Note: Descriptions are shown in the official language in which they were submitted.
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VESICLE ASSOCIATED PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of vesicle
associated proteins
and to the use of these sequences in the diagnosis, treatment, and prevention
of transport disorders,
autoimmunelintlammatory disorders, and cancer.
BACKGROUND OF THE INVENTION
Eukaryotic cells are bound by a lipid bilayer membrane and subdivided into
functionally
distinct, membrane-bound compartments. The membranes maintain the essential
differences between
the cytosol, the extracellular environment, and the lumenal space of each
intracellular organelle. As
lipid membranes are highly impermeable to most polar molecules, transport of
essential nutrients,
metabolic waste products, cell signaling molecules, macromolecules, and
proteins across lipid
membranes and between organelles must be mediated by a variety of transport-
associated molecules.
Integral membrane proteins, secreted proteins, and proteins destined for the
lumen of organelles
are synthesized within the endoplasmic reticulum (ER), delivered to the Golgi
complex for post-
transladonal processing and sorting, and then transported to specific
intracellular and extracellular
destinations. Material is internalized from the extracellular environment by
endocytosis, a process
essential for transmission of neuronal, metabolic, and proliferative signals;
uptake of many essential
nutrients; and defense against invading organisms. This intracellular and
extracellular movement of
protein molecules is termed vesicle trafficking. Trafficking is accomplished
by the packaging of protein
molecules into specialized vesicles which bud from the donor organelle
membrane and fuse to the target
membrane (Rothman, J.E and Wieland, F.T. (1996) Science 272:227-234).
Several steps in the transit of material along the secretory and endocytic
pathways requires the
formation of transport vesicles. Specifically, vesicles form at the
transitional endoplasmic reticulum
(tER), the rim of Golgi cisternae, the face of the Trans-Golgi Network (TGN),
the plasma membrane
(PM), and tubular extensions of the endosomes. Vesicle formation occurs when a
region of membrane
buds off from the donor organelle. The membrane-bound vesicle contains
proteins to be transported and
is surrounded by a proteinaceous coat, the components of which are recruited
from the cytosol. Vesicle
formation begins with the budding of a vesicle out of a donor organelle. The
initial budding and coating
processes are controlled by a cytosolic ras-like GTP-binding protein, ADP-
ribosylating factor (Arf),
and adapter proteins (AP). Different isoforms of both Arf and AP are involved
at different sites of
budding. For example, Arfs 1, 3, and 5 are required for Golgi budding, Arf4
for endosomal budding,
and Arf6 for plasma membrane budding. Two different classes of coat protein
have also been
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identified. Clathrin coats form on vesicles derived from the TGN and PM,
whereas coatomer (COP)
coats form on vesicles derived from the ER and Golgi. (Mellman, I. (1996)
Annu. Rev. Cell Dev. Biol.
12:575-625.)
Vesicle formation begins when an adapter protein (AP) interacts with cargo
proteins within the
donor membrane and recruits clathrin to the bud site. APs are heterotetrameric
complexes composed of
two large chains (a, 'y, 8, or s, and (3), a medium chain (~1), and a small
chain (a). Clathrin binds to
APs via the carboxy-terminal appendage domain of the (3-adaptin subunit (Le
Bourgne, R. and Hoflack,
B. (1998) Curr. Opin. Cell. Biol. 10:499-503). AP-1 functions in protein
sorting from the TGN and
endosomes to compartments of the endosomal/lysosomal system. AP-2 functions in
clathrin-mediated
endocytosis at the plasma membrane, while AP-3 is associated with endosomes
and/or the TGN and
recruits integral membrane proteins for transport to lysosomes and lysosome-
related organelles. The
recently isolated AP-4 complex localizes to the TGN or a neighboring
compartment and may play a role
in sorting events thought to take place in post-Golgi compartments
(Dell'Angelica, E. C. et al. (1999) J.
Biol. Chem. 274:7278-7285). Cytosolic GTP-bound Arf is also incorporated into
the vesicle as it
forms. Another GTP-binding protein, dynamin, forms a ring complex around the
neck of the forming
vesicle and provides the mechanochemical force required to release the vesicle
from the donor
membrane. The coated vesicle complex is then transported through the cytosol.
During the transport
process, Arf bound GTP is hydrolyzed to GDP and the coat dissociates from the
transport vesicle.
(West, M.A. et al. (1997) J. Cell Biol. 138:1239-1254.)
Coat protein (COP) coats form on the ER and Golgi. COP coats can further be
distinguished
as COPI, involved in retrograde traffic through the Golgi to the ER, and
COPII, involved in anterograde
traffic from the ER to the Golgi. The COP coat consists of two major
components, a GTP-binding
protein (Arf or Sar) and coat protomer (coatomer). Coatomer is an equimolar
complex of seven
proteins, termed alpha-, beta-, beta'-, gamma-, delta-, epsilon- and zeta-COP.
The coatomer complex
binds to dilysine motifs contained on the cytoplasmic tails of integral
membrane proteins. These include
the dilysine-containing retrieval motif of membrane proteins of the ER and
dibasic/diphenylamine
motifs of members of the p24 family. The p24 family of type I membrane
proteins represent the major
membrane proteins of COPI vesicles. (Harter, C. and Wieland, F.T. (1998) Proc.
Natl. Acad. Sci.
USA 95:11649-11654.)
Vesicles can undergo homotypic or heterotypic fusion. Molecules required for
appropriate
targeting and fusion of vesicles include proteins in the vesicle membrane, the
target membrane, and
proteins recruited from the cytosol. During budding of the vesicle from the
donor compartment, an
integral membrane protein. VAMP (vesicle-associated membrane protein) is
incorporated into the
vesicle. Soon after the vesicle uncoats, a cytosolic prenylated GTP-binding
protein, Rab, is inserted
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into the vesicle membrane. The amino acid sequence of Rab proteins reveals
conserved GTP-binding
domains characteristic of Ras superfamily members. In the vesicle membrane,
GTP-bound Rab
interacts with VAMP. Once the vesicle reaches the target membrane, a GTPase
activating protein
(GAP) in the target membrane converts the Rab protein to the GDP-bound form. A
cytosolic protein,
guanine-nucleotide dissociation inhibitor (GDI) then removes GDP-bound Rab
from the vesicle
membrane. Several Rab isoforms have been identified and appear to associate
with specific
compartments within the cell. For example, Rabs 4, 5, and 11 are associated
with the early endosome,
whereas Rabs 7 and 9 associate with the late endosome. These differences may
provide selectivity in
the association between vesicles and their target membranes. (Novick, P., and
Zerial, M. (1997) Cur.
