ser
140 145 150
Glu His Phe Leu Gln Glu Ala Lys Leu Leu His Trp Asn Gly Arg
155 160 165
His Lys Pro Trp Asp Phe Pro Ser Val His Asn Asp Leu Trp Glu
170 175 180
Ser Trp Phe Val Pro Asp Pro Ala Gly Ile Phe Lys Leu Asn His
185 190 195
His Ser
<210> 3
<211> 378
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 130117CD1
<400> 3
Met Asp Leu Ala Gly Leu Leu Lys Ser Gln Phe Leu Cys His Leu
1 5 10 15
Val Phe Cys Tyr Val Phe Ile Ala Ser Gly Leu Ile Ile Asn Thr
20 25 30
Ile Gln Leu Phe Thr Leu Leu Leu Trp Pro Ile Asn Lys Gln Leu
35 40 45
Phe Arg Lys Ile Asn Cys Arg Leu Ser Tyr Cys Ile Ser Ser Gln
50 55 60
Leu Val Met Leu Leu Glu Trp Trp Ser Gly Thr Glu Cys Thr Ile
65 70 75
2/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
Phe Thr Asp Pro Arg Ala Tyr Leu Lys Tyr Gly Lys Glu Asn Ala
80 85 90
Ile Val Val Leu Asn His Lys Phe Glu Ile Asp Phe Leu Cys Gly
95 100 105
Trp Ser Leu Ser Glu Arg Phe Gly Leu Leu Gly Gly Ser Lys Val
110 115 120
Leu Ala Lys Lys Glu Leu Ala Tyr Val Pro Ile Ile Gly Trp Met
125 130 135
Trp Tyr Phe Thr Glu Met Val Phe Cys Ser Arg Lys Trp Glu Gln
140 145 150
Asp Arg Lys Thr Val Ala Thr Ser Leu Gln His Leu Arg Asp Tyr
155 160 165
Pro Glu Lys Tyr Phe Phe Leu Ile His Cys Glu Gly Thr Arg Phe
170 175 180
Thr Glu Lys Lys His Glu Ile Ser Met Gln Val Ala Arg Ala Lys
185 190 195
Gly Leu Pro Arg Leu Lys His His Leu Leu Pro Arg Thr Lys Gly
200 205 210
Phe Ala Ile Thr Val Arg Ser Leu Arg Asn Val Val Ser Ala Val
215 220 225
Tyr Asp Cys Thr Leu Asn Phe Arg Asn Asn Glu Asn Pro Thr Leu
230 235 240
Leu Gly Val Leu Asn Gly Lys Lys Tyr His Ala Asp Leu Tyr Val
245 250 255
Arg Arg Ile Pro Leu Glu Asp Ile Pro Glu Asp Asp Asp Glu Cys
260 265 270
Ser Ala Trp Leu His Lys Leu Tyr Gln Glu Lys Asp Ala Phe Gln
275 280 285
Glu Glu Tyr Tyr Arg Thr Gly Thr Phe Pro Glu Thr Pro Met Val
290 295 300
Pro Pro Arg Arg Pro Trp Thr Leu Val Asn Trp Leu Phe Trp Ala
305 310 315
Ser Leu Val Leu Tyr Pro Phe Phe Gln Phe Leu Val Ser Met Ile
320 325 330
Arg Ser Gly Ser Ser Leu Thr Leu Ala Ser Phe Ile Leu Val Phe
335 340 345
Phe Val Ala Ser Val Gly Val Arg Trp Met Ile Gly Val Thr Glu
350 355 360
Ile Asp Lys Gly Ser Ala Tyr Gly Asn Ser Asp Ser Lys Gln Lys
365 370 375
Leu Asn Asp
<210> 4
<211> 285
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 267495CD1
<400> 4
Met Leu Tyr Leu Ile Gly Leu Gly Leu Gly Asp Ala Lys Asp Ile
1 5 10 15
Thr Val Lys Gly Leu Glu Val Val Arg Arg Cys Ser Arg Val Tyr
20 25 30
Leu Glu Ala Tyr Thr Ser Val Leu Thr Val Gly Lys Glu Ala Leu
35 40 45
Glu Glu Phe Tyr Gly Arg Lys Leu Val Val Ala Asp Arg Glu Glu
50 55 60
Val Glu Gln Glu Ala Asp Asn Ile Leu Lys Asp Ala Asp Ile Ser
65 70 75
Asp Val Ala Phe Leu Val Val Gly Asp Pro Phe Gly Ala Thr Thr
80 85 90
His Ser Asp Leu Val Leu Arg Ala Thr Lys Leu Gly Ile Pro Tyr
95 100 105
3/55
CA 02387785 2002-04-17
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Arg Val Ile His Asn Ala Ser Ile Met Asn Ala Val Gly Cys Cys
110 115 120
Gly Leu Gln Leu Tyr Lys Phe Gly Glu Thr Val Ser Ile Val Phe
125 130 135
Trp Thr Asp Thr Trp Arg Pro Glu Ser Phe Phe Asp Lys Val Lys
140 145 150
Lys Asn Arg Gln Asn Gly Met His Thr Leu Cys Leu Leu Asp Ile
155 160 165
Lys Val Lys Glu Gln Ser Leu Glu Asn Leu Ile Lys Gly Arg Lys
170 175 180
Ile Tyr Glu Pro Pro Arg Tyr Met Ser Val Asn Gln Ala Ala Gln
185 190 195
Gln Leu Leu Glu Ile Val Gln Asn Gln Arg Ile Arg Gly Glu Glu
200 205 210
Pro Ala Val Thr Glu Glu Thr Leu Cys Val Gly Leu Ala Arg Val
215 220 225
Gly Ala Asp Asp Gln Lys Ile Ala Ala Gly Thr Leu Arg Gln Met
230 235 240
Cys Thr Val Asp Leu Gly Glu Pro Leu His Ser Leu Ile Ile Thr
245 250 255
Gly Gly Ser Ile His Pro Met Glu Met Glu Met Leu Ser Leu Phe
260 265 270
Ser Ile Pro Glu Asn Ser Ser Glu Ser Gln Ser Ile Asn Gly Leu
275 280 285
<210> 5
<211> 301
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 410533CD1
<400> 5
Met Leu Ala Leu Arg Val Ala Arg Gly Ser Trp Gly Ala Leu Arg
1 5 10 15
Gly Ala Ala Trp Ala Pro Gly Thr Arg Pro Ser Lys Arg Arg Ala
20 25 30
Cys Trp Ala Leu Leu Pro Pro Val Pro Cys Cys Leu Gly Cys Leu
35 40 45
Ala Glu Arg Trp Arg Leu Arg Pro Ala Ala Leu Gly Leu Arg Leu
50 55 60
Pro Gly Ile Gly Gln Arg Asn His Cys Ser Gly Ala Gly Lys Ala
65 70 75
Ala Pro Arg Pro Ala Ala Gly Ala Gly Ala Ala Ala Glu Ala Pro
80 85 90
Gly Gly Gln Trp Gly Pro Ala Ser Thr Pro Ser Leu Tyr Glu Asn
95 100 105
Pro Trp Thr Ile Pro Asn Met Leu Ser Met Thr Arg Ile Gly Leu
110 115 120
Ala Pro Val Leu Gly Tyr Leu Ile Ile Glu Glu Asp Phe Asn Ile
125 130 135
Ala Leu Gly Val Phe Ala Leu Ala Gly Leu Thr Asp Leu Leu Asp
140 145 150
Gly Phe Ile Ala Arg Asn Trp Ala Asn Gln Arg Ser Ala Leu Gly
155 160 165
Ser Ala Leu Asp Pro Leu Ala Asp Lys Ile Leu Ile Ser Ile Leu
170 175 180
Tyr Val Ser Leu Thr Tyr Ala Asp Leu Ile Pro Val Pro Leu Thr
185 190 195
Tyr Met Ile Ile Ser Arg Asp Val Met Leu Ile Ala Ala Val Phe
200 205 210
Tyr Val Arg Tyr Arg Thr Leu Pro Thr Pro Arg Thr Leu Ala Lys
215 220 225
Tyr Phe Asn Pro Cys Tyr Ala Thr Ala Arg Leu Lys Pro Thr Phe
4/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
230 235 240
Ile Ser Lys Val Asn Thr Ala Val Gln Leu Ile Leu Val Ala Ala
245 250 255
Ser Leu Ala Ala Pro Val Phe Asn Tyr Ala Asp Ser Ile Tyr Leu
260 265 270
Gln Ile Leu Trp Cys Phe Thr Ala Phe Thr Thr Ala Ala Ser Ala
275 280 285
Tyr Ser Tyr Tyr His Tyr Gly Arg Lys Thr Val Gln Val Ile Lys
290 295 300
Asp
<210> 6
<211> 253
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 852708CD1
<400> 6
Met Ala Thr Ser Gly Asp Cys Pro Arg Ser Glu Ser Gln Gly Glu
1 5 10 15
Glu Pro Ala Glu Cys Ser Glu Ala Gly Leu Leu Gln Glu Gly Val
20 25 30
Gln Pro Glu Glu Phe Val Ala Ile Ala Asp Tyr Ala Ala Thr Asp
35 40 45
Glu Thr Gln Leu Ser Phe Leu Arg Gly Glu Lys Ile Leu Ile Leu
50 55 60
Arg Gln Thr Thr Ala Asp Trp Trp Trp Gly Glu Arg Ala Gly Cys
65 70 75
Cys Gly Tyr Ile Pro Ala Asn His Val Gly Lys His Val Asp Glu
80 85 90
Tyr Asp Pro Glu Asp Thr Trp Gln Asp Glu Glu Tyr Phe Gly Ser
95 100 105
Tyr Gly Thr Leu Lys Leu His Leu Glu Met Leu Ala Asp Gln Pro
110 115 120
Arg Thr Thr Lys Tyr His Ser Val Ile Leu Gln Asn Lys Glu Ser
125 130 135
Leu Thr Asp Lys Val Ile Leu Asp Val Gly Cys Gly Thr Gly Ile
140 145 150
Ile Ser Leu Phe Cys Ala His Tyr Ala Arg Pro Arg Ala Val Tyr
155 160 165
Ala Val Glu Ala Ser Glu Met Ala Gln His Thr Gly Gln Leu Val
170 175 180
Leu Gln Asn Gly Phe Ala Asp Ile Ile Thr Val Tyr Gln Gln Lys
185 190 195
Val Glu Asp Val Val Leu Pro Glu Lys Val Asp Val Leu Val Ser
200 205 210
Glu Trp Met Gly Thr Cys Leu Leu Val Arg Ala Gly Val Arg Ala
215 220 225
Ala Gly Gly Arg Ser Trp Gly Ala Ser Glu His Gly Leu Gly Trp
230 235 240
Ala Asn Leu Arg Ile Ser Arg Val Val Arg Asp Ser Phe
245 250
<210> 7
<211> 390
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 972944CD1
<400> 7
5/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
Met Ser Val Arg Val Ala Arg Val Ala Trp Val Arg Gly Leu Gly
1 5 10 15
Ala Ser Tyr Arg Arg Gly Ala Ser Ser Phe Pro Val Pro Pro Pro
20 25 30
Gly Ala Gln Gly Val Ala Glu Leu Leu Arg Asp Ala Thr Gly Ala
35 40 45
Glu Glu Glu Ala Pro Trp Ala Ala Thr Glu Arg Arg Met Pro Gly
50 55 60
Gln Cys Ser Val Leu Leu Phe Pro Gly Gln Gly Ser Gln Val Val
65 70 75
Gly Met Gly Arg Gly Leu Leu Asn Tyr Pro Arg Val Arg Glu Leu
80 85 90
Tyr Ala Ala Ala Arg Arg Val Leu Gly Tyr Asp Leu Leu Glu Leu
95 100 105
Ser Leu His Gly Pro Gln Glu Thr Leu Asp Arg Thr Val His Cys
110 115 120
Gln Pro Ala Ile Phe Val Ala Ser Leu Ala Ala Val Glu Lys Leu
125 130 135
His His Leu Gln Pro Ser Val Ile Glu Asn Cys Val Ala Ala Ala
140 145 150
Gly Phe Ser Val Gly Glu Phe Ala Ala Leu Val Phe Ala Gly Ala
155 160 165
Met Glu Phe Ala Glu Gly Leu Tyr Ala Val Lys Ile Arg Ala Glu
170 175 180
Ala Met Gln Glu Ala Ser Glu Ala Val Pro Ser Gly Met Leu Ser
185 190 195
Val Leu Gly Gln Pro Gln Ser Lys Phe Asn Phe Ala Cys Leu Glu
200 205 210
Ala Arg Glu His Cys Lys Ser Leu Gly Ile Glu Asn Pro Val Cys
215 220 225
Glu Val Ser Asn Tyr Leu Phe Pro Asp Cys Arg Val Ile Ser Gly
230 235 240
His Gln Glu Ala Leu Arg Phe Leu Gln Lys Asn Ser Ser Lys Phe
245 250 255
His Phe Arg Arg Thr Arg Met Leu Pro Val Ser Gly Ala Phe His
260 265 270
Thr Arg Leu Met Glu Pro Ala Val Glu Pro Leu Thr Gln Ala Leu
275 280 285
Lys Ala Val Asp Ile Lys Lys Pro Leu Val Ser Val Tyr Ser Asn
290 295 300
Val His Ala His Arg Tyr Arg His Pro Gly His Ile His Lys Leu
305 310 315
Leu Ala Gln Gln Leu Val Ser Pro Val Lys Trp Glu Gln Thr Met
320 325 330
His Ala Ile Tyr Glu Arg Lys Lys Gly Arg Gly Phe Pro Gln Thr
335 340 345
Phe Glu Val Gly Pro Gly Arg Gln Leu Gly Ala Ile Leu Lys Ser
350 355 360
Cys Asn Met Gln Ala Trp Lys Ser Tyr Ser Ala Val Asp Val Leu
365 370 375
Gln Thr Leu Glu His Val Asp Leu Asp Pro Gln Glu Pro Pro Arg
380 385 390
<210> 8
<211> 373
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 997730CD1
<400> 8
Met Leu Ile Pro Phe Ser Met Lys Asn Cys Phe Gln Leu Leu Cys
1 5 10 15
Asn Cys Gln Val Pro Ala Ala Gly Phe Lys Lys Thr Val Lys Asn
6/5S
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
20 25 30
Gly Leu Ile Leu Gln Ser Ile Ser Asn Asp Val Tyr Gln Asn Leu
35 40 45
Ala Val Glu Asp Trp Ile His Asp His Met Asn Leu Glu Gly Lys
50 55 60
Pro Ile Leu Phe Phe Trp Gln Asn Ser Pro Ser Val Val Ile Gly
65 70 75
Arg His Gln Asn Pro Trp Gln Glu Cys Asn Leu Asn Leu Met Arg
80 85 90
Glu Glu Gly Ile Lys Leu Ala Arg Arg Arg Ser Gly Gly Gly Thr
95 100 105
Val Tyr His Asp Met Gly Asn Ile Asn Leu Thr Phe Phe Thr Thr
110 115 120
Lys Lys Lys Tyr Asp Arg Met Glu Asn Leu Lys Leu Ile Val Arg
125 130 135
Ala Leu Asn Ala Val Gln Pro Gln Leu Asp Val Gln Ala Thr Lys
140 145 150
Arg Phe Asp Leu Leu Leu Asp Gly Gln Phe Lys Ile Ser Gly Thr
155 160 165
Ala Ser Lys Ile Gly Arg Thr Thr Ala Tyr His His Cys Thr Leu
170 175 180
Leu Cys Ser Thr Asp Gly Thr Phe Leu Ser Ser Leu Leu Lys Ser
185 190 195
Pro Tyr Gln Gly Ile Arg Ser Asn Ala Thr Ala Ser Ile Pro Ser
200 205 210
Leu Val Lys Asn Leu Leu Glu Lys Asp Pro Thr Leu Thr Cys Glu
215 220 225
Val Leu Met Asn Ala Val Ala Thr Glu Tyr Ala Ala Tyr His Gln
230 235 240
Ile Asp Asn His Ile His Leu Ile Asn Pro Thr Asp Glu Thr Leu
245 250 255
Phe Pro Gly Ile Asn Ser Lys Ala Lys Glu Leu Gln Thr Trp Glu
260 265 270
Trp Ile Tyr Gly Lys Thr Pro Lys Phe Ser Ile Asn Thr Ser Phe
275 280 285
His Val Leu Tyr Glu Gln 5er His Leu Glu Ile Lys Val Phe Ile
290 295 300
Asp Ile Lys Asn Gly Arg Ile Glu Ile Cys Asn Ile Glu Ala Pro
305 310 315
Asp His Trp Leu Pro Leu Glu Ile Arg Asp Lys Leu Asn Ser Ser
320 325 330
Leu Ile Gly Ser Lys Phe Cys Pro Thr Glu Thr Thr Met Leu Thr
335 340 345
Asn Ile Leu Leu Arg Thr Cys Pro Gln Asp His Lys Leu Asn Ser
350 355 360
Lys Trp Asn Ile Leu Cys Glu Lys Ile Lys Gly Ile Met
365 370
<210> 9
<211> 371
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1285944CD1
<400> 9
Met Ser Phe Arg Lys Val Asn Ile Ile Ile Leu Val Leu Ala Val
1 5 10 15
Ala Leu Phe Leu Leu Val Leu His His Asn Phe Leu Ser Leu Ser
20 25 30
Ser Leu Leu Arg Asn Glu Val Thr Asp Ser Gly Ile Val Gly Pro
35 40 45
Gln Pro Ile Asp Phe Val Pro Asn Ala Leu Arg His Ala Val Asp
50 55 60
Gly Arg Gln Glu Glu Ile Pro Val Val Ile Ala Ala Ser Glu Asp
7/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
65 70 75
Arg Leu Gly Gly Ala Ile Ala Ala Ile Asn Ser Ile Gln His Asn
80 85 90
Thr Arg Ser Asn Val Ile Phe Tyr Ile Val Thr Leu Asn Asn Thr
95 100 105
Ala Asp His Leu Arg Ser Trp Leu Asn Ser Asp Ser Leu Lys Ser
110 115 120
Ile Arg Tyr Lys Ile Val Asn Phe Asp Pro Lys Leu Leu Glu Gly
125 130 135
Lys Val Lys Glu Asp Pro Asp Gln Gly Glu Ser Met Lys Pro Leu
140 145 150
Thr Phe Ala Arg Phe Tyr Leu Pro Ile Leu Val Pro Ser Ala Lys
155 160 165
Lys Ala Ile Tyr Met Asp Asp Asp Val Ile Val Gln Gly Asp Ile
170 175 180
Leu Ala Leu Tyr Asn Thr Ala Leu Lys Pro Gly His Ala Ala Ala
185 190 195
Phe Ser Glu Asp Cys Asp Ser Ala Ser Thr Lys Val Val Ile Arg
200 205 210
Gly Ala Gly Asn Gln Tyr Asn Tyr Ile Gly Tyr Leu Asp Tyr Lys
215 220 225
Lys Glu Arg Ile Arg Lys Leu Ser Met Lys Ala Ser Thr Cys Ser
230 235 240
Phe Asn Pro Gly Val Phe Val Ala Asn Leu Thr Glu Trp Lys Arg
245 250 255
Gln Asn Ile Thr Asn Gln Leu Glu Lys Trp Met Lys Leu Asn Val
260 265 270
Glu Glu Gly Leu Tyr Ser Arg Thr Leu Ala Gly Ser Ile Thr Thr
275 280 285
Pro Pro Leu Leu Ile Val Phe Tyr Gln Gln His Ser Thr Ile Asp
290 295 300
Pro Met Trp Asn Val Arg His Leu Gly Ser Ser Ala Gly Lys Arg
305 310 315
Tyr Ser Pro Gln Phe Val Lys Ala Ala Lys Leu Leu His Trp Asn
320 325 330
Gly His Leu Lys Pro Trp Gly Arg Thr Ala Ser Tyr Thr Asp Val
335 340 345
Trp Glu Lys Trp Tyr Ile Pro Asp Pro Thr Gly Lys Phe Asn Leu
350 355 360
Ile Arg Arg Tyr Thr Glu Ile Ser Asn Ile Lys
365 370
<210> 10
<211> 123
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1293207CD1
<400> 10
Met Phe Asn Phe Asp Thr Phe Trp Lys Asn Phe Lys Ser Lys Leu
1 5 10 15
Gly Phe Ile Asn Trp Asp Ala Ile Asn Lys Asn Gln Val Pro Pro
20 25 30
Pro Ser Thr Arg Ala Leu Leu Tyr Phe Ser Arg Leu Trp Glu Asp
35 40 45
Phe Lys Gln Asn Thr Pro Phe Leu Asn Trp Lys Ala Ile Ile Glu
50 55 60
Gly Ala Asp Ala Ser Ser Leu Gln Lys Arg Ala Gly Arg Ala Asp
65 70 75
Gln Asn Tyr Asn Tyr Asn Gln His Ala Tyr Pro Thr Ala Tyr Gly
80 85 90
Gly Lys Tyr Ser Val Lys Thr Pro Ala Lys Gly Gly Val Ser Pro
95 100 105
Ser Ser Ser Ala Ser Arg Val Gln Pro Gly Leu Leu Gln Trp Val
8/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
Lys Phe Trp
110 115 120
<210> 11
<211> 85
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1308125CD1
<400> 11
Met Ser Ser Ser Arg Met Glu Gly Lys Ala Lys Tyr Ile Leu Pro
1 5 10 15
Thr Glu Thr Ile Tyr Val Gly Glu Met Lys Asp Gly Met Phe His
20 25 30
Gly Glu Gly Thr Leu Tyr Phe Pro Ser Gly Ser Gln Tyr Asp Ala
35 40 45
Ile Trp Glu Asn Gly Leu Ala Ile Lys Val Trp Leu Asn Ser Pro
50 55 60
Ile Trp Thr His Leu Glu Lys Ser Pro Arg Ala Ile Thr Ile Val
65 70 75
Glu Thr Ala Ser Ile Thr Gln Ser Arg Gly
80 85
<210> 12
<211> 184
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1439670CD1
<400> 12
Met Lys Pro Asp Glu Thr Pro Met Phe Asp Pro Ser Leu Leu Lys
1 5 10 15
Glu Val Asp Trp Ser Gln Asn Thr Ala Thr Phe Ser Pro Ala Ile
20 25 30
Ser Pro Thr His Pro Gly Glu Gly Leu Val Leu Arg Pro Leu Cys
35 40 45
Thr Ala Asp Leu Asn Arg Gly Phe Phe Lys Val Leu Gly Gln Leu
50 55 60
Thr Glu Thr Gly Val Val Ser Pro Glu Gln Phe Met Lys Ser Phe
65 70 75
Glu His Met Lys Lys Ser Gly Asp Tyr Tyr Val Thr Val Val Glu
80 85 90
Asp Val Thr Leu Gly Gln Ile Val Ala Thr Ala Thr Leu Ile Ile
95 100 105
Glu His Lys Phe Ile His Ser Cys Ala Lys Arg Gly Arg Val Glu
110 115 120
Asp Val Val Val Ser Asp Glu Cys Arg Gly Lys Gln Leu Gly Lys
125 130 13S
Leu Leu Leu Ser Thr Leu Thr Leu Leu Ser Lys Lys Leu Asn Cys
140 145 150
Tyr Lys Ile Thr Leu Glu Cys Leu Pro Gln Asn Val Gly Phe Tyr
155 160 165
Lys Lys Phe Gly Tyr Thr Val Ser Glu Glu Asn Tyr Met Cys Arg
170 175 180
Arg Phe Leu Lys
<210> 13
<211> 169
<212> PRT .