Opin. Cell Biol. 9:496-504.)
Docking of the transport vesicle with the target membrane involves the
formation of a complex
between the vesicle SNAP receptor (v-SNARE), target membrane (t-) SNARES, and
certain other
membrane and cytosolic proteins. Many of these other proteins have been
identified although their
exact functions in the docking complex remain uncertain. (Tellam, J.T. et al.
(1995) J. Biol. Chem
270:5857-63; Hata, Y. and Sudhof, T.C. (1995) J. Biol. Chem. 270:13022-28.) N-
ethylmaleimide
sensitive factor (NSF) and soluble NSF-attachment protein (a-SNAP and ~i-SNAP)
are two such
proteins that are conserved from yeast to man and function in most
intracellular membrane fusion
reactions. Sec 1 represents a family of yeast proteins that function at many
different stages in the
secretory pathway including membrane fusion. Recently, mammalian homologs of
Secl, called
Munc-18 proteins, have been identified. (Katagiri, H. et al. (1995) J. Biol.
Chem. 270:4963-66; Hata
et al. supra.)
The SNARE complex involves three SNARE molecules, one in the vesicular
membrane and
two in the target membrane. Together they form a rod-shaped complex of four a-
helical coiled-coils.
The membrane anchoring domains of all three SNARES project from one end of the
rod. This complex
is similar to the rod-like structures formed by fusion proteins characteristic
of the enveloped viruses,
such as myxovirus, influenza, filovirus (Ebola), and the HIV and SIV
retroviruses. (Skehel, J.J., and
Wiley, D.C. (1998) Cell 95:871-874.) It has been proposed that the SNARE
complex is sufficient for
membrane fusion, suggesting that the proteins which associate with the complex
provide regulation over
the fusion event. (Weber, T. et al. (1998) Cell 92:759-772.) For example, in
neurons, which exhibit
regulated exocytosis, docked vesicles do not fuse with the presynaptic
membrane until depolarization,
which leads to an influx of calcium. (Bennett, M.K., and Scheller, R.H. (1994)
Annu. Rev. Biochem.
63:63-100.) Synaptotagmin, an integral membrane protein in the synaptic
vesicle, associates with the t-
SNARE syntaxin in the docking complex. Synaptotagmin binds calcium in a
complex with negatively
charged phospholipids, which allows the cytosolic SNAP protein to displace
synaptotagmin from
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syntaxin and fusion to occur. Thus, synaptotagmin is a negative regulator of
fusion in the neuron.
(Littleton, J.T. et al. (1993) Cell 74:1125-1134.) The most abundant membrane
protein of synaptic
vesicles appears to be the glycoprotein synaptophysin" a 38 kDa protein with
four transmembrane
domains. Although the function of synaptophysin is not known, its calcium-
binding ability, tyrosine
phosphorylation, and widespread distribution in neural tissues suggest a
potential role in neurosecretion.
(Bennett, supra.)
Correct trafficking of proteins is of particular importance for the proper
function of epithelial
cells, which are polarized into distinct apical and basolateral domains
containing different cell
membrane components such as lipids and membrane-associated proteins. Certain
proteins are flexible
and may be sorted to the basolateral or apical side depending upon cell type
or growth conditions. For
example, the kidney anion exchanger (kAEl) can be retargeted from the apical
to the basolateral
domain if cells are plated at higher density. The protein kanadaptin was
isolated as a protein which
binds to the cytoplasmic domain of kAEl. It also colocalizes with kAEl in
vesicles, but not in the
membrane, suggesting that kanadaptin's function is to guide kAEl-containing
vesicles to the basolateral
target membrane (Chen, J. et al. (1998) J. Biol. Chem. 273:1038-1043).
The etiology of numerous human diseases and disorders can be attributed to
defects in the
trafficking of proteins to organelles or the cell surface. Defects in the
trafficking of membrane-bound
receptors and ion channels are associated with cystic fibrosis (cystic
fibrosis transmembrane
conductance regulator; CFTR), glucose-galactose malabsorption syndrome
(Na+/glucose cotransporter),
hypercholesterolemia (low-density lipoprotein (LDL) receptor), and forms of
diabetes mellitus (insulin
receptor). Abnormal hormonal secretion is linked to disorders including
diabetes insipidus
(vasopressin), hyper- and hypoglycemia (insulin, glucagon), Grave's disease
and goiter (thyroid
hormone), and Cushing's and Addison's diseases (adrenocorticotropic hormone;
ACTH).
Cancer cells secrete excessive amounts of hormones or other biologically
active peptides.
Disorders related to excessive secretion of biologically active peptides by
tumor cells include: fasting
hypoglycemia due to increased insulin secretion from insulinoma-islet cell
tumors; hypertension due to
increased epinephrine and norepinephrine secreted from pheochromocytomas of
the adrenal medulla and
sympathetic paraganglia; and carcinoid syndrome, which includes abdominal
cramps, diarrhea, and
valvular heart disease, caused by excessive amounts of vasoactive substances
(serotonin, bradykinin,
histamine, prostaglandins, and polypeptide hormones) secreted from intestinal
tumors. Ectopic
synthesis and secretion of biologically active peptides (peptides not expected
from a tumor) includes
ACTH and vasopressin in lung and pancreatic cancers; parathyroid hormone in
lung and bladder
cancers; calcitonin in lung and breast cancers; and thyroid-stimulating
hormone in medullary thyroid
carcinoma.
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Various human pathogens alter host cell protein trafficking pathways to their
own advantage.