9/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1444281CD1
<400> 13
Met Ala Asn Tyr Ile His Val Pro Pro Gly Ser Pro Glu Val Pro
1 5 10 15
Lys Leu Asn Val Thr Val Gln Asp Gln Glu Glu His Arg Cys Arg
20 25 30
Glu Gly Ala Leu Ser Leu Leu Gln His Leu Arg Pro His Trp Asp
35 40 45
Pro Gln Glu Val Thr Leu Gln Leu Phe Thr Asp Gly Ile Thr Asn
50 55 60
Lys Leu Ile Gly Cys Tyr Val Gly Asn Thr Met Glu Asp Val Val
65 70 75
Leu Val Arg Ile Tyr Gly Asn Lys Thr Glu Leu Leu Val Asp Arg
80 85 90
Asp Glu Glu Val Lys Ser Phe Arg Val Leu Gln Ala His Gly Cys
95 100 105
Ala Pro Gln Leu Tyr Cys Thr Phe Asn Asn Gly Leu Cys Tyr Glu
110 115 120
Phe Ile Gln Gly Glu Ala Leu Asp Pro Lys His Val Cys Asn Pro
125 130 135
Ala Ile Phe Ser Leu Ser Ser Leu Thr Leu Cys Lys Gly Lys Thr
140 145 150
Thr Arg Cys Phe Gly Leu Thr Gly Cys Arg Gly Ser Arg Leu Leu
155 160 165
Leu Ser Phe Phe
<210> 14
<211> 357
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1450140CD1
<400> 14
Met Gly Gly Ala Val Ser Ala Gly Glu Asp Asn Asp Asp Leu Ile
1 5 10 15
Asp Asn Leu Lys Glu Ala Gln Tyr Ile Arg Thr Glu Arg Val Glu
20 25 30
Gln Ala Phe Arg Ala Ile Asp Arg Gly Asp Tyr Tyr Leu Glu Gly
35 40 45
Tyr Arg Asp Asn Ala Tyr Lys Asp Leu Ala Trp Lys His Gly Asn
50 55 60
Ile His Leu Ser Ala Pro Cys Ile Tyr Ser Glu Val Met Glu Ala
65 70 75
Leu Lys Leu Gln Pro Gly Leu Ser Phe Leu Asn Leu Gly Ser Gly
80 85 90
Thr Gly Tyr Leu Ser Thr Met Val Gly Leu Ile Leu Gly Pro Phe
95 100 105
Gly Ile Asn His Gly Ile Glu Leu His Ser Asp Val Val Glu Tyr
110 115 120
Ala Lys Glu Lys Leu Glu Ser Phe Ile Lys Asn Ser Asp Ser Phe
125 13 0 13 5
Asp Lys Phe Glu Phe Cys Glu Pro Ala Phe Val Val Gly Asn Cys
140 145 150
Leu Gln Ile Ala Ser Asp Ser His Gln Tyr Asp Arg Ile Tyr Cys
155 160 165
Gly Ala Gly Val Gln Lys Asp His Glu Asn Tyr Met Lys Ile Leu
170 175 180
Leu Lys Val Gly Gly Ile Leu Val Met Pro Ile Glu Asp Gln Leu
10/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
185 190 195
Thr Gln Ile Met Arg Thr Gly Gln Asn Thr Trp Glu Ser Lys Asn
200 205 210
Ile Leu Ala Val Ser Phe Ala Pro Leu Val Gln Pro Ser Lys Asn
215 220 225
Asp Asn Gly Lys Pro Asp Ser Val Gly Leu Pro Pro Cys Ala Val
230 235 240
Arg Asn Leu Gln Asp Leu Ala Arg Ile Tyr Ile Arg Arg Thr Leu
245 250 255
Arg Asn Phe Ile Asn Asp Glu Met Gln Ala Lys Gly Ile Pro Gln
260 265 270
Arg Ala Pro Pro Lys Arg Lys Arg Lys Arg Val Lys Gln Arg Ile
275 280 285
Asn Thr Tyr Val Phe Val Gly Asn Gln Leu Ile Pro Gln Pro Leu
290 295 300
Asp Ser Glu Glu Asp Glu Lys Met Glu Glu Asp Ile Lys Glu Glu
305 310 315
Glu Glu Lys Asp His Asn Glu Ala Met Lys Pro Glu Glu Pro Pro
320 325 330
Gln Asn Leu Leu Arg Glu Lys Ile Met Lys Leu Pro Leu Pro Glu
335 340 345
Ser Leu Lys Ala Tyr Leu Thr Tyr Phe Arg Asp Lys
350 355
<210> 15
<211> 100
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1604828CD1
<400> 15
Met Asn Val Arg Gly Lys Val Ile Leu Ser Met Leu Val Val Ser
1 5 10 15
Thr Val Ile Ile Val Phe Trp Glu Phe Ile Asn Ser Thr Glu Asp
20 25 30
Ser Phe Leu Trp Ile Tyr His Ser Lys Asn Pro Glu Val Asp Asp
35 40 45
Ser Ser Ala Gln Lys Gly Trp Trp Phe Leu Ser Trp Phe Asn Asn
50 55 60
Gly Ile His Asn Tyr Gln Gln Gly Glu Glu Asp Ile Asp Lys Glu
65 70 75
Lys Gly Arg Glu Glu Thr Lys Gly Arg Lys Met Thr Gln Gln Ser
80 85 90
Phe Gly Tyr Gly Thr Gly Leu Ile Gln Thr
95 100
<210> 16
<211> 199
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1644023CD1
<400> 16
Met Lys Thr Phe Ile Ile Gly Ile Ser Gly Val Thr Asn Ser Gly
1 5 10 15
Lys Thr Thr Leu Ala Lys Asn Leu Gln Lys His Leu Pro Asn Cys
20 25 30
Ser Val Ile Ser Gln Asp Asp Phe Phe Lys Pro Glu Ser Glu Ile
35 40 45
Glu Thr Asp Lys Asn Gly Phe Leu Gln Tyr Asp Val Leu Glu Ala
50 55 60
11/$5
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
Leu Asn Met Glu Lys Met Met Ser Ala Ile Ser Cys Trp Met Glu
65 70 75
Ser Ala Arg His Ser Val Val Ser Thr Asp Gln Glu Ser Ala Glu
80 85 90
Glu Ile Pro Ile Leu Ile Ile Glu Gly Phe Leu Leu Phe Asn Tyr
95 100 105
Lys Pro Leu Asp Thr Ile Trp Asn Arg Ser Tyr Phe Leu Thr Ile
110 115 120
Pro Tyr Glu Glu Cys Lys Arg Arg Arg Ser Thr Arg Val Tyr Gln
125 130 135
Pro Pro Asp Ser Pro Gly Tyr Phe Asp Gly His Val Trp Pro Met
140 145 150
Tyr Leu Lys Tyr Arg Gln Glu Met Gln Asp Ile Thr Trp Glu Val
155 160 165
Val Tyr Leu Asp Gly Thr Lys Ser Glu Glu Asp Leu Phe Leu Gln
170 175 180
Val Tyr Glu Asp Leu Ile Gln Glu Leu Ala Lys Gln Lys Cys Leu
185 190 195
Gln Val Thr Ala
<210> 17
<211> 244
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1723402CD1
<400> 17
Met Glu Leu Thr Ile Phe Ile Leu Arg Leu Ala Ile Tyr Ile Leu
1 5 10 15
Thr Phe Pro Leu Tyr Leu Leu Asn Phe Leu Gly Leu Trp Ser Trp
20 25 30
Ile Cys Lys Lys Trp Phe Pro Tyr Phe Leu Val Arg Phe Thr Val
35 40 45
Ile Tyr Asn Glu Gln Met Ala Ser Lys Lys Arg Glu Leu Phe Ser
50 55 60
Asn Leu Gln Glu Phe Ala Gly Pro Ser Gly Lys Leu Ser Leu Leu
65 70 75
Glu Val Gly Cys Gly Thr Gly Ala Asn Phe Lys Phe Tyr Pro Pro
80 85 90
Gly Cys Arg Val Thr Cys Ile Asp Pro Asn Pro Asn Phe Glu Lys
95 100 105
Phe Leu Ile Lys Ser Ile Ala Glu Asn Arg His Leu Gln Phe Glu
~ 110 115 120
Arg Phe Val Val Ala Ala Gly Glu Asn Met His Gln Val Ala Asp
125 130 135
Gly Ser Val Asp Val Val Val Cys Thr Leu Val Leu Cys Ser Val
140 145 150
Lys Asn Gln Glu Arg Ile Leu Arg Glu Val Cys Arg Val Leu Arg
155 160 165
Pro Gly Gly Ala Phe Tyr Phe Met Glu His Val Ala Ala Glu Cys
170 175 180
Ser Thr Trp Asn Tyr Phe Trp Gln Gln Val Leu Asp Pro Ala Trp
185 190 195
His Leu Leu Phe Asp Gly Cys Asn Leu Thr Arg Glu Ser Trp Lys
200 205 210
Ala Leu Glu Arg Ala Ser Phe Ser Lys Leu Lys Leu Gln His Ile
215 220 225
Gln Ala Pro Leu Ser Trp Glu Leu Val Arg Pro His Ile Tyr Gly
230 235 240
Tyr Ala Val Lys
<210> 18
12/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
<211> 358
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1740585CD1
<400> 18
Met Lys Thr Ala Glu Asn Ile Arg Gly Thr Gly Ser Asp Gly Pro
1 5 10 15
Arg Lys Arg Gly Leu Cys Val Leu Cys Gly Leu Pro Ala Ala Gly
20 25 30
Lys Ser Thr Phe Ala Arg Ala Leu Ala His Arg Leu Gln Gln Glu
35 40 45
Gln Gly Trp Ala Ile Gly Val Val Ala Tyr Asp Asp Val Met Pro
50 55 60
Asp Ala Phe Leu Ala Gly Ala Arg Ala Arg Pro Ala Pro Ser Gln
65 70 75
Trp Lys Leu Leu Arg Gln Glu Leu Leu Lys Tyr Leu Glu Tyr Phe
80 85 90
Leu Met Ala Val Ile Asn Gly Cys Gln Met Ser Val Pro Pro Asn
95 100 105
Arg Thr Glu Ala Met Trp Glu Asp Phe Ile Thr Cys Leu Lys Asp
110 115 120
Gln Asp Leu Ile Phe Ser Ala Ala Phe Glu Ala Gln Ser Cys Tyr
125 130 135
Leu Leu Thr Lys Thr Ala Val Ser Arg Pro Leu Phe Leu Val Leu
140 145 150
Asp Asp Asn Phe Tyr Tyr Gln Ser Met Arg Tyr Glu Val Tyr Gln
155 160 165
Leu Ala Arg Lys Tyr Ser Leu Gly Phe Cys Gln Leu Phe Leu Asp
170 175 180
Cys Pro Leu Glu Thr Cys Leu Gln Arg Asn Gly Gln Arg Pro Gln
185 190 195
Ala Leu Pro Pro Glu Thr Ile His Leu Met Arg Arg Lys Leu Glu
200 205 210
Lys Pro Asn Pro Glu Lys Asn Ala Trp Glu His Asn Ser Leu Thr
215 220 225
Ile Pro Ser Pro Ala Cys Ala Ser Glu Ala Ser Leu Glu Val Thr
230 235 240
Asp Leu Leu Leu Thr Ala Leu Glu Asn Pro Val Lys Tyr Ala Glu
245 250 255
Asp Asn Met Glu Gln Lys Asp Thr Asp Arg Ile Ile Cys Ser Thr
260 265 270
Asn Ile Leu His Lys Thr Asp Gln Thr Leu Arg Arg Ile Val Ser
275 280 285
Gln Thr Met Lys Glu Ala Lys Asp Glu Gln Val Leu Pro His Asn
290 295 300
Leu Lys Leu Leu Ala Glu Glu Leu Asn Lys Leu Lys Ala Glu Phe
305 310 315
Leu Glu Asp Leu Lys Gln Gly Asn Lys Lys Tyr Leu Cys Phe Gln
320 325 330
Gln Thr Ile Asp Ile Pro Asp Val Ile Ser Phe Phe His Tyr Glu
335 340 345
Lys Asp Asn Ile Val Gln Lys Tyr Phe Ser Lys Gln His
350 355
<210> 19
<211> 302
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1810925CD1
13/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
<400> 19
Met Ile Leu Leu Asn Asn Ser His Lys Leu Leu Ala Leu Tyr Lys
1 5 10 15
Ser Leu Ala Arg Ser Ile Pro Glu Ser Leu Lys Val Tyr Gly Ser
20 25 30
Val Tyr His Ile Asn His Gly Asn Pro Phe Asn Met Glu Val Leu
35 40 45
Val Asp Ser Trp Pro Glu Tyr Gln Met Val Ile Ile Arg Pro Gln
50 55 60
Lys Gln Glu Met Thr Asp Asp Met Asp Ser Tyr Thr Asn Val Tyr
65 70 75
Arg Met Phe Ser Lys Glu Pro Gln Lys Ser Glu Glu Val Leu Lys
80 85 90
Asn Cys Glu Ile Val Asn Trp Lys Gln Arg Leu Gln Ile Gln Gly
95 100 105
Leu Gln Glu Ser Leu Gly Glu Gly Ile Arg Val Ala Thr Phe Ser
110 115 120
Lys Ser Val Lys Val Glu His Ser Arg Ala Leu Leu Leu Val Thr
125 130 135
Glu Asp Ile Leu Lys Leu Asn Ala Ser Ser Lys Ser Lys Leu Gly
140 145 150
Ser Trp Ala Glu Thr Gly His Pro Asp Asp Glu Phe Glu Ser Glu
155 160 165
Thr Pro Asn Phe Lys Tyr Ala Gln Leu Asp Val Ser Tyr Ser Gly
170 175 180
Leu Val Asn Asp Asn Trp Lys Arg Gly Lys Asn Glu Arg Ser Leu
185 190 195
His Tyr Ile Lys Arg Cys Ile Glu Asp Leu Pro Ala Ala Cys Met
200 205 210
Leu Gly Pro Glu Gly Val Pro Val Ser Trp Val Thr Met Asp Pro
215 220 225
Ser Cys Glu Val Gly Met Ala Tyr Ser Met Glu Lys Tyr Arg Arg
230 235 240
Thr Gly Asn Met Ala Arg Val Met Val Arg Tyr Met Lys Tyr Leu
245 250 255
Arg Gln Lys Asn Ile Pro Phe Tyr Ile Ser Val Leu Glu Glu Asn
260 265 270
Glu Asp Ser Arg Arg Phe Val Gly Gln Phe Gly Phe Phe Glu Ala
275 280 285
Ser Cys Glu Trp His Gln Trp Thr Cys Tyr Pro Gln Asn Leu Val
290 295 300
Pro Phe
<210> 20
<211> 234
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1915064CD1
<400> 20
Met Ser Ser Glu Val Ser Ala Arg Arg Asp Ala Lys Lys Leu Val
1 5 10 15
Arg Ser Pro Ser Gly Leu Arg Met Val Pro Glu His Arg Ala Phe
20 25 30
Gly Ser Pro Phe Gly Leu Glu Glu Pro Gln Trp Val Pro Asp Lys
35 40 45
Glu Cys Arg Arg Cys Met Gln Cys Asp Ala Lys Phe Asp Phe Leu
50 55 60
Thr Arg Lys His His Cys Arg Arg Cys Gly Lys Cys Phe Cys Asp
65 70 75
Arg Cys Cys Ser Gln Lys Val Pro Leu Arg Arg Met Cys Phe Val
80 85 90
Asp Pro Val Arg Gln Cys Ala Glu Cys Ala Leu Val Ser Leu Lys
14/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
95 100 105
Glu Ala Glu Phe Tyr Asp Lys Gln Leu Lys Val Leu Leu Ser Gly
110 115 120
Ala Thr Phe Leu Val Thr Phe Gly Asn Ser Glu Lys Pro Glu Thr
125 130 135
Met Thr Cys Arg Leu Ser Asn Asn Gln Arg Tyr Leu Phe Leu Asp
140 145 150
Gly Asp Ser His Tyr Glu Ile Glu Ile Val His Ile Ser Thr Val
155 160 165
Gln Ile Leu Thr Glu Gly Phe Pro Pro Gly Gly Gly Asn Ala Arg
170 175 180
Ala Thr Gly Met Phe Leu Gln Tyr Thr Val Pro Gly Thr Glu Gly
185 190 195
Val Thr Gln Leu Lys Leu Thr Val Val Glu Asp Val Thr Val Gly
200 205 210
Arg Arg Gln Ala Val Ala Trp Leu Val Ala Met His Lys Ala Ala
215 220 225
Lys Leu Leu Tyr Glu Ser Arg Asp Gln
230
<210> 21
<211> 403
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2185608CD1
<400> 21
Met Ala Gly Ala Ala Thr Gln Ala Ser Leu Glu Ser Ala Pro Arg
1 5 10 15
Ile Met Arg Leu Val Ala Glu Cys Ser Arg Ser Arg Ala Arg Ala
20 25 30
Gly Glu Leu Trp Leu Pro His Gly Thr Val Ala Thr Pro Val Phe
35 40 45
Met Pro Val Gly Thr Gln Ala Thr Met Lys Gly Ile Thr Thr Glu
50 55 60
Gln Leu Asp Ala Leu Gly Cys Arg Ile Cys Leu Gly Asn Thr Tyr
65 70 75
His Leu Gly Leu Arg Pro Gly Pro Glu Leu Ile Gln Lys Ala Asn
80 85 90
Gly Leu His Gly Phe Met Asn Trp Pro His Asn Leu Leu Thr Asp
95 100 105
Ser Gly Gly Phe Gln Met Val Ser Leu Val Ser Leu Ser Glu Val
110 115 120
Thr Glu Glu Gly Val Arg Phe Arg Ser Pro Tyr Asp Gly Asn Glu
125 130 135
Thr Leu Leu Ser Pro Glu Lys Ser Val Gln Ile Gln Asn Ala Leu
140 145 150
Gly Ser Asp Ile Ile Met Gln Leu Asp Asp Val Val Ser Ser Thr
155 160 165
Val Thr Gly Pro Arg Val Glu Glu Ala Met Tyr Arg Ser Ile Arg
170 175 180
Trp Leu Asp Arg Cys Ile Ala Ala His Gln Arg Pro Asp Lys Gln
185 190 195
Asn Leu Phe Ala Ile Ile Gln Gly Gly Leu Asp Ala Asp Leu Arg
200 205 210
Ala Thr Cys Leu Glu Glu Met Thr Lys Arg Asp Val Pro Gly Phe
215 220 225
Ala Ile Gly Gly Leu Ser Gly Gly Glu Ser Lys Ser Gln Phe Trp
230 235 240
Arg Met Val Ala Leu Ser Thr Ser Arg Leu Pro Lys Asp Lys Pro
245 250 255
Arg Tyr Leu Met Gly Val Gly Tyr Ala Thr Asp Leu Val Val Cys
260 265 270
Val Ala Leu Gly Cys Asp Met Phe Asp Cys Val Phe Pro Thr Arg
15/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
275 280 285
Thr Ala Arg Phe Gly Ser Ala Leu Val Pro Thr Gly Asn Leu Gln
290 295 300
Leu Arg Lys Lys Val Phe Glu Lys Asp Phe Gly Pro Ile Asp Pro
305 310 315
Glu Cys Thr Cys Pro Thr Cys Gln Lys His Ser Arg Ala Phe Leu
320 325 330
His Ala Leu Leu His Ser Asp Asn Thr Ala Ala Leu His His Leu
335 340 345
Thr Val His Asn Ile Ala Tyr Gln Leu Gln Leu Met Ser Ala Val
350 355 360
Arg Thr Ser Ile Val Glu Lys Arg Phe Pro Asp Phe Val Arg Asp
365 370 375
Phe Met Gly Ala Met Tyr Gly Asp Pro Thr Leu Cys Pro Thr Trp
380 385 390
Ala Thr Asp Ala Leu Ala Ser Val Gly Ile Thr Leu Gly
395 400
<210> 22
<211> 487
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2228862CD1
<400> 22
Met Arg Arg Gly Glu Arg Arg Asp Ala Gly Arg Pro Arg Pro Glu
1 5 10 15
Ser Pro Val Pro Ala Gly Arg Ala Ser Leu Glu Glu Pro Pro Asp
20 25 30
Gly Pro Ser Ala Gly Gln Ala Thr Gly Pro Gly Glu Gly Arg Arg
35 40 45
Ser Thr Glu Ser Glu Val Tyr Asp Asp Gly Thr Asn Thr Phe Phe
50 55 60
Trp Arg Ala His Thr Leu Thr Val Leu Phe Ile Leu Thr Cys Thr
65 70 75
Leu Gly Tyr Val Thr Leu Leu Glu Glu Thr Pro Gln Asp Thr Ala
80 85 90
Tyr Asn Thr Lys Arg Gly Ile Val Ala Ser Ile Leu Val Phe Leu
95 100 105
Cys Phe Gly Val Thr Gln Ala Lys Asp Gly Pro Phe Ser Arg Pro
110 115 120
His Pro Ala Tyr Trp Arg Phe Trp Leu Cys Val Ser Val Val Tyr
125 130 135
Glu Leu Phe Leu Ile Phe Ile Leu Phe Gln Thr Val Gln Asp Gly
140 145 150
Arg Gln Phe Leu Lys Tyr Val Asp Pro Lys Leu Gly Val Pro Leu
155 160 165
Pro Glu Arg Asp Tyr Gly Gly Asn Cys Leu Ile Tyr Asp Pro Asp
170 175 180
Asn Glu Thr Asp Pro Phe His Asn Ile Trp Asp Lys Leu Asp Gly
185 190 195
Phe Val Pro Ala His Phe Leu Gly Trp Tyr Leu Lys Thr Leu Met
200 205 210
Ile Arg Asp Trp Trp Met Cys Met Ile Ile Ser Val Met Phe Glu
215 220 225
Phe Leu Glu Tyr Ser Leu Glu His Gln Leu Pro Asn Phe Ser Glu
230 235 240
Cys Trp Trp Asp His Trp Ile Met Asp Val Leu Val Cys Asn Gly
245 250 255
Leu Gly Ile Tyr Cys Gly Met Lys Thr Leu Glu Trp Leu Ser Leu
260 265 270
Lys Thr Tyr Lys Trp Gln Gly Leu Trp Asn Ile Pro Thr Tyr Lys
275 280 285
Gly Lys Met Lys Arg Ile Ala Phe Gln Phe Thr Pro Tyr Ser Trp
16/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
290 295 300
Val Arg Phe Glu Trp Lys Pro Ala Ser Ser Leu Arg Arg Trp Leu
305 310 315
Ala Val Cys Gly Ile Ile Leu Val Phe Leu Leu Ala Glu Leu Asn
320 325 330
Thr Phe Tyr Leu Lys Phe Val Leu Trp Met Pro Pro Glu His Tyr
335 340 345
Leu Val Leu Leu Arg Leu Val Phe Phe Val Asn Val Gly Gly Val
350 355 360
Ala Met Arg Glu Ile Tyr Asp Phe Met Asp Asp Pro Lys Pro His
365 370 375
Lys Lys Leu Gly Pro Gln Ala Trp Leu Val Ala Ala Ile Thr Ala
380 385 390
Thr Glu Leu Leu Ile Val Val Lys Tyr Asp Pro His Thr Leu Thr
395 400 405
Leu Ser Leu Pro Phe Tyr Ile Ser Gln Cys Trp Thr Leu Gly Ser
410 415 420
Val Leu Ala Leu Thr Trp Thr Val Trp Arg Phe Phe Leu Arg Asp
425 430 435
Ile Thr Leu Arg Tyr Lys Glu Thr Arg Trp Gln Lys Trp Gln Asn
440 445 450
Lys Asp Asp Gln Gly Ser Thr Val Gly Asn Gly Asp Gln His Pro
455 460 465
Leu Gly Leu Asp Glu Asp Leu Leu Gly Pro Gly Val Ala Glu Gly
470 475 480
Glu Gly Ala Pro Thr Pro Asn
485
<210> 23
<211> 246
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2235577CD1
<400> 23
Met Asn Asp Met Met Ser Leu Gly Ile His Arg Val Trp Lys Asp
1 5 10 15
Leu Leu Leu Trp Lys Met His Pro Leu Pro Gly Thr Gln Leu Leu
20 25 30
Asp Val Ala Gly Gly Thr Gly Asp Ile Ala Phe Arg Phe Leu Asn
35 40 45
Tyr Val Gln Ser Gln His Gln Arg Lys Gln Lys Arg Gln Leu Arg
50 55 60
Ala Gln Gln Asn Leu Ser Trp Glu Glu Ile Ala Lys Glu Tyr Gln
65 70 75
Asn Glu Glu Asp Ser Leu Gly Gly Ser Arg Val Val Val Cys Asp
80 85 90
Ile Asn Lys Glu Met Leu Lys Val Gly Lys Gln Lys Ala Leu Ala
95 100 105
Gln Gly Tyr Arg Ala Gly Leu Ala Trp Val Leu Gly Asp Ala Glu
110 115 120
Glu Leu Pro Phe Asp Asp Asp Lys Phe Asp Ile Tyr Thr Ile Ala
125 130 135
Phe Gly Ile Arg Asn Val Thr His Ile Asp Gln Ala Leu Gln Glu
140 145 150
Ala His Arg Val Leu Lys Pro Gly Gly Arg Phe Leu Cys Leu Glu
155 160 165
Phe Ser Gln Val Asn Asn Pro Leu Ile Ser Arg Leu Tyr Asp Leu
170 175 180
Tyr Ser Phe Gln Val Ile Pro Val Leu Gly Glu Val Ile Ala Gly
185 190 195
Asp Trp Lys Ser Tyr Gln Tyr Leu Val Glu Ser Ile Arg Arg Phe
200 205 210
Pro Ser Gln Glu Glu Phe Lys Asp Met Ile Glu Asp Ala Gly Phe
17/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
215 220 225
His Lys Val Thr Tyr Glu Ser Leu Thr Ser Gly Ile Val Ala Ile
230 235 240
His Ser Gly Phe Lys Leu
245
<210> 24
<211> 410
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2271680CD1