For example, the HIV protein Nef downregulates cell-surface expression of CD4
molecules by
accelerating their endocvtosis through clathrin coated pits. This function of
Nef is important for the
spread of HIV from the infected cell (Harris, M. (1999) Curr. Biol. 9:8449-
8461). A recently
identified human protein, Nef associated factor 1 (Nafl), a protein with four
extended coiled-coil
domains, has been found to associate with Nef. Overexpression of Nafl
increased cell surface
expression of CD4, an effect which could be suppressed by Nef (Fukushi, M. et
al. (1999) FEBS Lett.
442:83-88).
The discovery of new vesicle associated proteins 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 transport disorders, autoimmune/inflammatory disorders, and
cancer.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, vesicle associated proteins,
referred to collectively
as "VEAS" and individually as ''VEAS-I," "VEAS-2," "VEAS-3," "VEAS-4," "VEAS-
5," "VEAS-
6," "VEAS-7," "VEAS-8," "YEAS-9," "YEAS-10," "VEAS-11," "VEAS-12," "VEAS-13,"
"VEAS-
14," "VEAS-15," "YEAS-16," "VEAS-17," "VEAS-18," and "VEAS-19." In one aspect,
the
invention provides an isolated polypeptide comprising a) an amino acid
sequence selected from the
group consisting of SEQ ID NO:1-19, 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-
19, c) a biologically active fragment of an amino acid sequence selected from
the group consisting of
SEQ ID NO:1-19, or d) an immunogenic fragment of an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-19. In one alternative, the invention provides an
isolated polypeptide
comprising the amino acid sequence of SEQ ID NO:1-19.
The invention further provides an isolated polynucleotide encoding a
polypeptide comprising a)
an amino acid sequence selected from the group consisting of SEQ ID NO:I-19,
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:1-19, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-19, or d) an
immunogenic fragment
of an amino acid sequence selected from the group consisting of SEQ ID NO:I-
19. In one alternative,
the polynucleotide is selected from the group consisting of SEQ ID N0:20-38.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide comprising
a) an amino acid
sequence selected from the group consisting of SEQ ID NO:1-19, b) a naturally
occurring amino acid
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sequence having at least 90% sequence identity to an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-19, c) a biologically active fragment of an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-19, or d) an immunogenic fragment of
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-19. 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 a)
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-19, 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:1-19, c) a biologically active fragment of an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-19, or d) an immunogenic fragment of
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-19. The method
comprises a) culturing a
cell under conditions suitable for expression of the polypeptide, wherein said
cell is transformed with a
recombinant polynucleotide comprising a promoter sequence operably linked to a
polynucleotide
encoding the polypeptide, and b) recovering the polypeptide so expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypepdde comprising a) an amino acid sequence selected from the group
consisting of SEQ ID
NO:1-19, 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:1-19, c) a
biologically active
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:I-19, or d) an
immunogenic fragment of an amino acid sequence selected from the group
consisting of SEQ ID NO:1-
19.
The invention further provides an isolated polynucleotide comprising a) a
polynucleotide
sequence selected from the group consisting of SEQ ID N0:20-38, b) a naturally
occurring
polynucleotide sequence having at least 90% sequence identity to a
polynucleotide sequence selected
from the group consisting of SEQ ID N0:20-38, c) a polynucleotide sequence
complementary to a), or
d) a polynucleodde sequence complementary to b). 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)
a polynucleotide
sequence selected from the group consisting of SEQ ID N0:20-38, b) a naturally
occurring
polynucleotide sequence having at least 90% sequence identity to a
polynucleotide sequence selected
from the group consisting of SEQ ID N0:20-38, c) a polynucleotide sequence
complementary to a), or
d) a polynucleotide sequence complementary to b). The method comprises a)
hybridizing the sample
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with a probe comprising at least 16 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, 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 30 contiguous
nucleotides. In another alternative, the probe comprises at least 60
contiguous nucleotides.
The invention further provides a pharmaceutical composition comprising an
effective amount of
a polypeptide comprising a) an amino acid sequence selected from the group
consisting of SEQ ID
NO:1-19, 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:1-19, c) a
biologically active
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-19, or d) an
immunogenic fragment of an amino acid sequence selected from the group
consisting of SEQ ID NO:1-
19, and a pharmaceutically acceptable excipient. The invention additionally
provides a method of
treating a disease or condition associated with decreased expression of
functional YEAS, comprising
administering to a patient in need of such treatment the pharmaceutical
composition.
The invention also provides a method for screening a compound for
effectiveness as an agonist
of a polypeptide comprising a) an amino acid sequence selected from the group
consisting of SEQ ID
NO:1-19, 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:1-19, c) a
biologically active
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-19, or d) an
immunogenic fragment of an amino acid sequence selected from the group
consisting of SEQ ID NO:1-
19. The method comprises a) exposing a sample comprising the polypeptide to a
compound, and b)
detecting agonist activity in the sample. In one alternative, the invention
provides a pharmaceutical
composition comprising an agonist compound identified by the method and a
pharmaceutically
acceptable excipient. In another alternative, the invention provides a method
of treating a disease or
condition associated with decreased expression of functional VEAS, comprising
administering to a
patient in need of such treatment the pharmaceutical composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as an
antagonist of a polypeptide comprising a) an amino acid sequence selected from
the group consisting of
SEQ ID NO:1-19, 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:l-
19, c) a biologically
active fragment of an amino acid sequence selected from the group consisting
of SEQ ID NO:l-19, or
d) an immunogenic fragment of an amino acid sequence selected from the group
consisting of SEQ ID
NO:l-19. The method comprises a) exposing a sample comprising the polypeptide
to a compound, and
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bj detecting antagonist activity in the sample. In one alternative, the
invention provides a
pharmaceutical composition comprising an antagonist compound identified by the
method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with overexpression of functional
YEAS, comprising
administering to a patient in need of such treatment the pharmaceutical
composition.
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 N0:20-38, the method comprising a)
exposing a sample
comprising the target polynucleotide to a compound, and b) detecting altered
expression of the target
polynucleotide.
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 YEAS.