<400> 24
Met Trp Ser Gly Arg Lys Leu Gly Ser Ser Gly Gly Trp Phe Leu
1 5 10 15
Arg Val Leu Gly Pro Gly Gly Cys Asn Thr Lys Ala Ala Arg Pro
20 25 30
Leu Ile Ser Ser Ala Val Tyr Val Lys Asn Gln Leu Ser Gly Thr
35 40 45
Leu Gln Ile Lys Pro Gly Val Phe Asn Glu Tyr Arg Thr Ile Trp
50 55 60
Phe Lys Ser Tyr Arg Thr Ile Phe Ser Cys Leu Asn Arg Ile Lys
65 70 75
Ser Phe Arg Trp Ser Phe Thr Ser Val Ala Gln Ala Gly Val Gln
80 85 90
Trp Cys Asp Leu Gly Ser Leu Gln Pro Pro Pro Pro Gly Phe Lys
95 100 105
Arg Phe Ser Cys Leu Ser Leu Leu Ser His Trp Asp Tyr Arg Tyr
110 115 120
Pro Trp Ala Arg Leu Tyr Ser Thr Ser Gln Thr Thr Val Asp Ser
125 130 135
Gly Glu Val Lys Thr Phe Leu Ala Leu Ala His Lys Trp Trp Asp
140 145 150
Glu Gln Gly Val Tyr Ala Pro Leu His Ser Met Asn Asp Leu Arg
155 160 165
Val Pro Phe Ile Arg Asp Asn Leu Leu Lys Thr Ile Pro Asn His
170 175 180
Gln Pro Gly Lys Pro Leu Leu Gly Met Lys Ile Leu Asp Val Gly
185 190 195
Cys Gly Gly Gly Leu Leu Thr Glu Pro Leu Gly Arg Leu Gly Ala
200 205 210
Ser Val Ile Gly Ile Asp Pro Val Asp Glu Asn Ile Lys Thr Ala
215 220 225
Gln Cys His Lys Ser Phe Asp Pro Val Leu Asp Lys Arg Ile Glu
230 235 240
Tyr Arg Val Cys Ser Leu Glu Glu Ile Val Glu Glu Thr Ala Glu
245 250 255
Thr Phe Asp Ala Val Val Ala Ser Glu Val Val Glu His Val Ile
260 265 270
Asp Leu Glu Thr Phe Leu Gln Cys Cys Cys Gln Val Leu Lys Pro
275 280 285
Gly Gly Ser Leu Phe Ile Thr Thr Ile Asn Lys Thr Gln Leu Ser
290 295 300
Tyr Ala Leu Gly Ile Val Phe Ser Glu Gln Ile Ala Gly Ile Val
305 310 315
Pro Lys Gly Thr His Thr Trp Glu Lys Phe Val Ser Pro Glu Thr
320 325 330
Leu Glu Ser Ile Leu Glu Ser Asn Gly Leu Ser Val Gln Thr Val
335 340 345
Val Gly Met Leu Tyr Asn Pro Phe Ser Gly Tyr Trp His Trp Ser
350 355 360
Glu Asn Thr Ser Leu Asn Tyr Ala Ala His Ala Val Lys Ser Arg
365 370 375
Val Gln Glu His Pro Ala Ser Ala Glu Phe Val Leu Lys Gly Glu
18/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
380 385 390
Thr Glu Glu Leu Gln Ala Asn Ala Cys Thr Asn Pro Ala Val His
395 400 405
Glu Lys Leu Lys Lys
410
<210> 25
<211> 253
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2325603CD1
<400> 25
Met Asn Phe Ser Gly Gly Gly Arg Gln Glu Ala Ala Gly Ser Arg
1 5 10 15
Ser Arg Arg Ala Pro Arg Pro Arg Glu Gln Asp Arg Asp Val Gln
20 25 30
Leu Ser Lys Ala Leu Ser Tyr Ala Leu Arg His Gly Ala Leu Lys
35 40 45
Leu Gly Leu Pro Met Gly Ala Asp Gly Phe Val Pro Leu Gly Thr
50 55 60
Leu Leu Gln Leu Pro Gln Phe Arg Gly Phe Ser Ala Glu Asp Val
65 70 75
Gln Arg Val Val Asp Thr Asn Arg Lys Gln Arg Phe Ala Leu Gln
80 85 90
Leu Gly Asp Pro Ser Thr Gly Leu Leu Ile Arg Ala Asn Gln Gly
95 100 105
His Ser Leu Gln Val Pro Lys Leu Glu Leu Met Pro Leu Glu Thr
110 115 120
Pro Gln Ala Leu Pro Pro Met Leu Val His Gly Thr Phe Trp Lys
125 130 135
His Trp Pro Ser Ile Leu Leu Lys Gly Leu Ser Cys Gln Gly Arg
140 145 150
Thr His Ile His Leu Ala Pro Gly Leu Pro Gly Asp Pro Gly Ile
155 160 165
Ile Ser Gly Met Arg Ser His Cys Glu Ile Ala Val Phe Ile Asp
170 175 180
Gly Pro Leu Ala Leu Ala Asp Gly Ile Pro Phe Phe Arg Ser Ala
185 190 195
Asn Gly Val Ile Leu Thr Pro Gly Asn Thr Asp Gly Phe Leu Leu
200 205 210
Pro Lys Tyr Phe Lys Glu Ala Leu Gln Leu Arg Pro Thr Arg Lys
215 220 225
Pro Leu Ser Leu Ala Gly Asp Glu Glu Thr Glu Cys Gln Ser Ser
230 235 240
Pro Lys His Ser Ser Arg Glu Arg Arg Arg Ile Gln Gln
245 250
<210> 26
<211> 303
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2356055CD1
<400> 26
Met Lys Gly Gly Phe Thr Gly Gly Asp Glu Tyr Gln Lys His Phe
1 5 10 15
Leu Pro Arg Asp Tyr Leu Ala Thr Tyr Tyr Ser Phe Asn Gly Ser
20 25 30
Pro Ser Pro Glu Ala Glu Met Leu Lys Phe Asn Leu Glu Cys Leu
35 40 45
19/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
His Lys Thr Phe Gly Pro Gly Gly Leu Gln Gly Asp Thr Leu Ile
50 55 60
Asp Ile Gly Ser Gly Pro Thr Ile Tyr Gln Val Leu Ala Ala Cys
65 70 75
Asp Ser Phe Gln Asp Ile Thr Leu Ser Asp Phe Thr Asp Arg Asn
80 85 90
Arg Glu Glu Leu Glu Lys Trp Leu Lys Lys Glu Pro Gly Ala Tyr
95 100 105
Asp Trp Thr Pro Ala Val Lys Phe Ala Cys Glu Leu Glu Gly Asn
110 115 120
Ser Gly Arg Trp Glu Glu Lys Glu Glu Lys Leu Arg Ala Ala Val
125 130 135
Lys Arg Val Leu Lys Cys Asp Val His Leu Gly Asn Pro Leu Ala
140 145 150
Pro Ala Val Leu Pro Leu Ala Asp Cys Val Leu Thr Leu Leu Ala
155 160 165
Met Glu Cys Ala Cys Cys Ser Leu Asp Ala Tyr Arg Ala Ala Leu
170 175 180
Cys Asn Leu Ala Ser Leu Leu Lys Pro Gly Gly His Leu Val Thr
185 190 195
Thr Val Thr Leu Arg Leu Pro Ser Tyr Val Val Gly Lys Arg Glu
200 205 210
Phe Ser Cys Val Ala Leu Glu Lys Glu Glu Val Ala Ala Arg Gln
215 220 225
Cys Pro Gly Glu Glu Ile Ala Lys Glu Arg Arg Leu Gln Met Pro
230 235 240
Pro Pro Cys Asp Val Arg Thr Ser Leu Ser Glu Arg Ser Gly Gln
245 250 255
Asp Thr Gly Lys Arg His Arg Ile Gln Thr Arg Gly Ser Ala Pro
260 265 270
Trp Thr Ala Gln Cys Arg Glu Ser Ala Gly Cys Leu Glu Gly Glu
275 280 285
Ser Arg Gln Gly Cys Glu Gly Ile Phe Gly Cys Cys Gly Ser Cys
290 295 300
Ser Thr Leu
<210> 27
<211> 307
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2448909CD1
<400> 27
Met Gln Lys Gly Lys Gly Arg Thr Ser Arg Ile Arg Arg Arg Lys
1 5 10 15
Leu Cys Gly Ser Ser Glu Ser Arg Gly Val Asn Glu Ser His Lys
20 25 30
Ser Glu Phe Ile Glu Leu Arg Lys Trp Leu Lys Ala Arg Lys Phe
35 40 45
Gln Asp Ser Asn Leu Ala Pro Ala Cys Phe Pro Gly Thr Gly Arg
50 55 60
Gly Leu Met Ser Gln Thr Ser Leu Gln Glu Gly Gln Met Ile Ile
65 70 75
Ser Leu Pro Glu Ser Cys Leu Leu Thr Thr Asp Thr Val Ile Arg
80 85 90
Ser Tyr Leu Gly Ala Tyr Ile Thr Lys Trp Lys Pro Pro Pro Ser
95 100 105
Pro Leu Leu Ala Leu Cys Thr Phe Leu Val Ser Glu Lys His Ala
110 115 120
Gly His Arg Ser Leu Trp Lys Pro Tyr Leu Glu Ile Leu Pro Lys
125 130 135
Ala Tyr Thr Cys Pro Val Cys Leu Glu Pro Glu Val Val Asn Leu
140 145 150
20/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
Leu Pro Lys Ser Leu Lys Ala Lys Ala Glu Glu Gln Arg Ala His
155 160 165
Val Gln Glu Phe Phe Ala Ser Ser Arg Asp Phe Phe Ser Ser Leu
170 175 180
Gln Pro Leu Phe Ala Glu Ala Val Asp Ser Ile Phe Ser Tyr Ser
185 190 195
Ala Leu Leu Trp Ala Trp Cys Thr Val Asn Thr Arg Ala Val Tyr
200 205 210
Leu Arg Pro Arg Gln Arg Glu Cys Leu Ser Ala Glu Pro Asp Thr
215 220 225
Cys Ala Leu Ala Pro Tyr Leu Asp Leu Leu Asn His Ser Pro His
230 235 240
Val Gln Val Lys Ala Ala Phe Asn Glu Glu Thr His Ser Tyr Glu
245 250 255
Ile Arg Thr Thr Ser Arg Trp Arg Lys His Glu Glu Val Phe Ile
260 265 270
Cys Tyr Gly Pro His Asp Asn Gln Arg Leu Phe Leu Glu Tyr Gly
275 280 285
Phe Val Ser Val His Asn Pro His Ala Cys Val Tyr Val Ser Arg
290 295 300
Gly Trp Asn Gln Leu Cys Ser
305
<210> 28
<211> 169
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2631212CD1
<400> 28
Met Lys Gly Ser Arg Ile Glu Leu Gly Asp Val Thr Pro His Asn
1 5 10 15
Ile Lys Gln Leu Lys Arg Leu Asn Gln Val Ile Phe Pro Val Ser
20 25 30
Tyr Asn Asp Lys Phe Tyr Lys Asp Val Leu Glu Val Gly Glu Leu
35 40 45
Ala Lys Leu Ala Tyr Phe Asn Asp Ile Ala Val Gly Ala Val Cys
50 55 60
Cys Arg Val Asp His Ser Gln Asn Gln Lys Arg Leu Tyr Ile Met
65 70 75
Thr Leu Gly Cys Leu Ala Pro Tyr Arg Arg Leu Gly Ile Gly Thr
80 85 90
Lys Met Leu Asn His Val Leu Asn Ile Cys Glu Lys Asp Gly Thr
95 100 105
Phe Asp Asn Ile Tyr Leu His Val Gln Ile Ser Asn Glu Ser Ala
110 115 120
Ile Asp Phe Tyr Arg Lys Phe Gly Phe Glu Ile Ile Glu Thr Lys
125 130 135
Lys Asn Tyr Tyr Lys Arg Ile Glu Pro Ala Asp Ala His Val Leu
140 145 150
Gln Lys Asn Leu Lys Val Pro Ser Gly Gln Asn Ala Asp Val Gln
155 160 165
Lys Thr Asp Asn
<210> 29
<211> 389
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2678733CD1
21/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
<400> 29
Met Arg Val Leu Val Arg Arg Cys Trp Gly Pro Pro Leu Ala His
1 5 10 15
Gly Ala Arg Arg Gly Arg Pro Ser Pro Gln Trp Arg Ala Leu Ala
20 25 30
Arg Leu Gly Trp Glu Asp Cys Arg Asp Ser Arg Val Arg Glu Lys
35 40 45
Pro Pro Trp Arg Val Leu Phe Phe Gly Thr Asp Gln Phe Ala Arg
50 55 60
Glu Ala Leu Arg Ala Leu His Ala Ala Arg Glu Asn Lys Glu Glu
65 70 75
Glu Leu Ile Asp Lys Leu Glu Val Val Thr Met Pro Ser Pro Ser
80 85 90
Pro Lys Gly Leu Pro Val Lys Gln Tyr Ala Val Gln Ser Gln Leu
95 100 105
Pro Val Tyr Glu Trp Pro Asp Val Gly Ser Gly Glu Tyr Asp Val
110 115 120
Gly Val Val Ala Ser Phe Gly Arg Leu Leu Asn Glu Ala Leu Ile
125 130 135
Leu Lys Phe Pro Tyr Gly Ile Leu Asn Val His Pro Ser Cys Leu
140 145 150
Pro Arg Trp Arg Gly Pro Ala Pro Val Ile His Thr Val Leu His
155 160 165
Gly Asp Thr Val Thr Gly Val Thr Ile Met Gln Ile Arg Pro Lys
170 175 180
Arg Phe Asp Val Gly Pro Ile Leu Lys Gln Glu Thr Val Pro Val
185 190 195
Pro Pro Lys Ser Thr Ala Lys Glu Leu Glu Ala Val Leu Ser Arg
200 205 210
Leu Gly Ala Asn Met Leu Ile Ser Val Leu Lys Asn Leu Pro Glu
215 220 225
Ser Leu Ser Asn Gly Arg Gln Gln Pro Met Glu Gly Ala Thr Tyr
230 235 240
Ala Pro Lys Ile Ser Ala Gly Thr Ser Cys Ile Lys Trp Glu Glu
245 250 255
Gln Thr Ser Glu Gln Ile Phe Arg Leu Tyr Arg Ala Ile Gly Asn
260 265 270
Ile Ile Pro Leu Gln Thr Leu Trp Met Ala Asn Thr Ile Lys Leu
275 280 285
Leu Asp Leu Val Glu Val Asn Ser Ser Val Leu Ala Asp Pro Lys
290 295 300
Leu Thr Gly Gln Ala Leu Ile Pro Gly Ser Val Ile Tyr His Lys
305 310 315
Gln Ser Gln Ile Leu Leu Val Tyr Cys Lys Asp Gly Trp Ile Gly
320 325 330
Val Arg Ser Val Met Leu Lys Lys Ser Leu Thr Ala Thr Asp Phe
335 340 345
Tyr Asn Gly Tyr Leu His Pro Trp Tyr Gln Lys Asn Ser Gln Ala
350 355 360
Gln Pro Ser Gln Cys Arg Phe Gln Thr Leu Arg Leu Pro Thr Lys
365 370 375
Lys Lys Gln Lys Lys Thr Val Ala Met Gln Gln Cys Ile Glu
380 385
<210> 30
<211> 600
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2768571CD1
<400> 30
Met Arg Ser Cys Leu Trp Arg Cys Arg His Leu Ser Gln Gly Val
1 5 10 15
Gln Trp Ser Leu Leu Leu Ala Val Leu Val Phe Phe Leu Phe Ala
22/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
20 25 30
Leu Pro Ser Phe Ile Lys Glu Pro Gln Thr Lys Pro Ser Arg His
35 40 45
Gln Arg Thr Glu Asn Ile Lys Glu Arg Ser Leu Gln Ser Leu Ala
50 55 60
Lys Pro Lys Ser Gln Ala Pro Thr Arg Ala Arg Arg Thr Thr Ile
65 70 75
Tyr Ala Glu Pro Val Pro Glu Asn Asn Ala Leu Asn Thr Gln Thr
80 85 90
Gln Pro Lys Ala His Thr Thr Gly Asp Arg Gly Lys Glu Ala Asn
95 100 105
Gln Ala Pro Pro Glu Glu Gln Asp Lys Val Pro His Thr Ala Gln
110 115 120
Arg Ala Ala Trp Lys Ser Pro Glu Lys Glu Lys Thr Met Val Asn
125 130 135
Thr Leu Ser Pro Arg Gly Gln Asp Ala Gly Met Ala Ser Gly Arg
140 145 150
Thr Glu Ala Gln Ser Trp Lys Ser Gln Asp Thr Lys Thr Thr Gln
155 160 165
Gly Asn Gly Gly Gln Thr Arg Lys Leu Thr Ala Ser Arg Thr Val
170 175 180
Ser Glu Lys His Gln Gly Lys Ala Ala Thr Thr Ala Lys Thr Leu
185 190 195
Ile Pro Lys Ser Gln His Arg Met Leu Ala Pro Thr Gly Ala Val
200 205 210
Ser Thr Arg Thr Arg Gln Lys Gly Val Thr Thr Ala Val Ile Pro
215 220 225
Pro Lys Glu Lys Lys Pro Gln Ala Thr Pro Pro Pro Ala Pro Phe
230 235 240
Gln Ser Pro Thr Thr Gln Arg Asn Gln Arg Leu Lys Ala Ala Asn
245 250 255
Phe Lys Ser Glu Pro Arg Trp Asp Phe Glu Glu Lys Tyr Ser Phe
260 265 270
Glu Ile Gly Gly Leu Gln Thr Thr Cys Pro Asp Ser Val Lys Ile
275 280 285
Lys Ala Ser Lys Ser Leu Trp Leu Gln Lys Leu Phe Leu Pro Asn
290 295 300
Leu Thr Leu Phe Leu Asp Ser Arg His Phe Asn Gln Ser Glu Trp
305 310 315
Asp Arg Leu Glu His Phe Ala Pro Pro Phe Gly Phe Met Glu Leu
320 325 330
Asn Tyr Ser Leu Val Gln Lys Val Val Thr Arg Phe Pro Pro Val
335 340 345
Pro Gln Gln Gln Leu Leu Leu Ala Ser Leu Pro Ala Gly Ser Leu
350 355 360
Arg Cys Ile Thr Cys Ala Val Val Gly Asn Gly Gly Ile Leu Asn
365 370 375
Asn Ser His Met Gly Gln Glu Ile Asp Ser His Asp Tyr Val Phe
380 385 390
Arg Leu Ser Gly Ala Leu Ile Lys Gly Tyr Glu Gln Asp Val Gly
395 400 405
Thr Arg Thr Ser Phe Tyr Gly Phe Thr Ala Phe Ser Leu Thr Gln
410 415 420
Ser Leu Leu Ile Leu Gly Asn Arg Gly Phe Lys Asn Val Pro Leu
425 430 435
Gly Lys Asp Val Arg Tyr Leu His Phe Leu Glu Gly Thr Arg Asp
440 445 450
Tyr Glu Trp Leu Glu Ala Leu Leu Met Asn Gln Thr Val Met Ser
455 460 465
Lys Asn Leu Phe Trp Phe Arg His Arg Pro Gln Glu Ala Phe Arg
470 475 480
Glu Ala Leu His Met Asp Arg Tyr Leu Leu Leu His Pro Asp Phe
485 490 495
Leu Arg Tyr Met Lys Asn Arg Phe Leu Arg Ser Lys Thr Leu Asp
500 505 510
Gly Ala His Trp Arg Ile Tyr Arg Pro Thr Thr Gly Ala Leu Leu
515 520 525
23/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
Leu Leu Thr Ala Leu Gln Leu Cys Asp Gln Val Ser Ala Tyr Gly
530 535 540
Phe Ile Thr Glu Gly His Glu Arg Phe Ser Asp His Tyr Tyr Asp
545 550 555
Thr Ser Trp Lys Arg Leu Ile Phe Tyr Ile Asn His Asp Phe Lys
560 565 570
Leu Glu Arg Glu Val Trp Lys Arg Leu His Asp Glu Gly Ile Ile
575 580 585
Arg Leu Tyr Gln Arg Pro Gly Pro Gly Thr Ala Lys Ala Lys Asn
590 595 600
<210> 31
<211> 448
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3189062CD1
<400> 31
Met Arg Glu Asn Val Val Val Ser Asn Met Glu Arg Glu Ser Gly
1 5 10 15
Lys Pro Val Ala Val Val Ala Val Val Thr Glu Pro Trp Phe Thr
20 25 30
Gln Arg Tyr Arg Glu Tyr Leu Gln Arg Gln Lys Leu Phe Asp Thr
35 40 45
Gln His Arg Val Glu Lys Met Pro Asp Gly Ser Val Ala Leu Pro
50 55 60
Val Leu Gly Glu Thr Leu Pro Glu Gln His Leu Gln Glu Leu Arg
65 70 75
Asn Arg Val Ala Pro Gly Ser Pro Cys Met Leu Thr Gln Leu Pro
80 85 90
Asp Pro Val Pro Ser Lys Arg Ala Gln Gly Cys Ser Pro Ala Gln
95 100 105
Lys Leu Cys Leu Glu Val Ser Arg Trp Val Val Gly Arg Gly Val
110 115 120
Lys Trp Ser Ala Glu Leu Glu Ala Asp Leu Pro Arg Ser Trp Gln
125 130 135
Arg His Gly Asn Leu Leu Leu Leu Ser Glu Asp Cys Phe Gln Ala
140 145 150
Lys Gln Trp Lys Asn Leu Gly Pro Glu Leu Trp Glu Thr Val Ala
155 160 165
Leu Ala Leu Gly Val Gln Arg Leu Ala Lys Arg Gly Arg Val Ser
170 175 180
Pro Asp Gly Thr Arg Thr Pro Ala Val Thr Leu Leu Leu Gly Asp
185 190 195
His Gly Trp Val Glu His Val Asp Asn Gly Ile Arg Tyr Lys Phe
200 205 210
Asp Val Thr Gln Cys Met Phe Ser Phe Gly Asn Ile Thr Glu Lys
215 220 225
Leu Arg Val Ala Ser Leu Ser Cys Ala Gly Glu Val Leu Val Asp
230 235 240
Leu Tyr Ala Gly Ile Gly Tyr Phe Thr Leu Pro Phe Leu Val His
245 250 255
Ala Gly Ala Ala Phe Val His Ala Cys Glu Trp Asn Pro His Ala
260 265 270
Val Val Ala Leu Arg Asn Asn Leu Glu Ile Asn Gly Val Ala Asp
275 280 285
Arg Cys Gln Ile His Phe Gly Asp Asn Arg Lys Leu Lys Leu Ser
290 295 300
Asn Ile Ala Asp Arg Val Ile Leu Gly Leu Ile Pro Ser Ser Glu
305 310 315
Glu Gly Trp Pro Ile Ala Cys Gln Val Leu Arg Gln Asp Ala Gly
320 325 330
Gly Ile Leu His Ile His Gln Asn Val Glu Ser Phe Pro Gly Lys
24/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
335 340 345
Asn Leu Gln Ala Leu Gly Val Ser Lys Val Glu Lys Glu His Trp
350 355 360
Leu Tyr Pro Gln Gln Ile Thr Thr Asn Gln Trp Lys Asn Gly Ala
365 370 375
Thr Arg Asp Ser Arg Gly Lys Met Leu Ser Pro Ala Thr Lys Pro
380 385 390
Glu Trp Gln Arg Trp Ala Glu Ser Ala Glu Thr Arg Ile Ala Thr
395 400 405
Leu Leu Gln Gln Val His Gly Lys Pro Trp Lys Thr Gln Ile Leu
410 415 420
His Ile Gln Pro Val Lys Ser Tyr Ala Pro His Val Asp His Ile
425 430 435
Val Leu Asp Leu Glu Cys Cys Pro Cys Pro Ser Val Gly
440 445
<210> 32
<211> 346
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3243884CD1
<400> 32
Met Ala Ala Ser Gly Lys Leu Ser Thr Cys Arg Leu Pro Pro Leu
1 5 10 15
Pro Thr Ile Arg Glu Ile Ile Lys Leu Leu Arg Leu Gln Ala Ala
20 25 30
Lys Gln Leu Ser Gln Asn Phe Leu Leu Asp Leu Arg Leu Thr Asp
35 40 45
Lys Ile Val Arg Lys Ala Gly Asn Leu Thr Asn Ala Tyr Val Tyr
50 55 60
Glu Val Gly Pro Gly Pro Gly Gly Ile Thr Arg Ser Ile Leu Asn
65 70 75
Ala Asp Val Ala Glu Leu Leu Val Val Glu Lys Asp Thr Arg Phe
80 85 90
Ile Pro Gly Leu Gln Met Leu Ser Asp Ala Ala Pro Gly Lys Leu
95 100 105
Arg Ile Val His Gly Asp Val Leu Thr Phe Lys Val Glu Lys Ala
110 115 120
Phe Ser Glu Ser Leu Lys Arg Pro Trp Glu Asp Asp Pro Pro Asn
125 130 135
Val His Ile Ile Gly Asn Leu Pro Phe Ser Val Ser Thr Pro Leu
140 145 150
Ile Ile Lys Trp Leu Glu Asn Ile Ser Cys Arg Asp Gly Pro Phe
155 160 165
Val Tyr Gly Arg Thr Gln Met Thr Leu Thr Phe Gln Lys Glu Val
170 175 180
Ala Glu Arg Leu Ala Ala Asn Thr Gly Ser Lys Gln Arg Ser Arg
185 190 195
Leu Ser Val Met Ala Gln Tyr Leu Cys Asn Val Arg His Ile Phe
200 205 210
Thr Ile Pro Gly Gln Ala Phe Val Pro Lys Pro Glu Val Asp Val
215 220 225
Gly Val Val His Phe Thr Pro Leu Ile Gln Pro Lys Ile Glu Gln
230 235 240
Pro Phe Lys Leu Val Glu Lys Val Val Gln Asn Val Phe Gln Phe
245 250 255
Arg Arg Lys Tyr Cys His Arg Gly Leu Arg Met Leu Phe Pro Glu
260 265 270
Ala Gln Arg Leu Glu Ser Thr Gly Arg Leu Leu Glu Leu Ala Asp
275 280 285
Ile Asp Pro Thr Leu Arg Pro Arg Gln Leu Ser Ile Ser His Phe
290 295 300
Lys Ser Leu Cys Asp Val Tyr Arg Lys Met Cys Asp Glu Asp Pro
25/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
305 310 315
Gln Leu Phe Ala Tyr Asn Phe Arg Glu Glu Leu Lys Arg Arg Lys
320 325 330
Ser Lys Asn Glu Glu Lys Glu Glu Asp Asp Ala Glu Asn Tyr Arg
335 340 345
Leu
<210> 33
<211> 173
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3400578CD1
<400> 33
Met Ala Ser Ile Leu Arg Thr Pro Gln Ala Leu Gln Leu Thr Leu
1 5 10 15
Ala Leu Ile Lys Pro Asp Ala Val Ala His Pro Leu Ile Leu Glu
20 25 30
Ala Val His Gln Gln Ile Leu Ser Asn Lys Phe Leu Ile Val Arg
35 40 45
Met Arg Glu Leu Leu Trp Arg Lys Glu Asp Cys Gln Arg Phe Tyr
50 55 60
Arg Glu His Glu Ala Gly Pro Ile Arg Ala Tyr Ile Leu Ala His
65 70 75
Lys Asp Ala Ile Gln Leu Trp Arg Thr Leu Met Gly Pro Thr Arg
80 85 90
Val Phe Arg Ala Arg His Val Ala Pro Asp Ser Ile Arg Gly Ser
95 100 105
Phe Gly Leu Thr Asp Thr Arg Asn Thr Thr His Gly Ser Asp Ser
110 115 120
Val Val Ser Ala Ser Arg Glu Ile Ala Ala Phe Phe Pro Asp Phe
125 130 135
Ser Glu Gln Arg Trp Tyr Glu Glu Glu Glu Pro Gln Leu Arg Cys
140 145 150
Gly Pro Val Cys Tyr Ser Pro Glu Gly Gly Val His Tyr Val Ala
155 160 165
Gly Thr Gly Gly Leu Gly Pro Ala
170
<210> 34
<211> 445
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3422577CD1
<400> 34
Met Thr Glu Leu Arg Gln Arg Val Ala His Glu Pro Val Ala Pro
1 5 10 15
Pro Glu Asp Lys Glu Ser Glu Ser Glu Ala Lys Val Asp Gly Glu
20 25 30
Thr Ala Ser Asp Ser Glu Ser Arg Ala Glu Ser Ala Pro Leu Pro
35 40 45
Val Ser Ala Asp Asp Thr Pro Glu Val Leu Asn Arg Ala Leu Ser
50 55 60
Asn Leu Ser Ser Arg Trp Lys Asn Trp Trp Val Arg Gly Ile Leu
65 70 75
Thr Leu Ala Met Ile Ala Phe Phe Phe Ile Ile Ile Tyr Leu Gly
80 85 90
Pro Met Val Leu Met Ile Ile Val Met Cys Val Gln Ile Lys Cys
95 100 105
26/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
Phe His Glu Ile Ile Thr Ile Gly Tyr Asn Val Tyr His Ser Tyr
110 115 120
Asp Leu Pro Trp Phe Arg Thr Leu Ser Trp Tyr Phe Leu Leu Cys
125 130 135
Val Asn Tyr Phe Phe Tyr Gly Glu Thr Val Thr Asp Tyr Phe Phe
140 145 150
Thr Leu Val Gln Arg Glu Glu Pro Leu Arg Ile Leu Ser Lys Tyr
155 160 165
His Arg Phe Ile Ser Phe Thr Leu Tyr Leu Ile Gly Phe Cys Met
170 175 180
Phe Val Leu Ser Leu Val Lys Lys His Tyr Arg Leu Gln Phe Tyr
185 190 195
Met Phe Gly Trp Thr His Val Thr Leu Leu Ile Val Val Thr Gln
200 205 210
Ser His Leu Val Ile His Asn Leu Phe Glu Gly Met Ile Trp Phe
215 220 225
Ile Val Pro Ile Ser Cys Val Ile Cys Asn Asp Ile Met Ala Tyr
230 235 240
Met Phe Gly Phe Phe Phe Gly Arg Thr Pro Leu Ile Lys Leu Ser
245 250 255
Pro Lys Lys Thr Trp Glu Gly Phe Ile Gly Gly Phe Phe Ala Thr
260 265 270
Val Val Phe Gly Leu Leu Leu Ser Tyr Val Met Ser Gly Tyr Arg
275 280 285
Cys Phe Val Cys Pro Val Glu Tyr Asn Asn Asp Thr Asn Ser Phe
290 295 300
Thr Val Asp Cys Glu Pro Ser Asp Leu Phe Arg Leu Gln Glu Tyr
305 310 315
Asn Ile Pro Gly Val Ile Gln Ser Val Ile Gly Trp Lys Thr Val
320 325 330
Arg Met Tyr Pro Phe Gln Ile His Ser Ile Ala Leu Ser Thr Phe
335 340 345
Ala Ser Leu Ile Gly Pro Phe Gly Gly Phe Phe Ala Ser Gly Phe
350 355 360
Lys Arg Ala Phe Lys Ile Lys Asp Phe Ala Asn Thr Ile Pro Gly
365 370 375
His Gly Gly Ile Met Asp Arg Phe Asp Cys Gln Tyr Leu Met Ala
380 385 390
Thr Phe Val Asn Val Tyr Ile Ala Ser Phe Ile Arg Gly Pro Asn
395 400 405
Pro Ser Lys Leu Ile Gln Gln Phe Leu Thr Leu Arg Pro Asp Gln
410 415 420
Gln Leu His Ile Phe Asn Thr Leu Arg Ser His Leu Ile Asp Lys
425 430 435
Gly Met Leu Thr Ser Thr Thr Glu Asp Glu
440 445
<210> 35
<211> 420
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3706809CD1
<400> 35
Met Ala Ala Leu Val Arg Pro Ala Arg Phe Val Val Arg Pro Leu
1 5 10 15
Leu Gln Val Val Gln Ala Trp Asp Leu Asp Ala Arg Arg Trp Val
20 25 30
Arg Ala Leu Arg Arg Ser Pro Val Lys Val Val Phe Pro Ser Gly
35 40 45
Glu Val Val Glu Gln Lys Arg Ala Pro Gly Lys Gln Pro Arg Lys
50 55 60
Ala Pro Ser Glu Ala Ser Ala Gln Glu Gln Arg Glu Lys Gln Pro
65 70 75
27/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
Leu Glu Glu Ser Ala Ser Arg Ala Pro Ser Thr Trp Glu Glu Ser
80 85 90
Gly Leu Arg Tyr Asp Lys Ala Tyr Pro Gly Asp Arg Arg Leu Ser
95 100 105
Ser Val Met Thr Ile Val Lys Ser Arg Pro Phe Arg Glu Lys Gln
110 115 120
Gly Lys Ile Leu Leu Glu Gly Arg Arg Leu Ile Ser Asp Ala Leu
125 130 135
Lys Ala Gly Ala Val Pro Lys Met Phe Phe Phe Ser Arg Leu Glu
140 145 150
Tyr Leu Lys Glu Leu Pro Val Asp Lys Leu Lys Gly Val Ser Leu
155 160 165
Ile Lys Val Lys Phe Glu Asp Ile Lys Asp Trp Ser Asp Leu Val
170 175 180
Thr Pro Gln Gly Ile Met Gly Ile Phe Ala Lys Pro Asp His Val
185 190 195
Lys Met Thr Tyr Pro Lys Thr Gln Leu Gln His Ser Leu Pro Leu
200 205 210
Leu Leu Ile Cys Asp Asn Leu Arg Asp Pro Gly Asn Leu Gly Thr
215 220 225
Ile Leu Arg Ser Ala Ala Gly Ala Gly Cys Ser Lys Val Leu Leu
230 235 240
Thr Lys Gly Cys Val Asp Ala Trp Glu Pro Lys Val Leu Arg Ala
245 250 255
Gly Met Gly Ala His Phe Arg Met Pro Ile Ile Asn Asn Leu Glu
260 265 270
Trp Glu Thr Val Pro Asn Tyr Leu Pro Pro Asp Thr Arg Val Tyr
275 280 285
Val Ala Asp Asn Cys Gly Leu Tyr Ala Gln Ala Glu Met Ser Asn
290 295 300
Lys Ala Ser Asp His Gly Trp Val Cys Asp Gln Arg Val Met Lys
305 310 315
Phe His Lys Tyr Glu Glu Glu Glu Asp Val Glu Thr Gly Ala Ser
320 325 330
Gln Asp Trp Leu Pro His Val Glu Val Gln Ser Tyr Asp Ser Asp
335 340 345
Trp Thr Glu Ala Pro Ala Ala Val Val Ile Gly Gly Glu Thr Tyr
350 355 360
Gly Val Ser Leu Glu Ser Leu Gln Leu Ala Glu Ser Thr Gly Gly
365 370 375
Lys Arg Leu Leu Ile Pro Val Val Pro Gly Val Asp Ser Leu Asn
380 385 390
Ser Ala Met Ala Ala Ser Ile Leu Leu Phe Glu Gly Lys Arg Gln
395 400 405
Leu Arg Gly Arg Ala Glu Asp Leu Ser Arg Asp Arg Ser Tyr His
410 415 420
<210> 36
<211> 354
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3745914CD1
<400> 36
Met Ala Pro Ala Lys Ala Thr Asn Val Val Arg Leu Leu Leu Gly
1 5 10 15
Ser Thr Ala Leu Trp Leu Ser Gln Leu ~Gly Ser Gly Thr Val Ala
20 25 30
Ala Ser Lys Ser Val Thr Ala His Leu Ala Ala Lys Trp Pro Glu
35 40 45
Thr Pro Leu Leu Leu Glu Ala Ser Glu Phe Met Ala Glu Glu Ser
50 55 60
Asn Glu Lys Phe Trp Gln Phe Leu Glu Thr Val Gln Glu Leu Ala
28/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
65 70 75
Ile Tyr Lys Gln Thr Glu Ser Asp Tyr Ser Tyr Tyr Asn Leu Ile
80 85 90
Leu Lys Lys Ala Gly Gln Phe Leu Asp Asn Leu His Ile Asn Leu
95 100 105
Leu Lys Phe Ala Phe Ser Ile Arg Ala Tyr Ser Pro Ala Ile Gln
110 115 120
Met Phe Gln Gln Ile Ala Ala Asp Glu Pro Pro Pro Asp Gly Cys
125 130 135
Asn Ala Phe Val Val Ile His Lys Lys His Thr Cys Lys Ile Asn
140 145 150
Glu Ile Lys Lys Leu Leu Lys Lys Ala Ala Ser Arg Thr Arg Pro
155 160 165
Tyr Leu Phe Lys Gly Asp His Lys Phe Pro Thr Asn Lys Glu Asn
170 175 180
Leu Pro Val Val Ile Leu Tyr Ala Glu Met Gly Thr Arg Thr Phe
185 190 195
Ser Ala Phe His Lys Val Leu Ser Glu Lys Ala Gln Asn Glu Glu
200 205 210
Ile Leu Tyr Val Leu Arg His Tyr Ile Gln Lys Pro Ser Ser Arg
215 220 225
Lys Met Tyr Leu Ser Gly Tyr Gly Val Glu Leu Ala Ile Lys Ser
230 235 240
Thr Glu Tyr Lys Ala Leu Asp Asp Thr Gln Val Lys Thr Val Thr
245 250 255
Asn Thr Thr Val Glu Asp Glu Thr Glu Thr Asn Glu Val Gln Gly
260 265 270
Phe Leu Phe Gly Lys Leu Lys Glu Ile Tyr Ser Asp Leu Arg Asp
275 280 285
Asn Leu Thr Ala Phe Gln Lys Tyr Leu Ile Glu Ser Asn Lys Gln
290 295 300
Met Met Pro Leu Lys Val Trp Glu Leu Gln Asp Leu Ser Phe Gln
305 310 315
Ala Ala Ser Gln Ile Met Ser Ala Pro Val Tyr Asp Ala Ile Lys
320 325 330
Leu Met Lys Asp Ile Ser Gln Asn Phe Pro Ile Lys Ala Arg Val
335 340 345
Gln Met Ile Gly Asn Val Leu Ile Gly
350
<210> 37
<211> 198
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4000776CD1
<400> 37
Met Ser Ser Lys Arg Ser His Tyr Asp Ser Ala Leu Lys Arg Lys
1 5 10 15
Val Ile Val Tyr Ala Glu Lys His Gly Asn Arg Ala Ala Gly Arg
20 25 30
Thr Phe Asp Ile Ser Glu Ala Asn Ile Arg Arg Trp Arg Asn Asp
35 40 45
Arg Asn Ser Ile Phe Ser Cys Lys Ala Thr Thr Lys Cys Phe Thr
50 55 60
Gly Pro Lys Lys Gly Arg Tyr Pro Gln Val Asp Glu Ala Val Leu
65 70 75
Arg Phe Val Ser Glu Thr Arg Ala Lys Gly Leu Pro Ile Thr Arg
80 85 90
Gln Ala Met Gln Leu Lys Ala Gly Glu Val Ala Lys Thr Leu Gly
95 100 105
Ile Asp Glu Thr Lys Phe Lys Ala Thr Arg Gly Trp Cys Asp Arg
110 115 120
Phe Met Arg Arg Ala Gly Leu Ser Leu Arg His Gln Thr Ser Phe
29/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
125 130 135
Cys Pro Lys Leu Pro Thr Ala Ile Lys Gln Lys Thr Val Leu Glu
140 145 150
His Ser Phe Lys Lys Cys Cys Ile Thr Ser Thr Leu Asp Asn Thr
155 160 165
Gly Arg Asp Val Leu Trp Lys Asn Ala Asp Ile Asn Asp Cys Gly
170 175 180
Leu Lys Ser Asp Ser Glu Glu Leu Asp Ser Glu Tyr Glu Val Ile
185 190 195
Ile Ile Thr
<210> 38
<211> 296
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4071304CD1
<400> 38
Met Met Leu Pro Leu Gln Gly Ala Gln Met Leu Gln Met Leu Glu
1 5 10 15
Lys Ser Leu Arg Lys Ser Leu Pro Ala Ser Leu Lys Val Tyr Gly
20 25 30
Thr Val Phe His Ile Asn His Gly Asn Pro Phe Asn Leu Lys Ala
35 40 45
Val Val Asp Lys Trp Pro Asp Phe Asn Thr Val Val Val Cys Pro
50 55 60
Gln Glu Gln Asp Met Thr Asp Asp Leu Asp His Tyr Thr Asn Thr
65 70 75
Tyr Gln Ile Tyr Ser Lys Asp Pro Gln Asn Cys Gln Glu Phe Leu
80 85 90
Gly Ser Pro Glu Leu Ile Asn Trp Lys Gln His Leu Gln Ile Gln
95 - 100 105
Ser Ser Gln Pro Ser Leu Asn Glu Ala Ile Gln Asn Leu Ala Ala
110 115 120
Ile Lys Ser Phe Lys Val Lys Gln Thr Gln Arg Ile Leu Tyr Met
125 130 135
Ala Ala Glu Thr Ala Lys Glu Leu Thr Pro Phe Leu Leu Lys Ser
140 145 150
Lys Ile Leu Ser Pro Ser Gly Gly Lys Pro Lys Ala Ile Asn Gln
155 160 165
Glu Met Phe Lys Leu Ser Ser Met Asp Val Thr His Ala His Leu
170 175 180
Val Asn Lys Phe Trp His Phe Gly Gly Asn Glu Arg Ser Gln Arg
185 190 195
Phe Ile Glu Arg Cys Ile Gln Thr Phe Pro Thr Cys Cys Leu Leu
200 205 210
Gly Pro Glu Gly Thr Pro Val Cys Trp Asp Leu Met Asp Gln Thr
215 220 225
Gly Glu Met Arg Met Ala Gly Thr Phe Ala Glu Tyr Arg Leu His
230 235 240
Gly Leu Val Thr Tyr Val Ile Tyr Ser His Ala Gln Lys Leu Gly
245 250 255
Lys Leu Gly Phe Pro Val Tyr Ser His Val Asp Tyr Ser Asn Glu
260 265 270
Ala Met Gln Lys Met Ser Tyr Thr Leu Gln His Val Pro Ile Pro
275 280 285
Arg Ser Trp Asn Gln Trp Asn Cys Val Pro Leu
290 295
<210> 39
<211> 214
<212> PRT
<213> Homo sapiens
30/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
<220>
<221> misc_feature
<223> Incyte ID No: 4344970CD1
<400> 39
Met Ala Gly Glu Asn Phe Ala Thr Pro Phe His Gly His Val Gly
1 5 10 15
Arg Gly Ala Phe Ser Asp Val Tyr Glu Pro Ala Glu Asp Thr Phe
20 25 30
Leu Leu Leu Asp Ala Leu Glu Ala Ala Ala Ala Glu Leu Ala Gly
35 40 45
Val Glu Ile Cys Leu Glu Val Gly Ser Gly Ser Gly Val Val Ser
50 55 60
Ala Phe Leu Ala Ser Met Ile Gly Pro Gln Ala Leu Tyr Met Cys
65 70 75
Thr Asp Ile Asn Pro Glu Ala Ala Ala Cys Thr Leu Glu Thr Ala
80 85 90
Arg Cys Asn Lys Val His Ile Gln Pro Val Ile Thr Asp Leu Val
95 100 105
Lys Gly Leu Leu Pro Arg Leu Thr Glu Lys Val Asp Leu Leu Val
110 115 120
Phe Asn Pro Pro Tyr Val Val Thr Pro Pro Gln Glu Val Gly Ser
125 130 135
His Gly Ile Glu Ala Ala Trp Ala Gly Gly Arg Asn Gly Arg Glu
140 145 150
Val Met Asp Arg Phe Phe Pro Leu Val Pro Asp Leu Leu Ser Pro
155 160 165
Arg Gly Leu Phe Tyr Leu Val Thr Ile Lys Glu Asn Asn Pro Glu
170 175 180
Glu Ile Leu Lys Ile Met Lys Thr Lys Gly Leu Gln Gly Thr Thr
185 190 195
Ala Leu Ser Arg Gln Ala Gly Gln Glu Thr Leu Ser Val Leu Lys
200 205 210
Phe Thr Lys Ser
<210> 40
<211> 322
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5392302CD1
<400> 40
Met Ala Ala Ser Gly Glu Pro Gln Arg Gln Trp Gln Glu Glu Val
1 5 10 15
Ala Ala Val Val Val Val Gly Ser Cys Met Thr Asp Leu Val Ser
20 25 30
Leu Thr Ser Arg Leu Pro Lys Thr Gly Glu Thr Ile His Gly His
35 40 45
Lys Phe Phe Ile Gly Phe Gly Gly Lys Gly Ala Asn Gln Cys Val
50 55 60
Gln Ala Ala Arg Leu Gly Ala Met Thr Ser Met Val Cys Lys Val
65 70 75
Gly Lys Asp Ser Phe Gly Asn Asp Tyr Ile Glu Asn Leu Lys Gln
80 85 90
Asn Asp Ile Ser Thr Glu Phe Thr Tyr Gln Thr Lys Asp Ala Ala
95 100 105
Thr Gly Thr Ala Ser Ile Ile Val Asn Asn Glu Gly Gln Asn Ile
110 115 120
Ile Val Ile Val Ala Gly Ala Asn Leu Leu Leu Asn Thr Glu Asp
125 130 135
Leu Arg Ala Ala Ala Asn Val Ile Ser Arg Ala Lys Val Met Val
140 145 150
Cys Gln Leu Glu Ile Thr Pro Ala Thr Ser Leu Glu Ala Leu Thr
31/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
155 160 165
Met Ala Arg Arg Ser Gly Val Lys Thr Leu Phe Asn Pro Ala Pro
170 175 180
Ala Ile Ala Asp Leu Asp Pro Gln Phe Tyr Thr Leu Ser Asp Val
185 190 195
Phe Cys Cys Asn Glu Ser Glu Ala Glu Ile Leu Thr Gly Leu Thr
200 205 210
Val Gly Ser Ala Ala Asp Ala Gly Glu Ala Ala Leu Val Leu Leu
215 220 225
Lys Arg Gly Cys Gln Val Val Ile Ile Thr Leu Gly Ala Glu Gly
230 235 240
Cys Val Val Leu Ser Gln Thr Glu Pro Glu Pro Lys His Ile Pro
245 250 255
Thr Glu Lys Val Lys Ala Val Asp Thr Thr Gly Ala Gly Asp Ser
260 265 270
Phe Val Gly Ala Leu Ala Phe Tyr Leu Ala Tyr Tyr Pro Asn Leu
275 280 285
Ser Leu Glu Asp Met Leu Asn Arg Ser Asn Phe Ile Ala Ala Val
290 295 300
Ser Val Gln Ala Ala Gly Thr Gln Ser Ser Tyr Pro Tyr Lys Lys
305 310 315
Asp Leu Pro Leu Thr Leu Phe
320
<210> 41
<211> 87
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5555235CD1
<400> 41
Met Ser Thr Ser Val Pro Gln Gly His Thr Trp Thr Gln Arg Val
1 5 10 15
Lys Lys Asp Asp Glu Glu Glu Asp Pro Leu Asp Gln Leu Ile Ser
20 25 30
Arg Ser Gly Cys Ala Ala Ser His Phe Ala Val Gln Glu Cys Met
35 40 45
Ala Gln His Gln Asp Trp Arg Gln Cys Gln Pro Gln Val Gln Ala
50 55 60
Phe Lys Asp Cys Met Ser Glu Gln Gln Ala Arg Arg Gln Glu Glu
65 70 75
Leu Gln Arg Arg Gln Glu Gln Ala Gly Ala His His
80 85
<210> 42
<211> 378
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5573296CD1
<400> 42
Met Asp Leu Ala Gly Leu Leu Lys Ser Gln Phe Leu Cys His Leu
1 5 10 15
Val Phe Cys Tyr Val Phe Ile Ala Ser Gly Leu Ile Ile Asn Thr
20 25 30
Ile Gln Leu Phe Thr Leu Leu Leu Trp Pro Ile Asn Lys Gln Leu
35 40 45
Phe Arg Lys Ile Asn Cys Arg Leu Ser Tyr Cys Ile Ser Ser Gln
50 55 60
Leu Val Met Leu Leu Glu Trp Trp Ser Gly Thr Glu Cys Thr Ile
65 70 75
32/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
Phe Thr Asp Pro Arg Ala Tyr Leu Lys Tyr Gly Lys Glu Asn Ala
80 85 90
Ile Val Val Leu Asn His Lys Phe Glu Ile Asp Phe Leu Cys Gly
95 100 105
Trp Ser Leu Ser Glu Arg Phe Gly Leu Leu Gly Gly Ser Lys Val
110 115 120
Leu Ala Lys Lys Glu Leu Ala Tyr Val Pro Ile Ile Gly Trp Met
125 130 135
Trp Tyr Phe Thr Glu Met Val Phe Cys Ser Arg Lys Trp Glu Gln
140 145 150
Asp Arg Lys Thr Val Ala Thr Ser Leu Gln His Leu Arg Asp Tyr
155 160 165
Pro Glu Lys Tyr Phe Phe Leu Ile His Cys Glu Gly Thr Arg Phe
170 175 180
Thr Glu Lys Lys His Glu Ile Ser Met Gln Val Ala Arg Ala Lys
185 190 195
Gly Leu Pro Arg Leu Lys His His Leu Leu Pro Arg Thr Lys Gly
200 205 210
Phe Ala Ile Thr Val Arg Ser Leu Arg Asn Val Val Ser Ala Val
215 220 225
Tyr Asp Cys Thr Leu Asn Phe Arg Asn Asn Glu Asn Pro Thr Leu
230 235 240
Leu Gly Val Leu Asn Gly Lys Lys Tyr His Ala Asp Leu Tyr Val
245 250 255
Arg Arg Ile Pro Leu Glu Asp Ile Pro Glu Asp Asp Asp Glu Cys
260 265 270
Ser Ala Trp Leu His Lys Leu Tyr Gln Glu Lys Asp Ala Phe Gln
275 280 285
Glu Glu Tyr Tyr Arg Thr Gly Thr Phe Pro Glu Thr Pro Met Val
290 295 300
Pro Pro Arg Arg Pro Trp Thr Leu Val Asn Trp Leu Phe Trp Ala
305 310 315
Ser Leu Val Leu Tyr Pro Phe Phe Gln Phe Leu Val Ser Met Ile
320 325 330
Arg Ser Gly Ser Ser Leu Thr Leu Ala Ser Phe Ile Leu Val Phe
335 340 345
Phe Val Ala Ser Val Gly Val Arg Trp Met Ile Gly Val Thr Glu
350 355 360
Ile Asp Lys Gly Ser Ala Tyr Gly Asn Ser Asp Ser Lys Gln Lys
365 370 375
Leu Asn Asp
<210> 43
<211> 1322
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 016233CB1
<400> 43
gcagcatcac gctgacctct gcctgggatg taaaccggac cagccgctgc gggcaaagga 60
aggctcttgg ctccttcggg aaacccagcc ccgtcaccgg gctccgagcg gctcgcaggc 120
gagcgacagc gacctcagcc ccggcagcgc ccagcggcgg ctgcggaaag cggagggagt 180
ccgacgcggg cgcgggcggg gagcgtgcgt ccgttcgcac aggcagcggg aggaggggcg 240
gcgcgaacca tggccgggga cagcgagcag accctgcaga accaccagca gcccaacggc 300
ggcgagccct tccttatagg cgtcagcggg ggaacagcta gcggcaagtc ttccgtgtgt 360
gctaagatcg tgcagctcct ggggcagaat gaggtggact atcgccagaa gcaggtggtc 420
atcctgagcc aggatagctt ctaccgtgtc cttacctcgg agcagaaggc caaagccctg 480
aagggccagt tcaactttga ccacccggat gcctttgaca atgaactcat tctcaaaaca 540
ctcaaagaaa tcactgaagg gaaaacagtc cagatccccg tgtatgactt tgtctcccat 600
tcccggaagg aggagacagt tactgtctat cccgcagacg tggtgctctt tgaagggatc 660