Table 2 shows features of each polypeptide sequence, including potential
motifs, homologous
sequences, and methods, algorithms, and searchable databases used for analysis
of YEAS.
Table 3 shows selected fragments of each nucleic acid sequence; the tissue-
specific expression
patterns of each nucleic acid sequence as determined by northern analysis;
diseases, disorders, or
conditions associated with these tissues; and the vector into which each cDNA
was cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which
cDNA clones
encoding YEAS were isolated.
Table ~ shows the tools, programs, and algorithms used to analyze VEAS, along
with
applicable descriptions, references, and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which will
be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
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
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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
"VEAS" refers .to the amino acid sequences of substantially purified VEAS
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
YEAS. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of VEAS either by
directly interacting with
YEAS or by acting on components of the biological pathway in which VEAS
participates.
An "allelic variant" is an alternative form of the gene encoding VEAS. 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 rimes in
a given sequence.
"Altered" nucleic acid sequences encoding VEAS include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as VEAS or a
polypeptide with at least one functional characteristic of VEAS. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe of
the polynucleotide encoding VEAS, and improper or unexpected hybridization to
allelic variants, with a
locus other than the normal chromosomal locus for the polynucleotide sequence
encoding VEAS. 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 VEAS. Deliberate
amino acid substitutions may be made on the basis of similarity in polarity,
charge, solubility,
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hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues,
as long as the biological
or immunological activity of VEAS 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 an amino acid
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
YEAS. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of YEAS either by
directly interacting with YEAS or by acting on components of the biological
pathway in which YEAS
participates.
The term ''antibody" refers to intact immunoglobulin molecules as well as to
fragments thereof,
such as Fab, F(ab')~, and Fv fragments, which are capable of binding an
epitopic determinant.
Antibodies that bind YEAS polypeptides can be prepared using intact
polypeptides or using fragments
containing small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide used
to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from
the translation of RNA, or
synthesized chemically, and can be conjugated to a carrier protein if desired.
Commonly used carriers
that are chemically coupled to peptides include bovine serum albumin,
thyroglobulin, and keyhole
limpet hemocyanin (KL,H). 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.
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The term "antisense'' refers to any composition capable of base-pairing with
the "sense' 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" refers to the capability
of the natural, recombinant, or synthetic VEAS, or of any oligopeptide
thereof, to induce a specific
immune response in appropriate animals or cells and to bind with specific
antibodies.
The terms ''complementary'' and "complementarity" refer to the natural binding
of
polynucleoddes by base pairing. For example, the sequence "5' A-G-T 3"' bonds
to the complementary
sequence "3' T-C-A 5'." Complementarity between two single-stranded molecules
may be "partial,"
such that only some of the nucleic acids bind, or it may be "complete," such
that total complementarity
exists between the single stranded molecules. The degree of complementarity
between nucleic acid
strands has significant effects on the efficiency and strength of the
hybridization between the nucleic
acid strands. This is of particular importance in amplification reactions,
which depend upon binding
between nucleic acid strands, and in the design and use of peptide nucleic
acid (PNA) molecules.
A "composition comprising a given polynucleodde 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 VEAS or fragments of
YEAS may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e. g., 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
resequenced to resolve
uncalled bases, extended using the XL-PCR kit (Perkin-Elmer, Norwalk CT) in
the 5' and/or the 3'
direction, and resequenced, or which has been assembled from the overlapping
sequences of one or
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more Incyte Clones and, in some cases, one or more public domain ESTs, using a
computer program
for fragment assembly, such as the GELVIEW Iragment assembly system (GCG,
Madison WI). Some
sequences have been both extended and assembled to produce the consensus
sequence.
''Conservative amino acid substitutions" are those substitutions that, when
made, 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 Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of the
side chain.
A ''deletion'' refers to a change in the amino acid or nucleotide sequence
that results in the
absence of one or more amino acid residues or nucleotides.
The term ''derivative'' refers to the chemical modification of a polypeptide
sequence, or a
polynucleotide sequence. Chemical modifications of a polynucleotide sequence
can include, for
example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group.
A derivative
polynucleotide encodes a polypeptide which retains at least one biological or
immunological function of
the natural molecule. A derivative polypeptide is one modified by
glycosylation, pegylation, or any
similar process that retains at least one biological or immunological function
of the polypeptide from
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which it was derived.
A ''fragment" is a unique portion of YEAS or the polynucleotide encoding YEAS
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,
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 N0:20-38 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:20-38, for example, as distinct from any
other sequence in the same
genome. A fragment of SEQ ID N0:20-38 is useful, for example, in hybridization
and amplification
technologies and in analogous methods that distinguish SEQ ID N0:20-38 from
related polynucleotide
sequences. The precise length of a fragment of SEQ ID N0:20-38 and the region
of SEQ ID N0:20-38
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:I-19 is encoded by a fragment of SEQ ID N0:20-38. A
fragment
of SEQ ID NO:1-19 comprises a region of unique amino acid sequence that
specifically identifies SEQ
ID NO:1-19. For example, a fragment of SEQ ID NO:1-19 is useful as an
immunogenic peptide for the
development of antibodies that specifically recognize SEQ ID NO:1-19. The
precise length of a
fragment of SEQ ID NO:1-19 and the region of SEQ ID NO:1-19 to which the
fragment corresponds
are routinely determinable by one of ordinary skill in the art based on the
intended purpose for the
fragment.
The term "similarity" refers to a degree of complementarity. There may be
partial similarity or
complete similarity. The word ''identity" may substitute for the word
"similarity." A partially
complementary sequence that at least partially inhibits an identical sequence
from hybridizing to a
target nucleic acid is referred to as "substantially similar." The inhibition
of hybridization of the
completely complementary sequence to the target sequence may be examined using
a hybridization
assay (Southern or northern blot, solution hybridization, and the like) under
conditions of reduced
stringency. A substantially similar sequence or hybridization probe will
compete for and inhibit the
binding of a completely similar (identical) sequence to the target sequence
under conditions of reduced
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stringency. This is not to say that conditions of reduced stringency are such
thai non-specific binding is
permitted, as reduced stringency conditions require that the binding of two
sequences to one another be
a specific (i.e., a selective) interaction. The absence of non-specific
binding may be tested by the use of
a second target sequence which lacks even a partial degree of complementarity
(e.g., less than about
30% similarity or identity). In the absence of non-specific binding, the
substantially similar sequence or
probe will not hybridize to the second non-complementary target sequence.