ctggccttct actcccagga ggtacgagac ctgttccaga tgaagctttt tgtggataca 720
gatgcggaca cccggctctc acgcagagta ttaagggaca tcagcgagag aggcagggat 780
cttgagcaga ttttatctca gtacattacg ttcgtcaagc ctgcctttga ggaattctgc 840
33/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
ttgccaacaa agaagtatgc tgatgtgatc atccctagag gtgcagataa tctggtggcc 900
atcaacctca tcgtgcagca catccaggac atcctgaatg gagggccctc caaacggcag 960
accaatggct gtctcaacgg ctacacccct tcacgcaaga ggcaggcatc ggagtccagc 1020
agcaggccgc attgacccgt ctccatcgga ccccagcccc tatctccaag agacagagga 1080
ggggtcagga ggcactgctc atctgtacat actgtttcct atgacattac tgtatttaag 1140
aaaacaccat ggagatgaaa tgcctttgat tttttttttc tttttgtact ttggaacgac 1200
aaaatgaaac agaacttgac cctgagctta aataacaaaa ctgtgccaac tactactggt 1260
gatgcctaat tatgaatcca acgtgtaacc agttataaat acatatatat ataaaaaaaa 1320
as 1322
<210> 44
<211> 1302
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 078336CB1
<400> 44
aagttttggt gtttaaatgt tgtgttctat ctcttccact acaaacagga tacgcattgg 60
tgtggtatct acttagcaac agaaataaag aaattagatc agtgccagtt tgaactcatc 120
atccagtggc tttctaaatc tgtaggcaag actcttaata aataaatagt caagccactt 180
gaaatcaggg atccaatact cttctcccat agcaaaatca agagctgttt aagctctaag 240
gctctatcta gcccaaaaac aaatgctata atgttttact tggtggtgtt tctaaattca 300
ggtgatatcc aagaactgta tgacaccacc ttggccctgg gccacgcggc ggctttctca 360
gatgactgcg atttgccctc tgctcaggac ataaacagac tcgtgggact tcagaacaca 420
tatatgggct atctggacta ccggaagaag gccatcaagg accttggcat cagccccagc 480
acctgctctt tcaatcctgg tgtgattgtt gccaacatga cagaatggaa gcaccagcgc 540
atcaccaagc aattggagaa atggatgcaa aagaatgtgg aggaaaacct ctatagcagc 600
tccctgggag gaggggtggc cacctcccca atgctgattg tgtttcatgg gaaatattcc 660
acaattaacc ccctgtggca cataaggcac ctgggctgga atccagatgc cagatattcg 720
gagcattttc tgcaggaagc taaattactc cactggaatg gaagacataa accttgggac 780
ttccctagtg ttcacaacga cttatgggaa agctggtttg ttcctgaccc tgcagggata 840
tttaaactca atcaccatag ctgatataac tctaccctta aaatattccc tgtatagaaa 900
tgtggaattg tccctttgta gccaactata acattgttct ttatgaatat tacctttgat 960
acatatgatc cacaatataa aaaccaaaaa ctactgtgtg caaattatac cttggaccat 1020
ataggcattg attaacttct ttaagtacat gtgataacta tggaaatcaa gattatgtga 1080
ctgaaaaaca taaaggaaga gacccatcta gataacagca atcaacctgc ttaattctga 1140
atgacaatta tatccacaaa tttttaaaac ttctacatgt atttttcaca tgaagatctc 1200
cttaacaggt tgccaacctt ttcttttata aaactattac atttaaaata tggacgtctg 1260
aaaaataaaa tattcatcat ttttatgaaa aaaaaaaaaa as 1302
<210> 45
<211> 1771
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 130117CB1
<400> 45
tgacttacag ctcttataaa ctagtggcaa tttctgaacc cagccggctc catctcagct 60
tctggtttct aagtccatgt gccaaaggct gccaggaagg agacgccttc ctgagtcctg 120
gatctttctt ccttctggaa atctttgact gtgggtagtt atttatttct gaataagagc 180
gtccacgcat catggacctc gcgggactgc tgaagtctca gttcctgtgc cacctggtct 240
tctgctacgt ctttattgcc tcagggctaa tcatcaacac cattcagctc ttcactctcc 300
tcctctggcc cattaacaag cagctcttcc ggaagatcaa ctgcagactg tcctattgca 360
tctcaagcca gctggtgatg ctgctggagt ggtggtcggg cacggaatgc accatcttca 420
cggacccgcg cgcctacctc aagtatggga aggaaaatgc catcgtggtt ctcaaccaca 480
agtttgaaat tgactttctg tgtggctgga gcctgtccga acgctttggg ctgttagggg 540
gctccaaggt cctggccaag aaagagctgg cctatgtccc aattatcggc tggatgtggt 600
acttcaccga gatggtcttc tgttcgcgca agtgggagca ggatcgcaag acggttgcca 660
ccagtttgca gcacctccgg gactaccccg agaagtattt tttcctgatt cactgtgagg 720
gcacacggtt cacggagaag aagcatgaga tcagcatgca ggtggcccgg gccaaggggc 780
tgcctcgcct caagcatcac ctgttgccac gaaccaaggg cttcgccatc accgtgagga 840
34/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
gcttgagaaa tgtagtttca gctgtatatg actgtacact caatttcaga aataatgaaa 900
atccaacact gctgggagtc ctaaacggaa agaaatacca tgcagatttg tatgttagga 960
ggatcccact ggaagacatc cctgaagacg atgacgagtg ctcggcctgg ctgcacaagc 1020
tctaccagga gaaggatgcc tttcaggagg agtactacag gacgggcacc ttcccagaga 1080
cgcccatggt gcccccccgg cggccctgga ccctcgtgaa ctggctgttt tgggcctcgc 1140
tggtgctcta ccctttcttc cagttcctgg tcagcatgat caggagcggg tcttccctga 1200
cgctggccag cttcatcctc gtcttctttg tggcctctgt gggagttcga tggatgattg 1260
gtgtgacgga aattgacaag ggctctgcct acggcaactc tgacagcaag cagaaactga 1320
atgactgact cagggaggtg tcaccatccg aagggaacct tggggaactg gtggcctctg 1380
catatcctcc ttagtgggac acggtgacaa aggctgggtg agcccctgct gggcacggcg 1440
gaagtcacga cctctccagc cagggagtct ggtctcaagg ccggatgggg aggaagatgt 1500
tttgtaatct ttttttcccc atgtgcttta gtgggctttg gttttctttt tgtgcgagtg 1560
tgtgtgagaa tggctgtgtg gtgagtgtga actttgttct gtgatcatag aaagggtatt 1620
ttaggctgca ggggagggca gggctgggga ccgaagggga caagttcccc tttcatcctt 1680
tggtgctgag ttttctgtaa cccttggttg ccagagataa agtgaaaagt gctttaggtg 1740
agatgactaa attatgcctc caagaaaaaa a 1771
<210> 46
<211> 1755
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 267495CB1
<400> 46
caatccgaag gacaggaagg ccatcttaga ttgataagcc atcttatgtt cccagtatct 60
gtctgaaata attatatcct gagagctctc gtagtgtgaa gctttcttct acatgtaaaa 120
tttcacaggg aaccatgttt ttcaaacaaa ggcataagaa cccaggctta ggaagcgggt 180
gtttaagcca tgggttagag ggctcgtgag cacgcccgat cctttccctg aggctatgaa 240
acggtggctc cacggactcc tctcggccga ctgacctttc gccgccctgc cccagcagcc 300
ggcgggtttc ttcagtggag ccgggctctg gtctccgcag cccagtagcc cgctagcccg 360
gccccctccc gctagtcgta aagtgcagta aaggcaccag cattttgcgg caccgtagtg 420
aggccggcgg cgtgggcctt ttctctgcac ggagccggcg cttttgcagt tgcttctgcg 480
gaaaggtggt agttaagaat ttgtaaaggc cagagaacta cctacgattc tctcagcggt 540
ctctcttctc ctcaagtttg aaatgcttta tctcatcggg ttgggcctgg gagatgccaa 600
ggacatcaca gtcaagggcc tggaagttgt tagacgctgc agtcgagtgt atctggaagc 660
ctacacctca gtcctaactg tagggaagga agccttggaa gagttttatg gaagaaaatt 720
ggttgttgct gatagagaag aagtggaaca agaagcagat aatattttaa aggatgctga 780
tatcagtgat gttgcattcc ttgtggttgg tgatccattt ggggccacaa cacacagtga 840
tcttgttcta agagcaacaa agctgggaat tccttataga gttattcaca atgcctccat 900
aatgaatgct gtaggctgct gtggtttaca gttatataag tttggagaga cagtttctat 960
tgttttttgg acagacactt ggagaccaga aagcttcttt gacaaagtga agaagaacag 1020
acaaaatggc atgcacacat tatgtttact agacatcaaa gtaaaggagc agtctttgga 1080
aaatctaatc aagggaagga agatctatga acctccacgg tatatgagtg taaaccaagc 1140
agcccagcag cttctggaga ttgttcaaaa tcaaagaata cgaggagaag aaccagcagt 1200
taccgaggag acactttgtg ttggcttagc cagggttgga gccgacgacc agaaaattgc 1260
agcaggcact ttaaggcaaa tgtgcactgt ggacttggga gaaccattgc attccttgat 1320
catcacagga ggcagcatac atccaatgga gatggagatg ctaagtctgt tttccatacc 1380
agaaaatagc tcagaatctc aaagcatcaa tggactttga acatagatat ttaccattgt 1440
ctgatgtaaa tttcagccat atatggattg atatggtttg gatgtatccc cacccaagtc 1500
tcatcttgaa ttttaatcct cataattccc aggtgttgtg gtaggtaatt gaatcatggg 1560
ggcagtttcc ctcatgctat tctcatgata gtgagctttc atgagatctg atggttttat 1620
aagtgcctgg catttcccct actggctctc attctcactc ttgccgccct gtgaagaggt 1680
gccttccacc gtgattgtta agtttcctga ggccttccca gccatgtgga actgtgagtc 1740
gaaaattaac cctct 1755
<210> 47
<211> 1811
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 410533CB1
35/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
<400> 47
gcggccgcag ggccatgcta gccttgcgcg tggcgcgcgg ctcgtggggg gccctgcgcg 60
gcgccgcttg ggctccggga acgcggccga gtaagcgacg cgcctgctgg gccctgctgc 120
cgcccgtgcc ctgctgcttg ggctgcctgg ccgaacgctg gaggctgcgt ccggccgctc 180
ttggcttgcg gctgcccggg atcggccagc ggaaccactg ttcgggcgcg gggaaggcgg 240
ctcccaggcc agcggccgga gcgggcgccg ctgccgaagc cccgggcggc cagtggggcc 300
cggcgagcac ccccagcctg tatgaaaacc catggacaat cccgaatatg ttgtcaatga 360
cgagaattgg cttggcccca gttctgggct atttgattat tgaagaagat tttaatattg 420
cactaggagt ttttgcttta gctggactaa cagatttgtt ggatggattt attgctcgaa 480
actgggccaa tcaaagatca gctttgggaa gtgctcttga tccacttgct gataaaatac 540
ttatcagtat cttatatgtt agcttgacct atgcagatct tattccagtt ccacttactt 600
acatgatcat ttcgagagat gtaatgttga ttgctgctgt tttttatgtc agataccgaa 660
ctcttccaac accacgaaca cttgccaagt atttcaatcc ttgctatgcc actgctaggt 720
taaaaccaac attcatcagc aaggtgaata cagcagtcca gttaatcttg gtggcagctt 780
ctttggcagc tccagttttc aactatgctg acagcattta tcttcagata ctatggtgtt 840
ttacagcttt caccacagct gcatcagctt atagttacta tcattatggc cggaagactg 900
ttcaggtgat aaaagactga tgaaagtcat ccctcactgt tagtaaggaa gcagtataca 960
tcaatgggaa cagggcccat ggaaatgtac aggagtttcc ctattttggt gttcagcttg 1020
aaaaaggact tgtcagaatc aactgtgtca tcaaaattta agtaatgtgc attgaaaata 1080
aggttgatca tgggaatatg cagaatttcc aatgtatttt taaatacaaa taaaattgta 1140
atttagaatt tttaatctta ggtttcttga ttaatttata agagatcaat tattgtcagt 1200
cttttttgta tgttttttaa aaacatagtc cagagcatgg gcagaattga cacctctctt 1260
ttaagtgaaa tttggattgc tcacaaagca ctaggaaatg tcatggggtt caaatatata 1320
tcctacacaa ctgggcaata catttttgtt tgatttttag gtctgtgtat acattaacag 1380
ttcatgtaat taatacctga tcatttggga taatgaaagt gaagttagtt gtagatgaag 1440
taaagttata aaagagatta aaaatgcggt aactttttaa gataataatc atacagaagg 1500
tatgaagttc attttcggta gtcttccaac ctctcaggtg cctaataatt tatgtttgag 1560
gataacaggt aacaaagata gtcgagatag gagaacgtgt ctattagtct ttgcatctaa 1620
aaggcagtga gttacgttcc tgccttccac tgtgtttctg acatagcaat gtttgtttga 1680
tattggaacc tggattcata ttttatgtaa ataatatcaa gctgtatatt tttcaaaggt 1740
tttttaaact ttggagactc tttcttttgt taagcagtta aaggaataaa agagctggaa 1800
aaaaaaaaaa a 1811
<210> 48
<211> 1003
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 852708CB1
<400> 48
tcccacgcgt ccgcggacgc gtgggcggac gcgtgggcgg acgcgtgggg cggtggctca 60
ggctcctgga aaggaccgtc cacccctccg cgctggcggt gtggacgcgg aactcagcgg 120
agaaacgcga ttgagaaatg gaaaagaaaa tgaaataaat cagcagttat gaggcagagc 180
ctaagagaac tatggcaaca tcaggtgact gtcccagaag tgaatcgcag ggagaagagc 240
ctgctgagtg cagtgaggcg ggtctcctgc aggagggagt acagccagag gagtttgtgg 300
ccatcgcgga ctacgctgcc accgatgaga cccagctcag ttttttgaga ggagaaaaaa 360
ttcttatcct gagacaaacc actgcagatt ggtggtgggg tgagcgtgcg ggctgctgtg 420
ggtacattcc ggcaaaccat gtggggaagc acgtggatga gtacgacccc gaggacacgt 480
ggcaggatga agagtacttc ggcagctatg gaactctgaa actccacttg gagatgttgg 540
cagaccagcc acgaacaact aaataccaca gtgtcatcct gcagaataaa gaatccctga 600
cggataaagt catcctggac gtgggctgtg ggactgggat catcagtctc ttctgtgcac 660
actatgcgcg gcctagagcg gtgtacgcgg tggaggccag tgagatggca cagcacacgg 720
ggcagctggt cctgcagaac ggctttgctg acatcatcac cgtgtaccag cagaaggtgg 780
aggatgtggt gctgcccgag aaggtggacg tgctggtgtc tgagtggatg gggacctgcc 840
tgctggtgag ggcgggcgtg cgggcagctg ggggccggag ctggggggct tctgagcacg 900
ggctcggctg ggccaacctc aggatctcaa gggtcgtgcg tgattcattt tgatgttttc 960
cctaatgtga ggtctaatta atttcttgtg tggaaaaaaa aaa 1003
<210> 49
<211> 1687
<212> DNA
<213> Homo Sapiens
<220>
36/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
<221> misc_feature
<223> Incyte ID No: 972944CB1
<400> 49
gtcgccacgg taacgcgcgc gggcaggtgt ccgaccatga gcgtccgggt cgcacgggta 60
gcgtgggtca ggggcttggg cgccagctac cgccgcggcg cctcgagctt cccggtgcct 120
ccgccgggcg cccagggtgt agcggagctg ctgcgagatg cgaccggggc ggaggaggag 180
gcgccctggg cggcgacgga gcggcgaatg ccgggccagt gctccgtgct gctcttcccg 240
ggccagggca gccaggtggt gggcatgggc cgcggtctgc tcaactaccc gcgcgtccgc 300
gaactctacg ccgccgcccg ccgcgtgctg ggctacgacc tgctggaact gagcctgcac 360
gggccgcagg agaccctgga ccgcaccgtg cactgtcagc ccgcgatctt cgtggcatcg 420
ctggccgctg tcgagaaact acatcacctg cagccctcgg tgattgagaa ctgtgttgct 480
gctgctggat tcagtgtggg agagtttgca gccctagtgt ttgccggagc catggaattt 540
gctgaaggtt tgtatgcagt gaaaatccga gctgaggcca tgcaggaagc ttcagaagct 600
gtccccagtg ggatgctgtc tgtcctcggc cagcctcagt ccaagttcaa cttcgcctgt 660
ttggaagccc gggaacactg caagtcttta ggcatagaga accccgtatg tgaagtgtcc 720
aactacctct ttccagattg cagggtgatt tcaggacacc aagaggctct acggtttctc 780
cagaagaatt cctctaagtt tcatttcaga cgcaccagga tgttgccggt tagtggcgca 840
ttccacaccc gcctcatgga gccagccgtg gagcccctga cgcaagcttt aaaggcagtc 900
gacattaaga agcctctggt ttctgtctac tccaacgtcc acgcgcatag atacaggcat 960
cccgggcaca tccacaagct gctggcccag cagctggtct ccccagtgaa gtgggagcag 1020
acgatgcatg ccatatacga aaggaaaaag ggcagggggt tcccccaaac tttcgaagta 1080
ggccctggca ggcagctggg agccatcctg aagagctgta acatgcaggc ctggaagtcc 1140
tacagcgccg tggatgtgct gcagaccctc gaacatgtgg acctggaccc tcaggagccc 1200
ccgagatgac tgcagggggc tcaaatgcga tgaccccctc tgtcctcctg aggagaggct 1260
gtaggctgtg cctgtcgccc cctaccttcc taatggctcc tcctctgagg agtgaaaggg 1320
atttgtttgc aacgtgcttt gaaggccaca taaaaagccc.taaaaatgag tatttcttta 1380
catgacccag tccatttctc ccccttggaa aaacgtttgg acgttgggaa gaatgatgcc 1440
acagggctgg tgtgtgggga agctggaatc ctggccccgc ctctgccagc cctgcaccat 1500
aggcaggtgt gccatctcag cgggaagggg aggactggct gctgcagcct gtcctgctct 1560
gtgagtgctg agaacatgct ggtgtggcct caatttcagc cagtgccagg ggagcccctc 1620
caccaccccc agcctgcccc ccagggctcc tttgtcacaa atctgaaagg ttttagaaaa 1680
aaaaaaa 1687
<210> 50
<211> 1239
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 997730CB1
<400> 50
ctcacgaggc cgtgggtacg accggaagcc gcagcatgct gatcccattt tcaatgaaga 60
attgcttcca gttactttgt aactgccagg tcccagcagc tggctttaaa aaaacagtaa 120
aaaatgggct cattttacag tcaatttcca atgatgtcta tcaaaatctg gctgtggaag 180
actggatcca tgaccatatg aatctagaag gcaaaccaat tctattcttt tggcagaatt 240
ctccctctgt tgtaattggt aggcatcaaa atccttggca ggaatgtaac ctgaatctaa 300
tgagagaaga aggtataaaa ctggctcgga gaagaagtgg aggaggaaca gtctaccatg 360
atatgggtaa tatcaatttg actttcttta caaccaaaaa aaagtatgat agaatggaaa 420
atctgaaatt aattgtgaga gctctgaatg ctgtccaacc ccagctggat gtgcaggcta 480
ccaaaagatt tgacctttta cttgatggac agtttaaaat ctcaggaaca gcttctaaga 540
tcggccggac tactgcctat caccattgca ctttattatg tagtactgat gggacgttcc 600
tgtcttcttt gctaaagagc ccttaccaag ggatcaggag caatgccact gctagcatac 660
cttccttagt gaaaaatctt ttggaaaagg atcccactct gacctgtgaa gtactaatga 720
atgctgttgc tacagagtat gctgcctatc atcaaattga taatcacatt cacctaataa 780
acccaacgga tgagacactg tttcctggaa taaatagcaa agccaaagaa ctgcaaactt 840
gggagtggat atatggcaaa actccaaagt ttagtataaa tacttccttt catgtgttat 900
atgaacagtc acacttggaa attaaagtat tcatagacat aaagaatgga agaattgaaa 960
tttgtaatat tgaagcacct gatcattggt tgccattgga aatacgtgac aaattaaatt 1020
caagtcttat tggcagtaag ttttgcccaa ctgaaactac catgctaaca aatatattac 1080
ttagaacatg tccacaagac cacaaactaa acagtaaatg gaatattctc tgtgaaaaaa 1140
ttaagggaat aatgtgattc caagtaaatg tcttaataca gtttcaatta gaaaataaaa 1200
tgtctcatac ttgcacattg tatgtcaaaa aaaaaaaaa 1239
<210> 51
37/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
<211> 1594
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1285944CB1
<400> 51
gtcggagccc taaccagggg tatctctgag cctggtggga tccccggagc gtcacatcac 60
tttccgatca cttcaaagtg gttaaaaact aatatttata tgacagaaga aaaagatgtc 120
attccgtaaa gtaaacatca tcatcttggt cctggctgtt gctctcttct tactggtttt 180
gcaccataac ttcctcagct tgagcagttt gttaaggaat gaggttacag attcaggaat 240
tgtagggcct caacctatag actttgtccc aaatgctctc cgacatgcag tagatgggag 300
acaagaggag attcctgtgg tcatcgctgc atctgaagac aggcttgggg gggccattgc 360
agctataaac agcattcagc acaacactcg ctccaatgtg attttctaca ttgttactct 420
caacaataca gcagaccatc tccggtcctg gctcaacagt gattccctga aaagcatcag 480
atacaaaatt gtcaattttg accctaaact tttggaagga aaagtaaagg aggatcctga 540
ccagggggaa tccatgaaac ctttaacctt tgcaaggttc tacttgccaa ttctggttcc 600
cagcgcaaag aaggccatat acatggatga tgatgtaatt gtgcaaggtg atattcttgc 660
cctttacaat acagcactga agccaggaca tgcagctgca ttttcagaag attgtgattc 720
agcctctact aaagttgtca tccgtggagc aggaaaccag tacaattaca ttggctatct 780
tgactataaa aaggaaagaa ttcgtaagct ttccatgaaa gccagcactt gctcatttaa 840
tcctggagtt tttgttgcaa acctgacgga atggaaacga cagaatataa ctaaccaact 900
ggaaaaatgg atgaaactca atgtagaaga gggactgtat agcagaaccc tggctggtag 960
catcacaaca cctcctctgc ttatcgtatt ttatcaacag cactctacca tcgatcctat 1020
gtggaatgtc cgccaccttg gttccagtgc tggaaaacga tattcacctc agtttgtaaa 1080
ggctgccaag ttactccatt ggaatggaca tttgaagcca tggggaagga ctgcttcata 1140
tactgatgtt tgggaaaaat ggtatattcc agacccaaca ggcaaattca acctaatccg 1200
aagatatacc gagatctcaa acataaagtg aaacagaatt tgaactgtaa gcaagcattt 1260
ctcaggaagt cctggaagat agcatgcgtg ggaagtaaca gttgctaggc ttcaatgcct 1320
atcggtagca agccatggaa aaagatgtgt cagctaggta aagatgacaa actgccctgt 1380
ctggcagtca gcttcccaga cagactatag actataaata tgtctccatc tgccttacca 1440
agtgttttct tactacaatg ctgaatgact ggaaagaaga actgatatgg ctagttcagc 1500
tagctggtac agataattca aaactgctgt tggttttaat tttgtaacct gtggcctgat 1560
ctgtaaataa aacttacatt tttcaaaaaa aaaa 1594
<210> 52
<211> 700
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1293207CB1
<400> 52
cccggcctgc ggtgggcagc agctcaggtt ctccaaatca ttgcgtagtt ccgaataccc 60
tcggccacac ctggccttct ccatgctcgg aataacttcc tgcagcgacc aacaggctaa 120
agagggggaa gggatccagc accggctcct cctccggcaa ccacggtggg agcggcggag 180
gaaatggaca taaacccggg tgtgaaaagc cagggaatga agcccgcggg agcggggaat 240
ctgggattca gaactctgag acgtctcctg ggatgtttaa ctttgacact ttctggaaga 300
attttaaatc caagctgggt ttcatcaact gggatgccat aaacaagaac caggtcccgc 360
cccccagcac ccgagccctc ctctacttca gccgactctg ggaggatttc aaacagaaca 420
ctcctttcct caactggaaa gcaattattg agggtgcgga cgcgtcatca ctgcagaaac 480
gtgcaggcag agccgatcag aactacaatt acaaccagca tgcgtatccc actgcctatg 540
gtgggaagta ctcagtcaag acccctgcaa aggggggagt ctcaccttct tcctcggctt 600
cccgggtgca acctggcctg ctgcagtggg tgaagttttg gtaggtgagt gtcagagtga 660
gccgacccag gccacatcct ggcagtggag gcacagtcac 700
<210> 53
<211> 536
<212> DNA
<213> Homo Sapiens
<220>
<221> misc feature
38/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
<223> Incyte ID No: 1308125CB1
<400> 53
gttttaggcg agttggagat gtctgtgaga ggtctgaaca gtgatgtcca gttcaaggat 60
ggagggcaaa gccaagtaca tcctccctac cgaaacaata tatgttgggg aaatgaagga 120
tggcatgttt cacggcgagg gaaccctgta cttccccagc ggaagccaat acgacgccat 180
ttgggaaaac ggattggcca taaaggtatg gctcaactca ccaatatgga cccacctaga 240
aaaatcccca agggctatta cgattgtgga gacggcttct ataacccagt cacgagggta 300
gtcaaggact ataggaaccg ctttctaaga aacgcagatg atgacgagca tgagtggatc 360
acccgtacct gtcgaaaggg ctaggatgag atcgtgggtc acaggcccga gccgtgaact 420
ctgtggctgc ctccaccaga ggtttccatc tgccctacta gcattggctg ccctggggga 480
cgggctgtag ttctagaacc tgattttaac tcaggaataa agactttctg cggtca 536
<210> 54
<211> 1130
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1439670CB1
<400> 54
agaggcgagg tggaggcgtt ggcgctgcca cgtctgggcc gcggttccca actgtggcgc 60
gggcggtgga ggaggaggtg gggctggcgc tgaagccgga tccggatccg gtgctgtgca 120
cactggtggg ggagagtccg acgcgcctgg ctaggagcgc cgaccgcagg gcctctacgg 180
accttactag aaaaatgaaa cctgatgaaa ctcctatgtt tgacccaagt ctactcaaag 240
aagtggactg gagtcagaat acagctacat tttctccagc catttcccca acacatcctg 300
gagaaggctt ggttttgagg cctctttgta ctgctgactt aaatagaggt ttttttaagg 360
tattgggtca gctaacagag actggagttg tcagccctga acaatttatg aaatcttttg 420
agcatatgaa gaaatctggg gattattatg ttacagttgt agaagatgtg actctaggac 480
agattgttgc tacggcaact ctgattatag aacataaatt catccattcc tgtgctaaga 540
gaggaagagt agaagatgtt gttgttagtg atgaatgcag aggaaagcag cttggcaaat 600
tgttattatc aacccttact ttgctaagca agaaactgaa ctgttacaag attacccttg 660
aatgtctacc acaaaatgtt ggtttctata aaaagtttgg atatactgta tctgaagaaa 720
actacatgtg tcggaggttt ctaaagtaaa aatcttgtaa gaaaattgtc aaaggggcta 780
atgctacaag gctacactct tcctagagtt gaaatatttt gttgctgcag ccgagtgacc 840
tccataaata ctggactgaa aaaacattgt aatactacaa gtataatgac atttagaaga 900
ttactttggg ctggtgggac atgctgtgaa tttagattac aaatgaatat tataacaggg 960
gatgattttt aaccaaacgg aatatatttt taacttggat cttttcttgc attgtatttt 1020
ttctaaaagg tttggcatcc ctatcttggg aaggtcagga ggtatggggt aaataaaggg 1080
agttaataat ggtctggcta atccggtggt ttggcctcat tttataaaaa 1130
<210> 55
<211> 930
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1444281CB1
<400> 55
ctccgagagc gggccgggct cagttcagct gctgtccaga cccggatcgg caacagtgcc 60
gcctccagac gttctcctgc cgctcgcccg cccgtcccag cgcccccagc cctcccgcga 120
gggcgccccg ggacggaagg atccaccagt ctgtcggcgc ccgccgttct cgtggtcgcc 180
gtcgccgtcg tcgtggtggt agtctccgcc gtcgcctggg ccatggccaa ttacatccac 240
gtccctcccg gctccccgga ggtgcccaag ctgaacgtca ccgttcagga tcaggaggag 300
catcgctgcc gggagggggc cctgagcctc ctgcaacacc tgcggcctca ctgggacccc 360
caggaggtga ccctgcagct cttcacagat ggaatcacaa ataaacttat tggctgttac 420
gtgggaaaca ccatggagga tgtagtcctg gtgagaattt atggcaataa gactgagtta 480
ttagtcgatc gagatgagga agtaaagagt tttcgagtgt tgcaggctca tgggtgtgca 540
ccacaactct actgtacctt caataatgga ctatgctatg aatttataca aggagaagca 600
ctggatccaa agcatgtctg caacccagcc attttcagtt tatcatcgtt gactctttgc 660
aaaggaaaaa ctacaagatg ttttggatta accggctgca gagggtcaag gcttctgctt 720
agttttttct agttagtgtt tgagttgaag tgttatacat ttcttactgc cgattgctta 780
ttacttaatt gttcatgttt tcagagttct tgcctattaa aattttatta atttgggtaa 840
39/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
caattctttg ttctttacgt tgttcattat ttaatttaat atattatgtt ttctataaag 900
aataatgtca tatatgcttg atcatttttg 930
<210> 56
<211> 1687
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1450140CB1
<400> 56
gtccgcgggg agctgcccct gtgcccgtgg ctctctgcgc atgcccggac cccggcggct 60
ccggagtccg ggaggctctg gctgctccag tcgcgggtcc gggccccgcc tctccactcg 120
gcggtggcgt ccgtgttgtt attgtggtgg tagcggcgtc tggctgctgc ggaccccgcc 180
gagtcctagc gcctggcctg cgcgccgctg cccgcgccac aggattaatt tatttttgga 240
aatcaagtgc aatattggaa gccatttcta ctaattttat ttcactttta aatttatgat 300
ttgatttgaa tactatcatg ggaggagctg tgagtgctgg ggaagataat gatgacttaa 360
ttgataattt aaaagaagct cagtatattc gtactgaaag agtggagcaa gccttcagag 420
cgattgatcg tggagattac tatttggaag gctacagaga caatgcttac aaagacttag 480
cctggaagca tggaaacatc cacttgtcag caccttgcat ttattctgaa gttatggaag 540
cattgaaact tcaaccagga ttgtcttttc ttaacctggg aagtggaacc ggatatttaa 600
gtacaatggt gggcttaatt ttaggtcctt ttggaataaa tcatgggatt gagcttcatt 660
cagatgtggt ggaatatgcc aaggaaaaac tggagagctt catcaaaaat agtgatagct 720
ttgataaatt tgagttctgt gaacctgcat ttgttgttgg taattgcctc cagatagctt 780
ctgacagtca tcagtatgat cgaatttatt gtggagctgg agtacagaaa gaccatgaaa 840
actacatgaa aatattacta aaagttggag gcatattagt catgcctata gaggatcagt 900
taacacagat tatgcgaact ggacagaaca cttgggaaag taaaaatatc cttgctgttt 960
catttgctcc acttgtgcaa ccaagtaaga atgataatgg caaaccagat tctgtgggac 1020
tccctccctg tgctgtcagg aatctacagg acttggctcg tatttacatt cgacgcacac 1080
ttagaaattt cataaatgat gagatgcagg ccaaggggat tcctcaaagg gctccaccca 1140
aaaggaaaag aaagagagtt aaacagagaa ttaacactta cgtatttgtg ggtaatcagc 1200
ttattcctca gcctctagac agtgaagagg atgaaaaaat ggaagaggat atcaaagaag 1260
aggaggaaaa agatcacaat gaagcaatga agccagagga gccacctcaa aatttactga 1320
gagaaaaaat catgaagctg cccctccctg aatctttaaa agcttacttg acatatttta 1380
gagacaaata acttagatca agaagaaaaa tgcctactga taattccttt agtcttgaaa 1440
atgtagcatt tgttaggagt taaaagagag aattatttct ttcatcagag caaattatag 1500
tggaaaaaaa aatcacttgt ttctgtcagt aacacaaatg atgtattcag tgaataaaag 1560
aatccctttt ataaaatcta tttttcttta aatcttggaa aaatgttgtt ttagctcaga 1620
gtgatttcaa agtggaatgc aacagtagtc aagacttgtg tactataaat ccttttctga 1680
ttcctta 1687
<210> 57
<211> 1262
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1604828CB1
<400> 57
ggacgccgcg cctcctctcg ggcggagcgg cgcggtggct gatcagagcg cgtagggctt 60
cgccggtgcc gggcgctggg cgcggtcctg ctcagcccag ctcaccgcgc gccggccctc 120
ggcgccctgg ttctgcggat caggagaaaa taatgaatgt cagaggaaaa gtaattctgt 180
caatgctggt tgtctcaact gtgatcattg tgttttggga atttatcaac agcacagaag 240
actctttctt gtggatatat cactcaaaaa acccagaagt tgatgacagc agtgctcaga 300
agggctggtg gtttctgagc tggtttaaca atgggatcca caattatcaa caaggggaag 360
aagacataga caaagaaaaa ggaagagagg agaccaaagg aaggaaaatg acacaacaga 420
gcttcggcta tgggactggt ttaatccaaa cttgaaggaa tccgaataac taaactggac 480
tctggttttc tgactcagtc cttctagaag acctggactg agagatcatg cggttaagga 540
gtgtgtaaca ggcggaccac ctgttgggac tgcgagattc tcaaggggaa ggactgggtc 600
tcatttctcc catctcagcg cttagcagga tgacctggta tagagcaggg aactgggaaa 660
tgtgggtcag gggatcagac actccagttg ggtcttttat ataaattaaa tggcaaaagg 720
ctccataccc ttctccttct ttcctaccct ccactttatc tgcaaaatgg gaatgatgat 780
aacacccact tcatagaatg gtcatgaaga tcaaatgaga gaataaaagt caagcactta 840
40/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
gcctctggtg cacaataagt attaaataag tatacctatt cctccttttc cttttttaaa 900
aataatatta ccaaatgtcc agcttataca catttacaag acttagctag tgggctatgt 960
tagagctact aaaagatctt tgacaagcta aaactaagat gcaatgaatg aggtgtaacg 1020
aacaagagag ttttaagttc agaaatggtt acagaagtat aagacagctg tgtgggtgtt 1080
ttttggtttt tggtttctgg tttacaatct cgtcattcaa caaagatggg agttttatag 1140
aactaaaagc accatgtaag ctactaaaaa caacaacaaa aaaggctcat catttctcag 1200
tctgaattga caaaaatgcc aatgcaaata aaaatgatta ctttttattt taaaaaaaaa 1260
as 1262
<210> 58
<211> 1330
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1644023CB1
<400> 58
gcgaagcggg cgtgcgccag tgcagtgccg cgggccgcgc gcgactcccg atagggaagg 60
gccggagctg cgcaggggcg cgcggaagag gccaagctag ccggaacccc gccccgcccc 120
gccccgcccc aacgggggct ggaacccggc gccgagagta gagaaaaggg gcctctggtg 180
accgccccta cctggcatcc ctctaaccca ggaggagcgt ggggaaaggg gctgtgggcc 240
tctcggggag cgagctgcgg gtagcggcgc actgggtaca ggcgcgcgct tggctgtcgc 300
ctctgccgct gtgtttggga ggactcgaac tggcgccagg aaatattagg aagctgtgat 360
tttcaaagct aattatgaaa acatttatca ttggaatcag tggtgtgaca aacagtggca 420
aaacaacact ggctaagaat ttgcagaaac acctcccaaa ttgcagtgtc atatctcagg 480
atgatttctt caagccagag tctgagatag agacagataa aaatggattt ttgcagtacg 540
atgtgcttga agcacttaac atggaaaaaa tgatgtcagc catttcctgc tggatggaaa 600
gcgcaagaca ctctgtggta tcaacagacc aggaaagtgc tgaggaaatt cccattttaa 660
tcatcgaagg ttttcttctt tttaattata agccccttga cactatatgg aatagaagct 720
atttcctgac gattccatat gaagaatgta aaaggaggag gagtacaagg gtctatcagc 780
ctccagactc tccgggatac tttgatggcc atgtgtggcc catgtatcta aagtacagac 840
aagaaatgca ggacatcaca tgggaagttg tgtacctgga tggaacaaaa tctgaagagg 900
acctcttttt gcaagtatat gaagatctaa tacaagaact agcaaagcaa aagtgtttgc 960
aagtgacagc ataaagacgg aacacaacaa atccttcctg aagtgaatta ggaaactcca 1020
aggagtaatt taagaacctt caccaagata caatgtatac tgtggtacaa tgacagccat 1080
tgtttcatat gtttgatttt tattgcacat ggttttccca acatgtggaa caataaatat 1140
ccatgccaat ggacaggact gtaccttagc aagttgctcc ctctccaggg agcgcataga 1200
tacagcagag ctcacagtga gtcagaaagt ctccactttc tgaacatagc tctataacaa 1260
tgattgtcaa acttttctaa ctggagctca gagtaagaaa taaagattac atcacaatcc 1320
aaaaaaaaaa 1330
<210> 59
<211> 1110
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1723402CB1
<400> 59
taagctgaat cgactgctgc caaacatcta ttaggcaaaa ttggcctctt gcccatgatt 60
tgactttcca gcacagccag ttcttttcct cctctgcagc tgattggctc tggagtgtgg 120
ccagaagcct ctctcctgca attaaaggag tcgggtctct aactgttgat ctgttttttt 180
cccttctgag caatggagct taccatcttt atcctgagac tggccattta catcctgaca 240
tttcccttgt acctgctgaa ctttctgggc ttgtggagct ggatatgcaa aaaatggttc 300
ccctacttct tggtgaggtt cactgtgata tacaacgaac agatggcaag caagaagcgg 360
gagctcttca gtaacctgca ggagtttgcg ggcccctccg ggaaactctc cctgctggaa 420
gtgggctgtg gcacgggggc caacttcaag ttctacccac ctgggtgcag ggtgacctgt 480
attgacccca accccaactt tgagaagttt ttgatcaaga gcattgcaga gaaccgacac 540
ctgcagtttg agcgctttgt ggtagctgcc ggggagaaca tgcaccaggt ggctgatggc 600
tctgtggatg tggtggtctg caccctggtg ctgtgctctg tgaagaacca ggagcggatt 660
ctccgcgagg tgtgcagagt gctgagaccg ggaggggctt tctatttcat ggagcatgtg 720
gcagctgagt gttcgacttg gaattacttc tggcaacaag tcctggatcc tgcctggcac 780
cttctgtttg atgggtgcaa cctgaccaga gagagctgga aggccctgga gcgggccagc 840
41/55
40/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
ttctctaagc tgaagctgca gcacatccag gccccactgt cctgggagtt ggtgcgccct 900
catatctatg gatatgctgt gaaatagtgt gagctggcag ttaagagctg aatggctcaa 960
agaatttaaa gcttcagttt tacatttaaa atgctaagtg ggagaagaga aacctttttt 1020
ttggggggcg gtttttttgg tttgttgttg gttttttttt tttttttggc ctgggtgaca 1080
agagcaagac tccgtctcaa aaaaaaaaaa 1110
<210> 60
<211> 1153
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1740585CB1
<400> 60
ggtagagcgg agacgacgct cccagactcc tccggtctcc ccgggcagca tgaagaccgc 60
cgagaacatc agaggaaccg gcagcgacgg gccgcggaaa cgaggcctct gcgtcctctg 120
tggcctcccc gcggcaggaa aatcgacttt cgcgcgcgcc ctcgcccacc ggctgcagca 180
ggagcagggt tgggccatcg gtgttgtcgc gtatgatgac gtcatgcccg acgcgtttct 240
cgccggggca agagcgcgac cggcgccatc ccaatggaaa ttgcttcgac aggaactgtt 300
gaagtacctg gaatacttct tgatggctgt cattaatggg tgtcagatgt ctgtcccacc 360
caacaggact gaagccatgt gggaagattt tataacctgc ttaaaggatc aagatctgat 420
attttctgca gcatttgagg cccagtcttg ctacctctta acaaaaactg ctgtttctag 480
acctttgttt ttggttttgg atgacaattt ttattatcag agtatgagat atgaagtcta 540
ccagctggct cggaaatatt cattgggctt ttgccagctc tttttagatt gtcctcttga 600
gacctgttta cagaggaatg gccagagacc acaggcactg cctcctgaga ccatccacct 660
gatgcgaaga aagctagaaa agcccaaccc tgagaaaaat gcttgggaac acaacagcct 720
cacaattccg agtccagcat gtgcttcgga ggccagcctg gaagtgactg atttattgct 780
cactgctttg gaaaatccag taaaatatgc tgaggacaat atggaacaaa aggacacaga 840
cagaattatt tgttcaacta acattcttca taaaactgat cagacactcc gaaggattgt 900
atctcagaca atgaaggaag caaaagatga acaagtgctt cctcacaact tgaagcttct 960
agcagaagaa cttaacaagc tcaaagcaga gtttttggaa gacctaaaac aaggaaacaa 1020
aaaatatctg tgctttcagc aaaccattga cataccagat gtcatttctt tttttcatta 1080
tgagaaagat aatattgtac agaagtattt ttcaaagcag cattaaaatt tctgaactgc 1140
caaaaaaaaa aaa 1153
<210> 61
<211> 1955
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1810925CB1
<400> 61
gggcaggcta agagatcttc ttttaattca gcctgcttaa gacgggaact gataactgta 60
gtgtatcctc tgcctttttt cttatctatt ggaggaagct cagatggtgt cacaagaagg 120
atctgaagtg gagcttctag tatccccagg agcgcgaagt gaacacggaa ggtacctgca 180
ggatccaatt gtgtccattg atctctcaga gtggctgagg ataatagagt ttcttcttca 240
aggtctcaag gtctgaagca tcccacagaa tgatcctact gaataactcc cataagctgc 300
tggccctata caaatccttg gccaggagca tccctgagtc cctgaaggtg tatggctctg 360
tgtatcacat caatcacggg aaccccttca acatggaggt gctggtggat tcctggcctg 420
aatatcagat ggttattatc cggcctcaaa agcaggagat gactgatgac atggattcat 480
acacaaacgt atatcgtatg ttctccaaag agcctcaaaa atcagaagaa gttttgaaaa 540
attgtgagat cgtaaactgg aaacagagac tccaaatcca aggtcttcaa gaaagtttag 600
gtgaggggat aagagtggct acattttcaa agtcagtgaa agtagagcat tcgagagcac 660
tcctcttggt tacggaagat attctgaagc tcaatgcctc cagtaaaagc aagcttggaa 720
gctgggctga gacaggccac ccagatgatg aatttgaaag tgaaactccc aactttaagt 780
atgcccagct ggatgtctct tattctgggc tggtaaatga caactggaag cgagggaaga 840
atgagaggag cctgcattac atcaagcgct gcatagaaga cctgccagca gcctgtatgc 900
tcggcccaga gggagtcccg gtctcatggg taaccatgga cccttcttgt gaagtaggaa 960
tggcctacag catggaaaaa taccgaagga caggcaacat ggcacgagtg atggtgcgat 1020
acatgaaata tctgcgtcag aagaatattc cattttacat ctctgtgttg gaagaaaatg 1080
aagactcccg cagatttgtg gggcagtttg gtttctttga ggcctcctgt gagtggcacc 1140
aatggacttg ctacccacag aatctagttc cattttagac aatgaagctg cttagtaatc 1200
42/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
tctgccaagc catctcttaa tattaaagca gacaccacag aatagatttc ttcacttaca 1260
aatgcatatt gggcacttat aatacagcag gaactcttct cacctggagc cttgatgtta 1320
aaagacacag ccatgctctt gaggagctta caatcctggc tggaggcagg ggagggtata 1380
ttctttaaat atgcttaagt gttataggga aagacggggt taccagtaaa catgtaacta 1440
gaaagccagg ctcagttctt acctctggga atcagaactc tttatgcaac ttggttaata 1500
gaatctacta tctggaagat aaatgaagga ttttaataaa attttcaata gaataaacct 1560
aatctgtatg gatactttat caaaaatgaa tgtccctgct atttctggat ttatgaggca 1620
atggtacact aaagaatgga atcagttcag tgagtagaaa ggtatccaag gtgaagcctg 1680
agacgaatgg ctttcccagg ctaccttcca tcactgttgt acagaaaaga aatccagaga 1740
atcaaatgga ctggccttgg gggtctctgc tatggaaatg ccattttttg tgtctccttt 1800
ctcctactct ttctcacatc ctcttcatga ttgacgcatg gcacaaggca aggtgtggcc 1860
tgcgagtctg gttgaaagtt cagcctttgg tgtttgcaca actgctaaca aaaggggaca 1920
ggggaattcc ggggaaaatt ttgcccctaa ggttg 1955
<210> 62
<211> 1434
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1915064CB1
<400> 62
tcgggcggtg ccccgggctg ggcgaggggc cgggtgcggg gccgctggcc gagaggctga 60
ggcggcgtca tgtcctccga ggtgtccgcg cgccgcgacg ccaagaagct ggtgcgctcc 120
ccgagcggcc tgcgcatggt gcccgaacac cgcgccttcg gaagcccgtt cggcctggag 180
gagccgcagt gggtcccgga caaggagtgt cggagatgta tgcagtgtga cgccaagttt 240
gactttctca ccagaaagca ccactgtcgc cgctgcggga agtgcttctg cgacaggtgc 300
tgcagccaga aggtgccgct gcggcgcatg tgctttgtgg accccgtgcg gcagtgcgcg 360
gagtgcgccc tggtgtccct caaggaggcg gagttctacg acaagcagct caaagtgctc 420
ctgagcggag ccaccttcct cgtcacgttt ggaaactcag agaaacctga aactatgact 480
tgtcgtcttt ccaataacca gagatacttg tttctggatg gagacagcca ctatgaaatc 540
gaaattgtac acatttccac cgtgcagatc ctcacagaag gcttccctcc tggaggaggc 600
aacgcacggg ccacaggcat gttcctgcag tatacagtgc cggggacgga gggtgtgacc 660
cagctgaagc tgacagtggt ggaggacgtg actgtgggca ggaggcaggc ggtggcgtgg 720
ctagtggcca tgcacaaggc tgccaagctc ctctatgaat ctcgggacca gtaactctac 780
gtggggctga gcttggagta cgtgtggtca ccaggactga gtcgcttgga acagcagagc 840
ctgctccttg cgtaccacag ggattaatcc tgcttgtgct gggaaatgca actcactcat 900.