The phrases "percent identity" and "% identity," as applied to polynucleotide
sequences, refer
to the percentage of residue matches between at least two polynucleodde
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
IS 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 sequence pairs.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms is
provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment Search
Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which
is available from several
sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence analysis
programs including "blastn," that is used to align a known polynucleotide
sequence with other
polynucleodde 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
2Ø9 (May-07-1999) set at default parameters. Such default parameters may be,
for example:
Matrix: BLOSUM62
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Reward for match: I
Penalty for mismatch: -2
Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off' S0
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 hydrophobicity and
acidity at the site of
substitution, thus preserving the structure (and therefore function) of the
polypepdde.
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: Ktupl~l, gap penalty=3,
window=5, and
"diagonals saved"=5. The PAM250 matrix is selected as the default residue
weight table. As with
polynucleotide alignments, the percent identity is reported by CLUSTAL V as
the ''percent similarity"
between aligned polypeptide sequence pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences''
tool Version 2Ø9
(May-07-1999) with blastp set at default parameters. Such default parameters
may be, for example:
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Matrix: BLOSUM62
Open Gap: Il and Extension Gap: 1 penalties
Gap x drop-off.' S0
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
stable mitotic chromosome segregation and maintenance.
The term ''humanized antibody'' refers to antibody molecules 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 identity. 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 ltg/ml 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. Generally, such wash temperatures
are selected to be about
5°C to 20°C lower than the thermal melting point (T"~ for the
specific sequence at a defined ionic
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suength and pH. The Tm is the temperature (under defined ionic suength and pH)
at which 50% of the
target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and conditions
for nucleic acid hybridization are well known and can be found in Sambrook et
al., 1989, Molecular
Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring Harbor Press,
Plainview NY; specifically
see volume 2, chapter 9.
High suingency 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 concenuation 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,
denatured salmon sperm DNA at about 100-200 ~g/ml. Organic solvent, such as
formamide at a
concenuation 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 suingency
conditions, may be
suggestive of evolutionary similarity between the nucleotides. Such similarity
is suongly 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 subsuate 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, uauma,
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 VEAS
which is
capable of eliciting an immune response when inuoduced into a living organism,
for example, a
mammal. The term "immunogenic fragment" also includes any polypeptide or
oligopeptide fragment of
YEAS 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 distinct polynucleotides
on a subsuate.
The terms "element" and "array element" in a microarray context, refer to
hybridizable
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polynucleotides arranged on the surface of a substrate.
The term "modulate" refers to a change in the activity of YEAS. For example,
modulation may
cause an increase or a decrease in protein activity, binding characteristics,
or any other biological,
functional, or immunological properties of YEAS.
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 the 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. Generally, 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.
"Probe'' refers to nucleic acid sequences encoding VEAS, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are isolated
oligonucleotides or polynucleotides attached to a detectable label or reporter
molecule. Typical labels
include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
"Primers" are short
nucleic acids, usually DNA oligonucleotides, which may be annealed to a target
polynucleotide by
complementary base-pairing. The primer may then be extended along the target
DNA strand by a DNA
polymerase enzyme. Primer pairs can be used for amplification (and
identification) of a nucleic acid
sequence, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers may
be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual,
2°d ed., vol. I-3, Cold
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Spring Harbor Press, Plainview NY; Ausubel et al.,1987, Current Protocols in
Molecular Biology,
Greene Publ. Assoc. & Wiley-Intersciences> New York NY; Innis et al., 1990,
PCR Protocols. A Guide
to Methods and Applications, Academic Press, San Diego CA. PCR primer pairs
can be derived from
a known sequence> for example, by using computer programs intended for that
purpose such as Primer
(Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to 5,000
nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection
programs have incorporated additional features for expanded capabilities. For
example, the PrimOU
primer selection program (available to the public from the Genome Center at
University of Texas South
West Medical Center, Dallas TX) is capable of choosing specific primers from
megabase sequences
and is thus useful for designing primers on a genome-wide scope. The Primer3
primer selection
program (available to the public from the Whitehead Institute/MIT Center for
Genome Research,
Cambridge MA) allows the user to input a "mispriming library," in which
sequences to avoid as primer
binding sites are user-specified. Primer3 is useful, in particular, for the
selection of oligonucleotides for
microarrays. (The source code for the latter two primer selection programs may
also be obtained from
their respective sources and modified to meet the user's specific 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
oligonucleoddes and
polynucleotide fragments. The oligonucleotides and polynucleotide fragments
identified by any of the
above selection methods are useful in hybridization technologies, for example,
as PCR or sequencing
primers, microarray elements, or specific probes to identify fully or
partially complementary
polynucleotides in a sample of nucleic acids. Methods of oligonucleotide
selection are not limited to
those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter sequence.
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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.
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 YEAS, or fragments thereof, or YEAS 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 containing the epitope
A, or the presence of free unlabeled A, in a reaction containing free labeled
A and the antibody will
reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 75% free, and most preferably at least 90% free from other
components with which
they are naturally associated.
A "substitution" refers to the replacement of one or more amino acids or
nucleotides by
different amino acids 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.
"Transformation'' describes a process by which exogenous DNA enters and
changes 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, viral
infection, electroporation,
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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., and-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 et al. (1989), supra.
A "variant'' of a particular nucleic acid sequence is defined as a nucleic
acid sequence having at
least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of the
nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version
2Ø9 (May-07-1999)
set at default parameters. Such a pair of nucleic acids may show, for example,
at least 50%, at least
60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or
at least 98% or greater
sequence identity over a certain defined length. A variant may be described
as, for example, an ''allelic"
(as defined above), "splice," "species,'' or ''polymorphic" variant. A splice
variant may have significant
identity to a reference molecule, but will generally have a greater or lesser
number of polynucleotides
due to alternate 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
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WO 00/60082 PCT/US00/09353
potypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version
2Ø9 (Nlay-07-1999)
set at default parameters. Such a pair of polypeptides may show, for example,
at least ~0%, at least
60070, at least 70%, at least 800. at least 90%, at least 95%, or at least 98%
or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human vesicle associated
proteins (VEAS), the
polynucleotides encoding VEAS, and the use of these compositions for the
diagnosis, treatment, or
prevention of transport disorders, autoimmune/inflammatory disorders, and
cancer.
Table I lists the Incyte clones used to assemble full length nucleotide
sequences encoding
VEAS. Columns I 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 VEAS 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. The
Incyte clones in column 5 were used to assemble the consensus nucleotide
sequence of each VEAS and
are useful as fragments in hybridization technologies.
The columns of Table 2 show various properties of each of the polypeptides of
the invention:
column 1 references the SEQ ID NO; column 2 shows the number of amino acid
residues in each
polypeptide; column 3 shows potential phosphorylation sites; column 4 shows
potential glycosylation
sites; column 5 shows the amino acid residues comprising signature sequences
and motifs; column 6
shows homologous sequences as identified by BLAST analysis; and column 7 shows
analytical methods
and in some cases, searchable databases to which the analytical methods were
applied. The methods of
column 7 were used to characterize each polypeptide through sequence homology
and protein motifs.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or
conditions
associated with nucleotide sequences encoding VEAS. The first column of Table
3 lists the nucleotide
SEQ ID NOs. Column 2 lists fragments of the nucleotide sequences of column 1.
These fragments are
useful, for example, in hybridization or amplification technologies to
identify SEQ ID N0:20-38 and to
distinguish between SEQ ID N0:20-38 and related polynucleotide sequences. The
polypeptides
encoded by these fragments are useful, for example, as immunogenic peptides.
Column 3 lists tissue
categories which express VEAS as a fraction of total tissues expressing YEAS.
Column 4 lists
diseases, disorders, or conditions associated with those tissues expressing
YEAS as a fraction of total
tissues expressing VEAS. Column 5 lists the vectors used to subclone each cDNA
library.
The columns of Table 4 show descriptions of the tissues used to conswct the
cDNA libraries
from which cDNA clones encoding VEAS were isolated. Column 1 references the
nucleotide SEQ ID
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WO 00/60082 PCT/US00/09353
NOs, column 2 shows the cDNA libraries from which these clones were isolated,
and culumn 3 shows
the tissue origins and other descriptive information relevant to the cDNA
libraries in column 2.
SEQ ID N0:38 maps to chromosome 6 within the interval from 73.9 to 78.8
centiMorgans, and
to chromosome 10 within the interval from 17.3 to 36.3 centiMorgans. The
interval on chromosome 6
from 73.9 to 78.8 centiMorgans also contains a gene associated with hemolytic
anemia due to gamma-
glutamylcysteine synthetase deficiency.
The invention also encompasses YEAS variants. A preferred YEAS variant is one
which has
at least about 80%, or alternatively at least about 90%, or even at least
about 95% amino acid sequence
identity to the VEAS amino acid sequence, and which contains at least one
functional or structural
characteristic of YEAS.
The invention also encompasses polynucleotides which encode VEAS. In a
particular
embodiment. the invention encompasses a polynucleotide sequence comprising a
sequence selected from
the group consisting of SEQ ID N0:20-38, which encodes VEAS. The
polynucleotide sequences of
SEQ ID N0:20-38, 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
YEAS. In
particular, such a variant polynucleotide sequence will have at least about
80%, or alternatively at least
about 85%, or even at least about 95% polynucleotide sequence identity to the
polynucleotide sequence
encoding YEAS. A particular aspect of the invention encompasses a variant of a
polynucleotide
sequence comprising a sequence selected from the group consisting of SEQ ID
N0:20-38 which has at
least about 80%, 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 N0:20-38.
Any one of the polynucleotide variants described above can encode an amino
acid sequence which
contains at least one functional or structural characteristic of YEAS.
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 VEAS, 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 VEAS, and all such variations are to be considered as being
specifically disclosed.
Although nucleotide sequences which encode YEAS and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally occurring YEAS under
appropriately selected
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WO 00/60082 PCT/US00/09353
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding YEAS 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
VEAS and its derivatives without altering the encoded amino acid sequences
include the production of
RNA transcripts having more desirable properties, such as a greater half life,
than transcripts produced
from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode VEAS
and VEAS
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 VEAS 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
N0:20-38 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
HIenow fragment of
DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(Perkin-Elmer),
thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or
combinations of
polymerases and proofreading exonucleases such as those found in the ELONGASE
amplification
system (Life Technologies, Gaithersburg MD). Preferably, sequence preparation
is automated with
machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV),
PTC200 thermal
cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Perkin-
Elmer).
Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing
system (Perkin-
Elmer), 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
Bioloey, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995)
Molecular Biolo~y and
Biotechnoloey> Wiley VCH, New York NY, pp. 856-853.)
The nucleic acid sequences encoding VEAS may be extended utilizing a partial
nucleotide
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WO 00/60082 PCT/US00/09353
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 tiagments comprising a
known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et al.
(1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR ampliticadon of DNA
fragments adjacent
to known sequences in human and yeast artificial chromosome DNA. (See, e.g.,
Lagerstrom, M. et al.
( 1991 ) PCR Methods Applic. 1:111-119.) In this method, multiple restriction
enzyme digestions and
ligations may be used to insert an engineered double-stranded sequence into a
region 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°l0 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 electrophoredc 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, Perkin-Elmer), 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.
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
In another embodiment of the invention, polynuclwtide sequences or fragments
thereof which
encode VEAS may be cloned in recombinant DNA molecules that direct expression
of VEAS, 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 YEAS.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter VEAS-encoding sequences for a variety of
purposes including, but not
limited to, modification of the cloning, processing, and/or expression of the
gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction sites,
alter glycosylation patterns, change codon preference; produce splice
variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such as
MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
Number
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or improve
the biological properties of YEAS, 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 YEAS may be synthesized, in whole or
in part,
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
al. (1980) Nucleic Acids
Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.) Alternatively,
VEAS itself or a fragment thereof may be synthesized using chemical methods.