gtatttggag aaacaggagt gttcacttat ctagtgcaat atgttcacag tttattaatg 960
ctttaaacag cttcatgttt tagaatttgt gtattgtcca tacttaattg ggggtgggag 1020
agactgagct acactactgc taaactattt ttagcataat atataccatt tttatgagtt 1080
cgcaggtcta ctagaaggtt ctggcccatc aatattcatt tcatttaatt cttccacaga 1140'
accagtttgg gcagtaggaa ctcaggcttc tggtctgcag tggagcctgt tcgcctctaa 1200
tagccagttt acagcacttg ccttagcctg tttcacagac ttgtccactt accttgtcac 1260
taatttgggg cttctgggct gtgagtgatc ctttgatact tcaccaaggg gaacgtgggg 1320
gctttgtgtt ttgtactttt cactcactat ttcactttat taagatgact gtacagcaat 1380
ttgtatataa agcttatgat taaaaactat tttgaacata aaaaaaaaaa aaaa 1434
<210> 63
<211> 1360
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2185608CB1
<400> 63
ccgataacga acgagactct ggcatgctaa ctagtgacag tcaagatggc gggagcagct 60
acccaggctt ccctggagtc ggccccacgg atcatgcggc tggtggccga atgcagccgc 120
tccagggccc gggcaggcga gctgtggctg ccgcatggga cagtggccac tcctgtgttc 180
atgccagtgg gcacgcaggc caccatgaag ggcatcacga ccgaacagct ggacgctctg 240
ggttgccgca tctgcctggg caatacctac catctgggtc taaggccggg acccgagctg 300
atccagaaag ccaacggtct ccacggcttc atgaattggc ctcataatct gctaacggac 360
agcggcggtt tccagatggt gtcgctggtg tctctgtccg aggtgacgga ggagggcgtc 420
cgcttccgct ccccctacga cggcaatgag accctgctga gcccggagaa atccgtgcag 480
43/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
atccagaatg cgctgggctc ggacatcatc atgcagctgg acgacgtggt tagcagtact 540
gtgactgggc cacgtgtgga ggaggccatg tacaggtcaa tccgctggct ggaccggtgc 600
attgcagccc atcagcggcc ggacaagcag aacctcttcg ccattatcca gggtgggctg 660
gacgcagatc tccgggccac ctgccttgaa gagatgacca agcgagacgt gcctggcttc 720
gccatcgggg gcctgagcgg gggtgagagc aagtcgcagt tctggcggat ggtggcgctg 780
agcacctctc ggctgccgaa ggacaagccc cgatatctga tgggggttgg ctatgccact 840
gatctggtag tctgcgtggc tcttggatgt gacatgttcg actgcgtctt ccccacacgg 900
acagcgcgct ttggctctgc cctggtgccc actgggaacc tgcagttgag gaagaaggtg 960
tttgagaagg acttcggccc catagacccg gagtgcacct gccccacgtg ccaaaagcac 1020
agccgcgcct tcctgcacgc actgctgcac agtgacaaca cggccgcgct gcaccacctc 1080
acggtccaca acatcgccta ccagctgcag ctcatgagcg ccgtccgcac cagcatcgtg 1140
gagaagcgct tcccggactt cgtgcgggac ttcatgggcg ccatgtacgg ggatcccacc 1200
ctctgtccca cctgggccac tgacgctctg gcctctgtgg gaatcacact gggctgacct 1260
ggcattggga gagggaggga ggaaggaagg gagggagggg ctggaagata ctgaaggatt 1320
cctttttgaa aggttttttt tattgtaact taaaaaaaaa 1360
<210> 64
<211> 2277
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2228862CB1
<400> 64
aaacgccatg cggaggggcg agcgcaggga cgccggacgt ccgcggcccg agtccccggt 60
gcccgcgggc agggcctcgc tggaggagcc gcctgacggg ccgtctgccg gccaagccac 120
cgggccgggc gagggccgcc gcagcaccga gtccgaggtc tacgacgacg gcaccaacac 180
cttcttctgg cgagcccaca ccttaaccgt gctcttcatc ctcacctgta cgcttggcta 240
tgtgacgctg ctggaggaaa cacctcagga cacggcctac aacaccaaga gaggtattgt 300
ggccagtatt ttggttttct tatgttttgg agtcacacaa gctaaagacg ggccattttc 360
cagacctcat ccagcttact ggaggttttg gctctgcgtg agtgtggtct acgagctgtt 420
tctcatcttt atactcttcc agactgtcca ggacggccgg cagtttctaa agtatgttga 480
ccccaagctg ggagtcccac tgccagagag agactacggg ggaaactgcc tcatctacga 540
cccagacaat gagactgacc cctttcacaa catctgggac aagttggatg gctttgttcc 600
cgcgcacttt cttggctggt acctgaagac cctgatgatc cgagactggt ggatgtgcat 660
gatcatcagc gtgatgttcg agttcctgga gtacagcctg gagcaccagc tgcccaactt 720
cagcgagtgc tggtgggatc actggatcat ggacgtgctc gtctgcaacg ggctgggcat 780
ctactgcggc atgaagaccc ttgagtggct gtccctgaag acgtacaagt ggcagggcct 840
ctggaacatt ccgacctaca agggcaagat gaagaggatc gccttccagt tcacgccgta 900
cagctgggtt cgcttcgagt ggaagccggc ctccagcctg cgtcgctggc tggccgtgtg 960
cggcatcatc ctggtgttcc tgttggcaga actgaacacg ttctacctga agtttgtgct 1020'
gtggatgccc ccggagcact acctggtcct cctgcggctc gtcttcttcg tgaacgtggg 1080
tggcgtggcc atgcgtgaga tctacgactt catggatgac ccgaagcccc acaagaagct 1140
gggcccgcag gcctggctgg tggcggccat cacggccacg gagctgctca tcgtggtgaa 1200
gtacgacccc cacacgctca ccctgtccct gcccttctac atctcccagt gctggaccct 1260
cggctccgtc ctggcgctca cctggaccgt ctggcgcttc ttcctgcggg acatcacatt 1320
gaggtacaag gagacccggt ggcagaagtg gcagaacaag gatgaccagg gcagcaccgt 1380
cggcaacggg gaccagcacc cactggggct ggacgaagac ctgctggggc ctggggtggc 1440
cgagggcgag ggagcaccaa ctccaaactg acctgggccg tggctgcctc gtgagcctcc 1500
cagagcccag gcctccgtgg cctcctcctg tgtgagtccc accaggagcc acgtgcccgg 1560
ccttgccctc aaggtttttt gcttttctcc tgtgcacctg gcgaggctga aggcgagggg 1620
tggaggaggc cccagcacag cctcatctcc atgtgtacac gtgtgtacgt gtgtatgcgt 1680
gtgtgtacgc gtgtgtacgc gcgtgtgtac acatgcgtgg ccgcctgtgg tgtgcacgtg 1740
tgctctgggc tccgaggctt ctccagagct gggagctggc tggcgtggca agggcatgct 1800
ctggggcagt gtgtccctca ggaaccaggg tcctccctcc cctttctgcc tggtcagccc 1860
cgtggcctct ggcccaccaa gctccctgtc acccagccat ggtgtggtcc aggcagggac 1920
atctcggtac cctttctgca ctccgtgggc cctgggtgcg ctgaggcctg gaggcgtcta 1980
cactggctcc acatccactt cccccgcagc tcgtgtgggc gctcgtccac aaacactccg 2040
tggctgagag gcagcggatc caggcagcga tgctgagcca cctcctccga gccttccttt 2100
cacacagacc accccggagg acacgtggat gatggggtca gagatcactg agctgcccct 2160
caagggggcc tggaacccgg gtgctggggt catgctgcct ccgtggctcc aaggtgaggg 2220
tcatcttcac gagcaaagag aaccaataaa gtgacaacga acgtcaaaaa aaaaaaa 2277
<210> 65
<211> 1592
44/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2235577CB1
<400> 65
ccaagatggc ggcccccggg agctgtgctc tatggagcta ttgcggccgt gggtggtcgc 60
gggcgatgcg gggctgccag ctcctcgggc ttcgtagctc ttggcccggg gacctactaa 120
gtgctcggct cttgtcccaa gagaagcggg cagcggaaac gcactttggg tttgagactg 180
tgtcggaaga ggagaagggg ggcaaagaac tcctttaatg tgaaagtttt tgctggcatc 240
acttcttctg ggacatcttc aacctttttg tcacagctcc atttcttgtt catgttgtct 300
atcaggtgtt tgaaagtgtg gctaagaagt atgatgtgat gaatgatatg atgagtcttg 360
gtatccatcg tgtttggaag gatttgctgc tctggaagat gcacccgctt cctgggaccc 420
agctgcttga tgttgctgga ggcacaggtg acattgcatt ccggttcctt aattatgttc 480
agtcccagca tcagagaaaa cagaagaggc agttaagggc ccaacaaaat ttatcctggg 540
aagaaattgc caaagagtac cagaatgaag aagattcctt gggcgggtct cgtgtcgtgg 600
tgtgtgacat caacaaggag atgctaaagg ttggaaagca gaaagccttg gctcaaggat 660
acagagctgg acttgcatgg gtattaggag atgctgaaga actgcccttt gatgatgaca 720
agtttgatat ttacaccatt gcctttggga tccggaatgt cacacacatt gatcaggcac 780
tccaggaagc tcatcgggtg ctgaaaccag gaggacggtt tctctgtctg gaatttagcc 840
aagtgaacaa tcccctcata tccaggcttt atgatctata tagcttccag gtcatccctg 900
tcctgggaga ggtcatcgct ggagactgga agtcctatca gtaccttgta gagagtatcc 960
gaaggtttcc gtctcaggaa gagttcaagg acatgataga agatgcaggc tttcacaagg 1020
tgacttacga aagtctaaca tcaggcattg tggccattca ttctggcttc aaactttaat 1080
tcctttccta tcatggagca tgaaccagtc atatcctgtt gaaagcctgg aactgaagga 1140
taatctggca aatgagacag cagcagagca tctcctctta aggatacgtg ccttggactc 1200
atgtttgaat cgaacagtct caaagtggaa gaacaaattc ttgtcacttt tttacagctt 1260
tctttggagc tgcttcagtc catctcccag aggcatttgg tctgtatctt tgctcaactg 1320
ctaatttctc ttggctgtag ggtgtgtggt taaggtacaa ccacccctaa agctcagttt 1380
tgaagtgagt gtatttatag cttctctgct ggtgctgcct tctagaggga tgatagatca 1440
tttgaaccca atgacaattt ttaaccagaa aatttaattg tacctgaatc aacctttcag 1500
cctaggacga agtctaggcc caagtcagag tattaatgat catgagaatt gtgtgctgaa 1560
ccagtaaacg agtttacctt ttaaaaaaaa as 1592
<210> 66
<211> 1390
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2271680CB1
<400> 66
gatcgtttgt cgcgatgtgg agtggccgta agctgggctc ctccgggggt tggtttttaa 60
gagtgctggg gcctggaggc tgtaatacaa aagctgcgcg tcccttaatt tcctcggcgg 120
tttatgtgaa gaaccagctc agtgggactc tacagattaa accaggggtt ttcaatgaat 180
acagaaccat atggttcaaa tcctacagga cgatcttttc ctgtttgaac agaataaaga 240
gtttcaggtg gagtttcact tctgttgccc aggctggagt acaatggtgc gatcttggct 300
cactgcaacc tccacctccc gggttcaagc gattctcctg cctgagcctc ctgagtcatt 360
gggattacag gtacccttgg gcgagactgt acagtacttc ccaaaccact gtcgacagcg 420
gtgaggtaaa aaccttcttg gccctggctc acaaatggtg ggatgaacaa ggagtatatg 480
cacctcttca ttccatgaat gacctgaggg tgccatttat tagggacaat cttctgaaaa 540
caattcctaa tcaccagcca ggaaaacctt tgttggggat gaagattctt gacgttggct 600
gtggtggtgg gctgttaact gaacctctag ggcggcttgg ggcttcagtt attggaatcg 660
accctgtgga tgagaacatt aaaacagcac aatgccataa atcatttgat ccagtcctgg 720
ataagagaat agagtacaga gtgtgttccc tggaagagat tgtggaagag actgcagaaa 780
catttgatgc tgttgtagct tctgaagttg tagaacatgt gattgatcta gaaacatttt 840
tacagtgctg ctgtcaagtg ttaaaacccg gtggttcttt attcattact acaatcaaca 900
aaacacaact ttcctatgcc ttgggaattg ttttttcaga gcaaattgca ggtattgtac 960
caaaaggtac tcatacatgg gagaagtttg tttcacctga aacactagag agcattctgg 1020
aatcaaatgg tctgtcagtt caaacagtgg taggaatgct ctataacccc ttctcaggtt 1080
actggcattg gagtgaaaat accagcctta actatgcagc tcatgctgtg aaatccaggg 1140
tccaggaaca cccagcctct gctgagtttg ttttaaaggg agaaacagaa gagctccaag 1200
ctaatgcctg caccaatcca gctgtgcatg aaaagctgaa gaaatgaatt gtttctgaga 1260
45/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
actatagtaa tatggcttgg atatctgatg ttttcaaata caagaaatgt acaatttatc 1320
ctttgagaga gaatcatgaa gaaaagaagg tcaataaaaa gggctaaaac cttggaaaaa 1380
aaaaaaaaaa 1390
<210> 67
<211> 899
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2325603CB1
<400> 67
ttcattggcg ggtccgccgc ggtcgaggat cctggaggtc ttaaccatga acttctctgg 60
aggagggagg caggaagcag cagggtccag gagtagaagg gctcccagac cccgagaaca 120
ggaccgagac gtgcagctgt ccaaggctct gtcctatgcc ctgcgccatg gggccttgaa 180
gctggggctt cccatgggag ctgatggctt cgtgcccctg ggcaccctcc tgcagttgcc 240
ccagttccgc ggcttctctg ctgaagatgt gcagcgcgtg gtggacacca ataggaagca 300
gcggttcgcc ctgcagctgg gggatcccag cactggcctt ctcatccggg ccaaccaggg 360
ccattccctg caggtaccta agttggagct gatgcccctg gagacaccgc aggccctgcc 420
cccgatgcta gtccatggta cattctggaa gcactggcca tccatcctac tcaaaggcct 480
gtcctgccag ggaaggacgc acattcacct ggccccagga ctgcctggag accccggtat 540
catcagtggc atgcggtccc attgtgaaat agctgtgttc atcgatggac ccctggctct 600
ggcagatgga atacccttct tccgctctgc caatggggtg attctgactc cagggaatac 660
tgatggcttc ctccttccca agtacttcaa ggaggccctg cagctacgcc ctacccgaaa 720
gcccctttcc ttggctggtg atgaagagac agagtgtcag agtagcccca agcacagctc 780
cagagaaagg aggaggatcc aacaataaaa tattaattta taaaaaagaa attttaaaaa 840
gtaacaagaa agaactcgtt tgaaaccatg tttcatcatc ctgtaaaaaa aaaaaaaaa 899
<210> 68
<211> 1041
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2356055CB1
<400> 68
gacgtaggac ctgatagcaa ggagggggca catttcaggg acaccatgaa gggtggcttc 60
actgggggtg atgagtacca gaagcacttc ctgcccaggg actacttggc tacttactac 120
agcttcaatg gcagcccctc acccgaggcc gagatgctga agtttaactt ggaatgtctc 180
cacaagacct tcggccctgg aggcctccaa ggggacacgc tgattgacat tggctcaggt 240
cctaccatct accaagttct tgctgcctgt gattccttcc aagacatcac tctctccgac 300
tttaccgacc gcaaccggga ggagctggaa aagtggctga agaaggagcc gggggcctat 360
gactggaccc cagcggtgaa attcgcctgt gagctggaag gaaacagcgg ccgatgggag 420
gagaaggagg agaagctgcg ggcagcggtg aagcgggtgc tcaagtgcga tgtccacctg 480
ggcaacccgc tggccccggc tgtgttgcct ctcgccgact gtgtgctcac cctgctggcc 540
atggagtgtg cctgctgtag ccttgatgcc taccgcgctg ccctgtgcaa ccttgcctca 600
ctgctcaagc cgggtggcca cctggtgacc actgtcacgc ttcggctccc gtcctacgtg 660
gtggggaagc gtgaattttc ctgcgtggcc ctggagaaag aggaggtggc agccaggcaa 720
tgtccaggag aggagattgc caaggaaagg aggctacaaa tgccccctcc ttgtgatgtc 780
aggacctccc ttagcgagcg atctggccaa gacacaggga aaagacacag gatccagacc 840
cggggctctg ctccttggac ggctcagtgc agagagtcag ctggctgcct ggaaggagag 900
agtcggcaag ggtgtgaggg aatctttggg tgctgtggaa gctgttctac cttatgaaat 960
ggggctggga tggactgagt gactatgctg tgctctgtca tttgtccgta agtactcgct 1020
gcatactctg atgcgctgat g 1041
<210> 69
<211> 1106
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2448909CB1
46/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
<400> 69
acggccagtg caagctaaaa ttaaccctca ctaaagggaa taagcttgcg gccgccgcgc 60
ctagaactgt atttcagaaa aaagaaacta cagttttagc atgcagaaag gaaaagggag 120
aacaagccgg atcagaagac gaaaactctg cggaagttct gaatcaagag gagtgaatga 180
gagccacaag tctgaattta tagagctgag gaagtggctg aaagctagga agtttcaaga 240
ttcaaactta gcgcctgctt gttttccagg tacaggaaga gggctgatga gtcaaacatc 300
cctgcaggag ggacagatga ttatttcgtt gcctgagagt tgcctgctca ccacggacac 360
agtgattcga agctacttag gggcatacat tactaagtgg aagcctcctc catctcctct 420
gctggcgctg tgcacctttt tagtttcaga aaagcatgct gggcaccgat ctctttggaa 480
gccttacctg gagattttac ccaaggcgta tacctgccct gtttgtttgg agccggaagt 540
ggtgaacctt cttcccaaat ctttaaaagc aaaggctgaa gagcagagag cccacgtgca 600
ggagttcttt gcttcctcca gagacttttt ctcttctctg cagcctctgt ttgcggaggc 660
tgttgacagc atcttcagct acagtgccct gctgtgggct tggtgcaccg tcaacaccag 720
agccgtgtac ctgaggccca ggcagcggga atgcctttct gcagagccgg acacctgtgc 780
actcgctccg tacctggacc tgctgaatca tagcccacat gtccaggtaa aagcagcgtt 840
taatgaagaa actcattctt acgaaattag aacgacttca cgttggagaa agcatgaaga 900
ggtattcatc tgttacggcc ctcacgataa tcaacggctg ttcctggaat acggatttgt 960
ttctgtccat aatcctcatg cttgtgttta tgtctcaaga ggttggaatc aactttgttc 1020
ttaacattaa cactatataa tttttttccc catttggaga tgtgtatttt cagttttaat 1080
aaaaatatca aaaccttaaa aaaaaa 1106
<210> 70
<211> 2405
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2631212CB1
<400> 70
cagcgggtct ggctggcggc agcggcggga gggagccgag agacccgagt gcacgtgtgg 60
agaagcggcg gcacaagcgc ggcggcggga gacactcccg cccccaccag actcaagccc 120
tcactcgact ctcgcggcct tcgttgctcg cacagctccc tgcccaggct aggaggccgg 180
cttgcggggt tgagtggccc gagctaaggg tgcggagacc taagggcggc gactacgacg 240
gcgttgatat cggtggtaac gacggcctca gcaggcgggg aagatgaaag gtagccggat 300
cgagctggga gatgtgacac cacacaatat taaacagttg aaaagattga atcaggtcat 360
ctttccagtc agctacaatg acaagttcta caaggatgtg ctggaggttg gcgagctagc 420
aaaacttgcc tatttcaatg atattgctgt aggtgcagta tgctgtaggg tggatcattc 480
acagaatcag aagagacttt acatcatgac actaggatgt ctggcacctt accgaaggct 540
aggaatagga actaaaatgt taaatcatgt cttaaacatc tgtgaaaaag atggtacttt 600
tgacaacatt tatctgcatg tccagatcag caatgagtcg gcaattgact tctacaggaa 660
gtttggcttt gagattattg agacaaagaa gaactactat aagaggatag agcccgcaga 720
tgctcatgtg ctgcagaaaa acctcaaagt tccttctggt cagaatgcag atgtgcaaaa 780.
gacagacaac tgaacaaatt acaaatgaac tttcttgcac ttgcttgtcg ccaaataaaa 840
gagaggccca ttgattcctc ccccacccca acacttttct tttaaagctt ttctccctcc 900
ttgttcttgt ttttctttct tcctttcctt ttctctgaga gttttaatac tttcaaggac 960
tttaaaaaaa taatcatgtt tgaattgttt tctcttattt ttgtgaggtg gtttgaagga 1020
aggacaaggt agatctgttt agttttgcag ttgaagttag atggtcctaa acatttaatt 1080
gtcaaataat ttcaaattta atgtcctgct ttcacattga agggcagagc ctacaaaaca 1140
ttgtatattt caaaagacaa aaagaagcag cagcagtatc ttgttctcta attcatagac 1200
aagttgagtg tgtttgtggt actttgggtt tttaaacact ttgggatact aatccctaga 1260
cattgccttc actccacctt tagtccttct gagcactctc tcgggagttg gaacattgtt 1320
atccttgtaa gaaatactaa gcttatgttg atttttaagt aattatatct tctcttcttg 1380
ctggtgggtg gggcagtttg gtttagtgtt atactttggt ctaagtattt gagttaaact 1440
gcttttttgc taatgagtgg gctggttgtt agcaggtttg tttttcctgc tgttgattgt 1500
tactagtggc attaactttt agaatttggg ctggtgagat taattttttt taatatccca 1560
gctagagata tggcctttaa ctgacctaaa gaggtgtgtt gtgatttaat tttttcccgt 1620
tcctttttct tcagtaaacc caacaatagt ctaaccttaa aaattgagtt gatgtcctta 1680
taggtcacta cccctaaata aacctgaagc aggtgttttc tcttggacat actaaaaaat 1740
acctaaaagg aagcttagat gggctgtgac acaaaaaatt caattactgt catctaatgc 1800
cagctgttaa aagtgtggcc actgagcatt tgattttata ggaaaaaata gtatttttga 1860
gaataacata gctgtgctat tgcacatctg ttggaggaca tcccagattt gcttatactc 1920
agtgcctgtg atattgagtt taaggatttg aggcaggggt aattattaaa catattgctt 1980
ctattcttgg aaaaatagaa gtgtaaaatg ttaataatac aaatgtcact gtgacctcct 2040
ccactgagag gactggttta tgccagatca ttttccggca cacacggagt ggctttgaca 2100
gattgataac tttgtaagat gggagacatc tgaaatattc atgttttcct tttgtagtcc 2160
47/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
catctccact atttagaaat gttctcagac tttaaaataa tgcacagggc ttgagctttc 2220
tgtcatttga ctttaaaagg aagtttcatt catatttatc ctcttatgta aaattgcggt 2280
ataaagtctc atttccaaat atgttaaatg acaaaattat tttataaaat gtttatgcac 2340
actttataac cttaagtttt tatttgagaa tgtgaaagta caaagtgcag tagacttcaa 2400
caatc 2405
<210> 71
<211> 1239
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2678733CB1
<400> 71
ggcgatgagg gtgttggtgc ggcgctgttg gggtcctccg ctggctcatg gcgccaggcg 60
tgggaggccg agtccccagt ggcgagcact ggcccgactc ggctgggagg actgccggga 120
ctccagagtc cgcgagaagc ctccctggcg ggtgctcttc ttcggcacgg accagttcgc 180
ccgcgaggcg ctgcgggcgc tgcacgccgc cagggaaaac aaagaagaag agttaatcga 240
caaactggag gtggtcacaa tgccttcccc atcaccaaaa ggactgccag tgaagcaata 300
tgctgtgcag tctcagcttc ccgtatatga gtggccggat gtgggatctg gagaatatga 360
tgttggagta gtggcttcgt ttggccgact tttgaatgag gctcttattc ttaaatttcc 420
ctatggcata ttgaatgttc atcccagttg cctcccgaga tggcgtggcc cagcccctgt 480
aatccataca gtgcttcacg gagacacagt tactggagta acaattatgc aaattagacc 540
taaaaggttt gatgtaggcc caattctcaa acaagaaact gttcctgtgc cacccaagag 600
cactgcaaag gaattggaag cagtgttgtc aagactgggt gccaacatgc tcatttcagt 660
tttgaaaaat ttgcctgaaa gtctgagcaa tggaaggcag cagccaatgg agggggcgac 720
ttacgcccct aagatttctg ctggtaccag ttgtataaaa tgggaggaac aaacttcaga 780
acaaatattc agactttacc gtgccattgg aaatataatt ccgttgcaga cgctctggat 840
ggcgaatacc attaaacttc tggatttggt agaagttaac agttcagtcc ttgctgatcc 900
aaaattaacg ggacaggctc ttattccagg atcagtaata taccacaaac agtcacaaat 960
actattggtt tattgcaagg atggttggat tggtgttcga tcagtgatgc tcaagaaatc 1020
actaacagct actgacttct acaatggata tttgcacccc tggtaccaga aaaattccca 1080
agctcaacca agccaatgca gatttcagac tctcagactt ccaacaaaga agaagcagaa 1140
aaaaactgtt gctatgcaac aatgcattga gtagttagga agaagatgga taaaaaccta 1200
ttacatattt gtaatttatt aaaaacctta tttacaagg 1239
<210> 72
<211> 2295
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2768571CB1
<400> 72
aaactcagca cttgccggag tggctcattg ttaagacaaa gggtgtgcac ttcctggcca 60
ggaaacctga gcggtgagac tcccagctgc ctacatcaag gccccaggac atgcagaacc 120
ttcctctaga acccgaccca ccaccatgag gtcctgcctg tggagatgca ggcacctgag 180
ccaaggcgtc cagtggtcct tgcttctggc tgtcctggtc ttctttctct tcgccttgcc 240
ctcttttatt aaggagcctc aaacaaagcc ttccaggcat caacgcacag agaacattaa 300
agaaaggtct ctacagtccc tggcaaagcc taagtcccag gcacccacaa gggcaaggag 360
gacaaccatc tatgcagagc cagtgccaga gaacaatgcc ctcaacacac aaacccagcc 420
caaggcccac accaccggag acagaggaaa ggaggccaac caggcaccgc cggaggagca 480
ggacaaggtg ccccacacag cacagagggc agcatggaag agcccagaaa aagagaaaac 540
catggtgaac acactgtcac ccagagggca agatgcaggg atggcctctg gcaggacaga 600
ggcacaatca tggaagagcc aggacacaaa gacgacccaa ggaaatgggg gccagaccag 660
gaagctgacg gcctccagga cggtgtcaga gaagcaccag ggcaaagcgg caaccacagc 720
caagacgctc attcccaaaa gtcagcacag aatgctggct cccacaggag cagtgtcaac 780
aaggacgaga cagaaaggag tgaccacagc agtcatccca cctaaggaga agaaacctca 840
ggccacccca ccccctgccc ctttccagag ccccacgacg cagagaaacc aaagactgaa 900
ggccgccaac ttcaaatctg agcctcggtg ggattttgag gaaaaataca gcttcgaaat 960
aggaggcctt cagacgactt gccctgactc tgtgaagatc aaagcctcca agtcgctgtg 1020
gctccagaaa ctctttctgc ccaacctcac tctcttcctg gactccagac acttcaacca 1080
gagtgagtgg gaccgcctgg aacactttgc accacccttt ggcttcatgg agctcaacta 1140
48/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
ctccttggtg cagaaggtcg tgacacgctt ccctccagtg ccccagcagc agctgctcct 1200
ggccagcctc cccgctggga gcctccggtg catcacctgt gccgtggtgg gcaacggggg 1260
catcctgaac aactcccaca tgggccagga gatagacagt cacgactacg tgttccgatt 1320
gagcggagct ctcattaaag gctacgaaca ggatgtgggg actcggacat ccttctacgg 1380
ctttaccgcc ttctccctga cccagtcact ccttatattg ggcaatcggg gtttcaagaa 1440
cgtgcctctt gggaaggacg tccgctactt gcacttcctg gaaggcaccc gggactatga 1500
gtggctggaa gcactgctta tgaatcagac ggtgatgtca aaaaaccttt tctggttcag 1560
gcacagaccc caggaagctt ttcgggaagc cctgcacatg gacaggtacc tgttgctgca 1620
cccagacttt ctccgataca tgaagaacag gtttctgagg tctaagaccc tggatggtgc 1680
ccactggagg atataccgcc ccaccactgg ggccctcctg ctgctcactg cccttcagct 1740
ctgtgaccag gtgagtgctt atggcttcat cactgagggc catgagcgct tttctgatca 1800
ctactatgat acatcatgga agcggctgat cttttacata aaccatgact tcaagctgga 1860
gagagaagtc tggaagcggc tacacgatga agggataatc cggctgtacc agcgtcctgg 1920
tcccggaact gccaaagcca agaactgacc ggggccaggg ctgccatggt ctccttgcct 1980
gctccaaggc acaggataca gtgggaatct tgagactctt tggccatttc ccatggctca 2040
gactaagctc caagcccttc aagagttcca agggaacact tgaaccatgg acaagactct 2100
ctcaagatgg caaatggcta attgaggttc tgaagttctt cagtacattg ctgtaggtcc 2160
tgaggccagg gatttttaat taaatggggt gatgggtggc caataccaca attcctgctg 2220
aaaaacactc ttccagtcca aaagcttctt gatacagaaa aaagagcctg gatttacaga 2280
aacatataga tctgg 2295
<210> 73
<211> 2182
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3189062CB1