For example, peptide
synthesis can be performed using various solid-phase techniques. (See, e.g.,
Roberge, J.Y. et al. (1995)
Science 269:202-204.) Automated synthesis may be achieved using the ABI 431A
peptide synthesizer
(Perkin-Elmer). Additionally, the amino acid sequence of YEAS, or any part
thereof, may be altered
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WO 00/60082 PCT/US00/09353
during direct synthesis and/or combined with sequences from other proteins, ~r
any part thereof, to
produce a variant polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by sequencing.
(See, e.g., Creighton, T. (1984) Proteins. Structures and Molecular
Properties, WH Freeman, New
York NY.)
In order to express a biologically active VEAS, the nucleotide sequences
encoding VEAS 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 VEAS. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
VEAS. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding YEAS 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 VEAS 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
Clonine. A Laboratory
Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel,
F.M. et al. (1995)
Current Protocols in Molecular BioloQV, John Wiley & Sons, New York NY, ch. 9,
13, and 16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding VEAS. 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
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tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or
animal cell systems. 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 YEAS. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding YEAS 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 VEAS into the vector's
multiple cloning site
disrupts the lacZ gene, allowing a colorimetric screening procedure for
identification of transformed
bacteria containing recombinant molecules. In addition, these vectors may be
useful for in vitro
transcription, dideoxy sequencing, single strand rescue with helper phage, and
creation of nested
deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M. Schuster
(1989) J. Biol. Chem.
264:5503-5509.) When large quantities of VEAS are needed, e.g. for the
production of antibodies,
vectors which direct high level expression of YEAS may be used. For example,
vectors containing the
strong, inducible T5 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of VEAS. 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
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable integration
of foreign sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, 1995, supra;
Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et
al. (1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of VEAS. Transcription of
sequences encoding
VEAS 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, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R.
et al. (1984) Science
224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-
105.) 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 Technoloey (1992) McGraw Hill,
New York NY, pp.
191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding VEAS
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
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infective virus which expresses VEAS 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. 1:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression of
VEAS in cell lines is preferred. For example, sequences encoding VEAS 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), I3
glucuronidase and its substrate 13-glucuronide, or luciferase and its
substrate luciferin may be used.
These markers can be used not only to identify transformants, but also to
quantify the amount of
transient or stable protein expression attributable to a specific vector
system. (See, e.g., Rhodes, C.A.
(1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
confirmed. For example, if the
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WO 00/60082 PCT/US00/09353
sequence encoding YEAS is inserted within a marker gene sequence, transformed
cells containing
sequences encoding VEAS can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding YEAS 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 VEAS
and that express
VEAS 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 VEAS 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 YEAS 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. 1V; Coligan, J.E.
et al. (1997) Current Protocols in ImmunoloQV, Greene Pub. Associates and
Wiley-Interscience, New
York NY; and Pound, J.D. (1998) Immunochemical Protocols, Humans 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 VEAS
include oligolabeling,
nick translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the
sequences encoding VEAS, or any fragments thereof, may be cloned into a vector
for the production of
an mRNA probe. Such vectors are known in the art, are commercially available,
and may be used to
synthesize RNA probes in vitro by addition of an appropriate RNA polymerase
such as T7, T3, or SP6
and labeled nucleotides. These procedures may be conducted using a variety of
commercially available
kits, such as those provided by Amersham Pharmacia Biotech, Promega (Madison
WI), and US
Biochemical. Suitable reporter molecules or labels which may be used for ease
of detection include
radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents,
as well as substrates,
cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with nucleotide sequences encoding YEAS may be cultured
under
conditions suitable for the expression and recovery of the protein fiom cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the sequence
CA 02365421 2001-09-26
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and/or the vector used. As will be understood by those of skill in the art,
expression vectors containing
polynucleotides which encode VEAS may be designed to contain signal sequences
which direct
secretion of VEAS 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 YEAS 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
VEAS protein containing a
heterologous moiety that can be recognized by a commercially available
antibody may facilitate the
screening of peptide libraries for inhibitors of VEAS 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 VEAS encoding sequence and the heterologous protein
sequence, so that VEAS
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 YEAS may
be achieved in
vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These systems
couple transcription and translation of protein-coding sequences operably
associated with the T7, T3, or
SP6 promoters. Translation takes place in the presence of a radiolabeled amino
acid precursor, for
example, 3'S-methionine.
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Fragments of VEAS may be produced not only by recombinant means, but also by
direct
peptide synthesis using solid-phase techniques. (See, e.g., Creighton, su ra
pp. 55-60.) Protein
synthesis may be performed by manual techniques or by automation. Automated
synthesis may be
achieved, for example, using the ABI 431A peptide synthesizer (Perkin-Elmer).
Various fragments of
VEAS may be synthesized separately and then combined to produce the full
length molecule.
THERAPEUTICS
Chemical and swctural similarity, e.g., in the context of sequences and
motifs, exists between
regions of VEAS and vesicle associated proteins. In addition, the expression
of VEAS is closely
associated with nervous tissue, cancer, inflammation/trauma and the immune
response. Therefore,
VEAS appears to play a role in transport disorders, autoimmune/intlammatory
disorders, and cancer.
In the treatment of disorders associated with increased VEAS expression or
activity, it is desirable to
decrease the expression or activity of VEAS. In the treatment of disorders
associated with decreased
YEAS expression or activity, it is desirable to increase the expression or
activity of VEAS.
Therefore, in one embodiment, VEAS 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 VEAS.