<400> 73
ccggcatggc cgggtgagct gcaggcttcc ttatttaaga ccgggaattt agtcagcctg 60
gtgagccgac tctgaggaga tggagtatcg ctgaggtgat gagagagaat gtggttgtta 120
gcaacatgga gagagaaagt gggaagcccg tggctgttgt cgcagttgtg actgagcctt 180
ggtttaccca gcgatacaga gaatatctcc agaggcagaa actctttgat acacagcacc 240
gtgtggaaaa gatgccggat ggctcggtgg cgctaccggt gctgggagag acgcttccag 300
agcagcacct gcaggagctg aggaatcgtg ttgccccagg cagtccctgt atgctcacgc 360
agctcccgga tcctgttcct tcgaagaggg cccagggttg ttcacctgcc caaaaattgt 420
gtcttgaggt gagtcgctgg gtggtgggtc ggggagtcaa gtggtcagcc gagttggagg 480
ctgatttgcc ccgatcatgg caacggcatg gtaatctctt gttgctgagt gaagactgtt 540
tccaagccaa gcagtggaaa aatctgggac cggaactctg ggagaccgtt gccttggcac 600
ttggcgtcca gcgtttggca aaacgagggc gggtatcacc ggatggtact cgaactccag 660
cagtgacact gctgctgggt gaccatggct gggtagagca tgtggataat ggtatccgtt 720
ataagtttga cgtgacccag tgtatgttct cctttggaaa catcactgag aagcttcgag 780
tggcatcgtt gtcctgtgct ggagaagtgc tggtggatct ctatgcaggg attggttatt 840
ttacattgcc tttcctagtt catgctggtg ctgccttcgt ccatgcttgt gagtggaatc 900
cccatgctgt agttgctctg agaaataacc ttgagatcaa tggagtagca gatcggtgcc 960
aaatacactt tggagataac agaaaactga agctctcaaa tattgcagat agggtgatcc 1020
tggggctgat tcccagctct gaagaaggct ggcccattgc ctgccaagtg ttaaggcagg 1080
atgctggagg cattttgcat atccaccaaa atgtggaatc tttcccaggg aagaatcttc 1140
aggctcttgg agtcagcaaa gtagagaaag agcattggct gtatcctcag caaattacca 1200
ccaaccaatg gaaaaatgga gctaccaggg attctagggg aaaaatgctg tcaccagcca 1260
ccaagccaga gtggcaaagg tgggcagaat ctgcagaaac tcgaatcgcc actcttcttc 1320
agcaggtgca tgggaaacca tggaagacac aaattctgca catccaacca gtgaaatcct 1380
atgctcccca tgtggatcac atagtcctgg atctggaatg ctgcccctgt ccttcagttg 1440
gctagaggag gtagatcctg ggacacatgg gatccacgtg cgagtggccc ttaaatgtat 1500
cagttcagtc caggttgtca tcccttttgt cccctggtga tcagtttttt tcatatttta 1560
tagccctgaa agcaggctct agatcaattc aaattatttc atttgtcttt cattgataac 1620
agaaaatgaa atacctgttt gggagaagca gcatggccca ttgaaatgag gctcatctgt 1680
gcaattatga attccaaatt ctgacctcag ttctggaatt gaagtttcag tatgttttgg 1740
cctcgggttt cgttatttgc aaaatgagag tttctttgaa ctgtctcacg tgactattaa 1800
gcaactatac acaggacatc ggttatttta gagtgaaaga cacagtgctt tttccaaatt 1860
gctctggcta ccatatagaa aattgactga aggagggcca agatggaaac agagagacca 1920
gtgaggaggc ttctgtggtt gtccaggtct gaggtgatgg taacttggac tcggatggtg 1980
gtaatgggag gtagattgat atgataaata aaattgacag accaagcaat ggaatcagaa 2040
ttaagtcctt aacatgaagc tgctttgtta ttatgactga ctaaattaga gagagaaggg 2100
aacaaaaatt attttagtgt atttccattc ccttactgtg gcattcctaa atgattgtga 2160
49/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
ggggttgtct tataaatttg gt 2182
<210> 74
<211> 1288
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3243884CB1
<400> 74
cgtttctttc ggagcggcgg tgaaggtcct gggtgaggta ggggtggatg gtgcttgccg 60
cgtatcatgg ctgcctccgg aaaactcagc acttgccgtc tccctccgtt gcccacgatt 120
cgagaaatca ttaagttgtt aagactgcaa gcagcgaagc agctatcaca gaatttcctc 180
ctggacttga ggctgacaga taagattgta aggaaagctg gcaatctgac aaatgcttat 240
gtttacgaag tgggccctgg gccaggggga atcacaagat ctattcttaa tgccgacgtc 300
gctgaacttc tggtggttga aaaggacact cgatttattc ctggattaca gatgctttct 360
gatgcagcac ctgggaaact gagaattgtt catggagatg tcttgacatt taaggtagaa 420
aaggcttttt cagaaagtct taaaagaccc tgggaagatg atcctccaaa tgtacatatt 480
attggaaatc tgccttttag tgtttcaact ccactgatta tcaagtggct tgaaaatatt 540
tcctgtagag atggaccttt tgtttatggc agaactcaga tgactttgac ttttcaaaag 600
gaagtggcag agagacttgc agccaataca ggaagcaaac agcgtagtcg cctctctgtt 660
atggctcagt acctctgcaa tgttcgacac atctttacaa ttccaggaca agcttttgtc 720
cccaaaccag aggtggacgt gggcgtggtg cacttcactc ccttgataca gcccaagata 780
gagcagccat tcaagctggt ggaaaaagtg gttcagaatg tatttcagtt ccgaaggaaa 840
tactgccatc gagggctcag aatgttattc cctgaagcgc agcgcttgga aagcacgggc 900
aggctgttag agttggcaga catagaccct actcttcggc cccgccagct ctccatctca 960
cactttaaga gcctctgtga tgtatacaga aaaatgtgtg atgaagaccc acaactcttt 1020
gcatataatt tcagagaaga actcaagcga agaaaaagca aaaatgaaga aaaagaagag 1080
gatgacgcag agaattacag actctagctg ctgcctgggg gcgagcagcc taccagatgt 1140
cgatttgcac tacgtggagc ttcttatata ggtactcttt tgtctttaca gaatgacgat 1200
acaaatgcca atgaccagat gtgacttatt ttccttttac tatacagctt ggcagagaaa 1260
ataaatatca tcaaataaga aaaaaaaa 1288
<210> 75
<211> 1130
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3400578CB1
<400> 75
ggtgaacccg cagccggact tctgctgcac tggggctccg aatgacccag aatctggggt 60
gggcttaggc cccgggagtg agatggcctc aatcttgcga acgcctcagg ctctccagct 120
cactctagcc ctgatcaagc ctgacgcagt cgcccatcca ctgattctgg aggctgttca 180
tcagcagatt ctaagcaaca agttcctgat tgtacgaatg agagaactac tgtggagaaa 240
ggaagattgc cagaggtttt accgagagca tgaagccggg ccaatccgag cctacatcct 300
tgcccacaag gatgccatcc agctctggag gacgctcatg ggacccacca gagtgttccg 360
agcacgccat gtggccccag attctatccg tgggagtttc ggcctcactg acacccgcaa 420
caccacccat ggttcggact ctgtggtttc agccagcaga gagattgcag ccttcttccc 480
tgacttcagt gaacagcgct ggtatgagga ggaagagccc cagttgcgct gtggccctgt 540
gtgctatagc ccagagggag gtgtccacta tgtagctgga acaggaggcc taggaccagc 600
ctgatgcagg tctatgaaga ccagtggtag tgcccagact tctcctagac atctagtcta 660
aaacattctc ctaggaccag ggaagcctgg cttacagtgc catttctgct gggcaccacc 720
acctgcctga gggcctagct caccacagca catcctccag gatctagcct tctatctacc 780
tcttctctgg aatgtttatg gtggttcaga agaatgatga ctcctctttg ctgagaactg 840
ttcatccttc ttcaagaaga agcttgccag gccgggcacg gtgctcacgc ctataatccc 900
agcactttgg gaggccgagg cgggcggatc acaaggtcag gaattcgaga ccagcctgac 960
caacatggtg aaaccccatc tctactaaaa atacaacaat tagccaggca tggtggtgca 1020
tgcctgtaat cccagctact cagaggctga ggcaggagaa ttgcttgatc ctgggaggca 1080
gagggtgcag tgagccgaga tcgtgccatt gcactccagc ctgagggagg 1130
<210> 76
<211> 1815
50/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3422577CB1
<400> 76
gacggcactg ggtggggccg agctccaggg ctggctgctg ggctgctaag ggaactgtga 60
gccgctcaga gccgcgcgcc tcccgggcgg ggcggggccg gccgtgggag tccgcgcgtg 120
cccgcgccga gctgcctgct ccggcggctt cgctgctagc tcgcggcgac gtcgggccga 180
ttttcccagg atgacagagc tgaggcagag ggtggcccat gagccggttg cgccacccga 240
ggacaaggag tcagagtcag aagcaaaggt agatggagag actgcatcgg acagtgagag 300
ccgggcagaa tccgcacccc tgccagtctc tgcagatgat accccggagg tcctcaatag 360
ggccctttcc aacttgtctt caagatggaa gaactggtgg gtgagaggca tcctgacttt 420
ggccatgatt gcatttttct tcatcatcat ttacctggga ccaatggttt tgatgataat 480
cgtgatgtgc gttcagatta agtgtttcca tgagataatc actattggct acaacgtcta 540
ccactcatat gatctgccct ggttcaggac gctcagctgg tactttctcc tgtgtgtaaa 600
ctatttcttc tatggtgaga cagtgacgga ttacttcttc accctggtcc agagagaaga 660
gcctttgcgg attctcagta aataccaccg gttcatttcc tttactctct atctaatagg 720
attctgcatg tttgtactga gtctggtcaa gaagcattat cgactgcagt tctacatgtt 780
tggctggacc catgtgacat tgctgattgt tgtaacacag tcacatcttg ttatccacaa 840
cctatttgaa ggaatgatct ggttcattgt ccccatatct tgtgtgatct gtaatgacat 900
catggcctat atgtttggct ttttctttgg tcggacccca ctcatcaagc tgtccccgaa 960
gaagacctgg gaaggcttca ttgggggctt ctttgctact gtggtgtttg gccttctgct 1020
gtcctatgtg atgtccgggt acagatgctt tgtctgccct gtggagtaca acaatgacac 1080
caacagcttc actgtggact gtgagccctc ggacctgttt cgcctgcagg agtacaacat 1140
tcctggggtg atccagtcag tcattggctg gaaaacggtc cggatgtacc ccttccagat 1200
tcacagcatc gctctctcca cctttgcctc gctcattggc ccctttggag gattcttcgc 1260
aagtggattc aaacgagcct ttaaaatcaa agactttgcc aataccattc ctggccatgg 1320
aggcatcatg gatcgctttg actgccagta tctgatggcc acctttgtca atgtatacat 1380
cgccagtttt atcagaggcc ctaacccaag caaactgatt cagcagttcc tgactttacg 1440
gccagatcag cagctccaca tcttcaacac gctgcggtct catctgatcg acaaagggat 1500
gctgacatcc accacagagg acgagtaggg gccacccagg gccaggagaa caggaacaga 1560
actgagcagg ggcaggtctc caaggcaagc ccagctggtg tgacttagac aatgacgagg 1620
cttcaactca ctgtcttttt tttttttttt tggagggtat tttttatttg tgggttcaaa 1680
aaatctgtat atacagtcta tgtgtttaga atttgtgttg taagtaaact acagctttga 1740
gttggaaaga agtcacgggt tgtaaaacca tttggatttt tttaaaacaa aagtattaat 1800
aatctggaag acggt
1815
<210> 77
<211> 1740
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3706809CB1
<400> 77
tgacgcagcc cgggtctcag ggaacatggc ggcgctggtg agacccgcga ggtttgtcgt 60
gcgaccgttg ctgcaggtgg tccaggcttg ggaccttgac gcgaggcgct gggtccgggc 120
gctgcggcgg agcccagtga aagtggtgtt tccttccgga gaggtggtgg aacagaagcg 180
cgctcctggg aagcagcccc gcaaggcacc atctgaggcc agtgcccagg agcaacgaga 240
gaaacaaccg ctcgaggagt ccgcatcccg cgctcccagc acctgggaag agtctgggct 300
tcgctacgat aaagcttatc ccggggacag gaggctgagc agtgtaatga caatagtaaa 360
gtccaggcca tttcgggaaa aacaagggaa gatcctgctg gaaggtcgca ggctcatttc 420
agacgctctc aaggctggag ctgtgccaaa aatgttcttc tttagccgtc tagaatacct 480
aaaggagttg ccagtcgata agctgaaagg tgtcagcctc attaaggtga aatttgagga 540
tatcaaggat tggtccgacc tcgtaacgcc acaaggaata atggggattt ttgccaagcc 600
tgaccatgtt aagatgacat atccaaagac tcagcttcag cattcactgc ctttattatt 660
gatttgtgac aatctccgtg accctgggaa cctggggaca attctgagat ctgcagctgg 720
ggcaggctgc agcaaagtgt tactcaccaa aggctgtgtg gatgcctggg agcccaaagt 780
gctccgggcg ggtatgggcg cacatttccg gatgcccatt atcaataatc tggaatggga 840
aaccgtgccc aattacctgc cccctgacac tcgggtctat gtggctgaca actgtggcct 900
ttatgcccag gctgagatgt ctaataaagc tagtgaccat ggctgggtgt gtgatcaacg 960
agtgatgaag tttcacaagt atgaggaaga ggaagatgta gaaaccggag ccagtcaaga 1020
51/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
ttggctgcct catgttgagg ttcagagtta cgactcggac tggacagagg cgccggcagc 1080
tgtggtgatt ggcggggaga cctacggcgt gagcctggag tccctgcagc tggccgagag 1140
cactggtggc aagaggctgc tgatccccgt tgtgcctggt gtggacagcc tcaactcggc 1200
catggcggca agcatcctgc ttttcgaagg gaaaagacag ctgcggggga gggcggagga 1260
cttgagcagg gacaggagtt accactgagg acgcagaagt gacttctgct tgaggacgtc 1320
tgcagctcct cctacaccag cacactggtg ggaggctggc ggagtcagtg actatggccc 1380
ccacgttcag gaggaaggtg tgatgccgtc atacagttac aggaaaaata agaacttcct 1440
cagaaagaac aggtccgaat tcttcctgtc gcgtcactga ttttgaggtt cttttttctc 1500
ttggtgacaa taggtgaccc acgtggctct gtgtgttttt aaaaattgtc caccaagaag 1560
cactttgtgc ccagaaagtt cctgaagcat catcctggca gggaggcgcc tgctccacca 1620
gctggtgggt gtttgtaatc gccaagcacc agctataggt cacagccaca tcactcacag 1680
ctgatcactg gttggtggaa aataaactat gagcagcaga ttacgttaaa aaaaaaaaaa 1740
<210> 78
<211> 1225
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3745914CB1
<400> 78
cgggaagagc cggccgaagc gtggcggcca cagactgtgg gtaccgggtc cgagggactc 60
gcgcttttct ctccgtgcca tggcgccagc gaaagccacg aacgtggtgc ggctgctact 120
aggctccaca gcgctgtggc tttcgcagct cggctccggg acggtcgccg cgtccaagtc 180
ggtgactgcc cacttggccg cgaagtggcc cgagaccccg ctgctgctgg aggcaagtga 240
atttatggca gaagaaagta atgaaaaatt ttggcagttt ttggaaactg tgcaagaatt 300
agcaatttat aagcaaacag aatcagatta ttcttattac aacttaatcc tgaagaaagc 360
tggacagttt ctagacaatt tacacatcaa ccttttaaag tttgctttct ctataagggc 420
atactcccca gctattcaga tgtttcagca gattgcagct gatgagccac caccagatgg 480
ttgtaatgca tttgtggtta ttcataagaa gcacacctgt aaaattaatg agattaaaaa 540
gctgctgaag aaagctgctt caaggactag accttatcta tttaaaggag atcacaaatt 600
tcctacaaac aaagagaact taccagtggt gattctctat gccgaaatgg gtactagaac 660
atttagtgca tttcacaaag tattgtctga aaaagctcaa aatgaggaaa ttctgtatgt 720
tcttcgccat tatattcaga aaccaagctc acggaaaatg tacttatctg ggtatggtgt 780
ggagctagca attaagagta cagaatacaa agcactggat gatacccaag ttaaaactgt 840
gactaatact actgtagagg atgagactga aacaaatgaa gttcaaggat ttctctttgg 900
gaaactaaaa gaaatatatt cagatcttag agataatctg acagcattcc aaaaatacct 960
gattgagagt aacaaacaaa tgatgccttt gaaagtctgg gaactacaag atcttagttt 1020
tcaagcagct tctcaaataa tgtccgctcc agtttatgat gccattaaat taatgaaaga 1080
catttcacag aacttcccca taaaagccag agtccaaatg attggtaatg tcttaattgg 1140
atgaatattg tgtggagtac ttttttgcca agaggatgtc tcgttgaact gcttccatga 1200
atactgatgt tacattaaac atata 1225
<210> 79
<211> 800
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4000776CB1
<400> 79
atttacagct tgtcatcgtg tcatcccttt aaccaacatg tgttttgttg gtatcaaatg 60
tgtcaggttt aattgacatt ttaaaatgtc ttcaaaaaga tcacactatg attcagcatt 120
gaaacgaaaa gttattgtgt atgcagaaaa gcatggaaac agagcagcag ggcgtacatt 180
tgatattagt gaagcaaata ttcgtcgctg gagaaatgat cgcaattcca tattttcctg 240
caaagcaaca acaaaatgtt ttacgggacc taagaaaggt aggtacccac aagtagatga 300
agctgtacta cgtttcgtca gtgagacacg tgcaaaagga ttgcctatca cacgccaagc 360
aatgcaattg aaggcaggag aagttgccaa aaccctagga attgatgaaa caaaattcaa 420
agccacaaga ggctggtgtg accgattcat gcgtcgagca ggactatcat taaggcatca 480
aacatcgttt tgtcccaagc tccccactgc cattaaacag aagacagtgt tggagcactc 540
tttcaagaag tgctgcataa ccagtactct tgacaacacc gggagagacg ttctgtggaa 600
aaatgcagac atcaatgact gtggtttgaa aagtgattca gaagagttgg attcagaata 660
tgaagttata attataactt aaccaattta tttcacacat tttctttaca tatgcacaag 720
52/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
attgatgtga taaaaatctg tttaactcaa agcgctgttt caataaatat aaacattttc 780
tgtgatatga aaaaaaaaaa 800
<210> 80
<211> 1163
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4071304CB1
<400> 80
atcaaaattt actctgcgag actctgctga actggctgca tcagggagaa atttcatctc 60
ccagaagggt gttgctcatc gtttcttccc ggaaacatct gcagagacta gcttttcagg 120
ctaaggtatc ctccatgatg ttaccattgc aaggtgccca gatgctgcag atgctggaga 180
aatccttgag gaagagcctc ccagcatcct taaaggttta tggaactgtc tttcacataa 240
accatggaaa tccattcaat ctgaaggctg tggtggacaa gtggcctgat tttaatacag 300
tggttgtctg ccctcaggag caggatatga cagatgacct tgatcactat accaatactt 360
accaaatcta ctccaaagat ccccaaaact gtcaggaatt ccttggatca ccagaactca 420
tcaactggaa acagcattta cagattcaaa gttcacagcc tagcctgaat gaggctatac 480
aaaatcttgc agccattaag tccttcaaag tcaaacaaac acaacgcatt ctctatatgg 540
cagctgaaac agccaaggaa ctgactcctt tcctgctgaa atcaaagatt ttatctccca 600
gtggtggcaa acccaaggcc atcaaccaag agatgtttaa actctcatcc atggatgtta 660
cccatgctca cttggtgaat aaattctggc attttggtgg taatgagagg agccagagat 720
tcattgagcg ctgcattcag acctttccca cctgctgtct cctggggcct gaggggaccc 780
ctgtgtgctg ggatctaatg gaccagactg gagagatgag aatggcaggc accttcgcgg 840
aataccggct ccatggcctt gtgacgtatg tcatctattc ccacgcccag aaattgggca 900
aacttgggtt tcctgtctat tctcatgtag actacagcaa tgaagctatg caaaaaatga 960
gttacacact gcaacatgtt cccattccca gaagctggaa ccagtggaac tgtgtacctc 1020
tgtgatgcca atcctgaaca taagacagtg ttgggcaggt ctgggcgtat aatttgagga 1080
gtggatggtg gatgggaaga attaattaag cggtcgcaag cttattccct ttagtgaggg 1140
ttaattttag cttgcaggct acg 1163
<210> 81
<211> 888
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4344970C81
<400> 81
aaggactatg gcaggggaga acttcgctac gccgttccac gggcacgtgg gccgcggcgc 60
cttcagcgac gtgtacgagc ccgcggagga cacgtttctg cttttggacg cgctcgaggc 120
agcggctgcc gaactggcag gagtggaaat atgcctggaa gtagggtcag ggtctggtgt 180
agtatctgca ttcctagcct ctatgatagg ccctcaggct ttgtacatgt gcactgatat 240
caaccctgag gcagcagctt gtaccctaga gacagcacgc tgtaacaaag ttcacattca 300
accagttatt acagatttgg tcaaaggctt gctaccaaga ttgaccgaaa aagttgatct 360
tctggtgttt aatcccccct atgtagtgac tccacctcaa gaggtaggaa gtcacggaat 420
agaggcagct tgggctggtg gcagaaatgg tcgggaagtc atggacaggt tttttcccct 480
ggttccagat ctcctttcac caagaggatt attctattta gttaccatta aagaaaacaa 540
cccagaagaa attttgaaaa taatgaagac aaaaggtctg caaggaacca ctgcactttc 600
cagacaagca ggccaagaaa ctctttcagt cctcaagttc accaagtctt agcatacagt 660
gtgtgcccag aactactgga aactgaatgc atttagcata ttttgaaact gaagtcattc 720
attaggtaac aaggaatttt atcagaaatt tgtcattaaa aaaaggtaga aaggtggaac 780
attccctttc agtcaggcct atagaattat ttgcacaagt acaagtaaat atgtgtatta 840
tatttatagt ttgcatttta aatatagaaa ttatgtatga aaaaaaaa 888
<210> 82
<211> 1163
<212> DNA
<213> Homo Sapiens
<220>
<221> misc feature
53/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
<223> Incyte ID No: 5392302CB1
<400> 82
gtggcgtctc tggttacggg gtcgggcaaa gggcagaggt caccgtccag gttggacagc 60
agcacctttg agcgatggcg gcgtctgggg aaccccagag gcagtggcaa gaggaggtgg 120
cggcggtggt agtggtgggc tcctgcatga ccgacctggt cagtcttact tctcgtttgc 180
caaaaactgg agaaaccatc catggacata agttttttat tggctttgga gggaaaggtg 240
ccaaccagtg tgtccaagct gctcggcttg gagcaatgac gtccatggtg tgtaaggttg 300
gcaaagattc ttttggcaat gattatatag aaaacttaaa acagaatgat atttctacag 360
aatttacata tcagactaaa gatgctgcta caggaactgc ttctataatt gtcaataatg 420
aaggccagaa tatcattgtc atagtggctg gagcaaattt acttttgaat acggaggatc 480
tgagggcagc agccaatgtc attagcagag ccaaagtcat ggtctgccag ctcgaaataa 540
ctccagcaac ttctttggaa gccctaacaa tggcccgcag gagtggagtg aaaaccttgt 600
tcaatccagc ccctgccatt gctgacctgg atccccagtt ctacaccctc tcagatgtgt 660
tctgctgcaa tgaaagtgag gctgagattt taactggcct cacggtgggc agcgctgcag 720
atgctgggga ggctgcatta gtgctcttga aaaggggctg ccaggtggta atcattacct 780
taggggctga aggatgtgtg gtgctgtcac agacagaacc tgagccaaag cacattccca 840
cagagaaagt caaggctgtg gataccacgg gtgctggtga cagctttgtg ggagctctgg 900
ccttctacct ggcttactat ccaaatctgt ccttggaaga catgctcaac agatccaatt 960
tcattgcagc agtcagtgtc caggctgcag gaacacagtc atcttaccct tacaaaaaag 1020
accttccgct tactctgttt tgattgctat tagtcccaaa ataaatatac ctgggaataa 1080
aatgtacttg ggggtggctg ctcctggcta atgcttatta gaaaatgtcc tcgtcccgta 1140
tctttgcaaa tattagtatt ggg 1163
<210> 83
<211> 931
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5555235CB1
<400> 83
gcgaggacag aggtgggctc aacaccaaga ggccaccaat gaaaacgtga ctttttcaca 60
ttccccgccc cttccgtgtg cacgctcccc acgaggggct ggttcctgaa aggatgtaca 120
gaggatcccc aaccgcctgc gaaacccaag ccgccgcgta ggagcgtgcg ttcgggccct 180
cttctcccac ctgttcgact ccccatcccc aggatgtcaa cctcagtccc tcaaggccat 240
acctggaccc aacgggtgaa gaaagacgat gaggaggagg acccgctgga ccagctgatc 300
tcccgctctg gctgtgctgc ctcccacttt gcagtgcagg agtgcatggc ccagcaccag 360
gactggcggc aatgccagcc acaggtgcag gcgttcaagg attgcatgag tgaacagcag 420
gcgaggcggc aagaggagct gcagaggagg caagaacaag ccggtgccca ccactgagac 480
cccaaaccac ctatccccag tagatggccc tgccaagacc agcacccagc aagattatag 540
aggaagaaat cctaaatgct ggtgtgggag gtctaaaaca tggggagagt ttttggatct 600
ggagttgaga gccatgggtt tggacatgac tggcacaaac agctgtcata tgttcatggt 660
cagatgtcat acattctcag ctgtcttgtt ccaccagtat ttaccaggaa aacaaagaat 720
gtgttaaggg atgctccccc accccacatc ttaagtcagt gtgccaagta ctgagatgat 780
tttagggaca ttttatttta aattaaattt acaatctaat ggtaaattga ttacctaatt 840
agtgcctttc tctttcatca cttgttctgg atgtttcaat cagtttagtg gaaggaaaaa 900
taaatgggga aactttttat tcaaaaaaaa a 931
<210> 84
<211> 1794
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5573296CB1
<400> 84
agctcttata aactagtggc aatttctgaa cccagccggc tccatctcag cttctggttt 60
ctaagtccat gtgccaaagg ctgccaggaa ggagacgcct tcctgagtcc tggatctttc 120
ttccttctgg aaatctttga ctgtgggtag ttatttattt ctgaataaga gcgtccacgc 180
atcatggacc tcgcgggact gctgaagtct cagttcctgt gccacctggt cttctgctac 240
gtctttattg cctcagggct aatcatcaac accattcagc tcttcactct cctcctctgg 300
cccattaaca agcagctctt ccggaagatc aactgcagac tgtcctattg catctcaagc 360
54/55
CA 02387785 2002-04-17
WO 01/32888 PCT/US00/30485
cagctggtga tgctgctgga gtggtggtcg ggcacggaat gcaccatctt cacggacccg 420
cgcgcctacc tcaagtatgg gaaggaaaat gccatcgtgg ttctcaacca caagtttgaa 480
attgactttc tgtgtggctg gagcctgtcc gaacgctttg ggctgttagg gggctccaag 540
gtcctggcca agaaagagct ggcctatgtc ccaattatcg gctggatgtg gtacttcacc 600
gagatggtct tctgttcgcg caagtgggag caggatcgca agacggttgc caccagtttg 660
cagcacctcc gggactaccc cgagaagtat tttttcctga ttcactgtga gggcacacgg 720
ttcacggaga agaagcatga gatcagcatg caggtggccc gggccaaggg gctgcctcgc 780
ctcaagcatc acctgttgcc acgaaccaag ggcttcgcca tcaccgtgag gagcttgaga 840
aatgtagttt cagctgtata tgactgtaca ctcaatttca gaaataatga aaatccaaca 900
ctgctgggag tcctaaacgg aaagaaatac catgcagatt tgtatgttag gaggatccca 960
ctggaagaca tccctgaaga cgatgacgag tgctcggcct ggctgcacaa gctctaccag 1020
gagaaggatg cctttcagga ggagtactac aggacgggca ccttcccaga gacgcccatg 1080
gtgccccccc ggcggccctg gaccctcgtg aactggctgt tttgggcctc gctggtgctc 1140
taccctttct tccagttcct ggtcagcatg atcaggagcg ggtcttccct gacgctggcc 1200
agcttcatcc tcgtcttctt tgtggcctct gtgggagttc gatggatgat tggtgtgacg 1260
gaaattgaca agggctctgc ctacggcaac tctgacagca agcagaaact gaatgactga 1320
ctcagggagg tgtcaccatc cgaagggaac cttggggaac tggtggcctc tgcatatcct 1380
ccttagtggg acacggtgac aaaggctggg tgagcccctg ctgggcacgg cggaagtcac 1440
gacctctcca gccagggagt ctggtctcaa ggccggatgg ggaggaagat gttttgtaat 1500
ctttttttcc ccatgtgctt tagtgggctt tggttttctt tttgtgcgag tgtgtgtgag 1560
aatggctgtg tggtgagtgt gaactttgtt ctgtgatcat agaaagggta ttttaggctg 1620
caggggaggg cagggctggg gaccgaaggg gacaagttcc cctttcatcc tttggtgctg 1680
agttttctgt aacccttggt tgccagagat aaagtgaaaa gtgctttagg tgagatgact 1740
aaattatgcc tccaagaaaa aaaaattaaa gtgcttttct gggtcaaaaa aaaa 1794
55/55