Examples of such disorders include, but are not limited to, a transport
disorder, such as akinesia,
amyotrophic lateral sclerosis, ataxia telangiectasia, cystic fibrosis,
Becker's muscular dystrophy, Bell's
palsy, Charcot-Marie Tooth disease, diabetes mellitus, diabetes insipidus,
diabetic neuropathy,
Duchenne muscular dystrophy, hyperkalemic periodic paralysis, normokalemic
periodic paralysis,
Parkinson's disease, malignant hyperthermia, multidrug resistance, myasthenia
gravis, myotonic
dystrophy, catatonia, tardive dyskinesia, dystonias, peripheral neuropathy,
cerebral neoplasms, prostate
cancer, cardiac disorders associated with transport, e.g., angina,
bradyarrythmia, tachyarrythmia,
hypertension, Long QT syndrome. myocarditis, cardiomyopathy, nemaline
myopathy, centronuclear
myopathy, lipid myopathy, mitochondrial myopathy, thyrotoxic myopathy, ethanol
myopathy,
dermatomyositis, inclusion body myositis, infectious myositis, polymyositis,
neurological disorders
associated with transport, e.g., Alzheimer's disease, amnesia, bipolar
disorder, dementia, depression,
epilepsy, Tourette's disorder, paranoid psychoses, and schizophrenia, and
other disorders associated
with transport, e.g., neurofibromatosis, postherpetic neuralgia, trigeminal
neuropathy, sarcoidosis,
sickle cell anemia, cataracts, infertility, pulmonary artery stenosis,
sensorineural autosomal deafness,
hyperglycemia, hypoglycemia, Grave's disease, goiter, glucose-galactose
malabsorption syndrome, and
hypercholesterolemia, Cushing's disease, and Addison's disease,
gastrointestinal disorders including
ulcerative colitis, gastric and duodenal ulcers, other conditions associated
with abnormal vesicle
trafficking, including acquired immunodeficiency syndrome (AIDS). allergies
including hay fever,
asthma, and urticaria (hives), autoimmune hemolytic anemia, proliferative
glomerulonephritis,
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inflammatory bowel disease, multiple sclerosis, rheumatoid and osteoarthritis,
sclercxierma,
Chediak-Higashi and Sjogren's syndromes, systemic lupus erythematosus, toxic
shock syndrome,
traumatic tissue damage, viral, bacterial, fungah helminthic, and protozoal
infections, cystinuria,
dibasicaminoaciduria, hypercystinuria, lysinuria, hartnup disease, tryptophan
malabsorption,
methionine malabsorpdon, histidinuria, iminoglycinuria,
dicarboxylicaminoaciduria, cystinosis, remal
glycosuria, hypouricemia, familial hypophophatemic rickets, congenital
chloridorrhea, distal renal
tubular acidosis, Menkes' disease, Wilson's disease, lethal diarrhea, juvenile
pernicious anemia, folate
malabsorption, adrenoleukodystrophy, hereditary myoglobinuria, and Zellweger
syndrome: an
autoimmune/inflammatory disorder, such as acquired immunodeficiency syndrome
(AIDS),
Addison's disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis,
anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune
thyroiditis,
autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED),
bronchitis,
cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis,
dermatomyositis, diabetes
mellitus, emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis fetalis,
erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's
syndrome, gout, Graves'
disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome,
multiple sclerosis,
myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis,
osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma,
Sjogren's syndrome,
systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura,
ulcerative colitis, uveitis, Werner syndrome, complications of cancer,
hemodialysis, and
extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal,
and helminthic infections, and
trauma: and a cancer, such as adenocarcinoma, leukemia, lymphoma, melanoma,
myeloma, sarcoma,
teratocareinoma, 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.
In another embodiment, a vector capable of expressing YEAS 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 VEAS including, but not limited to, those described
above.
In a further embodiment, a pharmaceutical composition comprising a
substantially purified
VEAS in conjunction with a suitable pharmaceutical carrier may be administered
to a subject to treat or
prevent a disorder associated with decreased expression or activity of VEAS
including, but not limited
to, those provided above.
In still another embodiment, an agonist which modulates the activity of VEAS
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or activity
of VEAS including, but not limited to, those listed above.
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WO 00/60082 PCT/US00/09353
In a further embodiment, an antagonist of YEAS may be administered to a
subjec;i to treat or
prevent a disorder associated with increased expression or activity of VEAS.
Examples of such
disorders include, but are not limited to, those transport disorders,
autoimmune~inflammatory disorders,
and cancers described above. In one aspect. an antibody which specifically
binds YEAS 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 YEAS.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding VEAS may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of YEAS 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 YEAS may be produced using methods which are generally known
in the art.
In particular, purified YEAS may be used to produce antibodies or to screen
libraries of pharmaceutical
agents to identify those which specifically bind VEAS. Antibodies to YEAS 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 YEAS 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, KL~I, and dinitrophenol. Among adjuvants
used in humans, BCG
(bacilli Calmette-Guerin) and Corynebacterium parvum are especially
preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to VEAS
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 and
contain the entire amino acid
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WO 00/60082 PCT/US00/09353
sequence of a small, naturally occurring molecule. Short stretches of 'v'EAS
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 VEAS 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 EB
V-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
YEAS-specific single
chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be
generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g., Burton,
D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte population
or by screening immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in
the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.
USA 86:3833-3837; Winter,
G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for VEAS 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
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
VEAS and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal
antibodies reactive to two
non-interfering YEAS epitopes is generally used, but a competitive binding
assay may also be employed
(Pound, supra).
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay techniques
may be used to assess the affinity of antibodies for VEAS. Affinity is
expressed as an association
constant, Ka, which is defined as the molar concentration of YEAS-antibody
complex divided by the
molar concentrations of free antigen and free antibody under equilibrium
conditions. The I~ determined
for a preparation of polyclonal antibodies, which are heterogeneous in their
affinities for multiple VEAS
epitopes, represents the average affinity, or avidity, of the antibodies for
VEAS. The Ka determined for
a preparation of monoclonal antibodies, which are monospecific for a
particular VEAS epitope,
represents a true measure of affinity. High-affinity antibody preparations
with I~ ranging from about
109 to 10'z L/mole are preferred for use in immunoassays in which the VEAS-
antibody complex must
withstand rigorous manipulations. Low-affinity antibody preparations with I~
ranging from about 106
to 10' L/mole are preferred for use in immunopurification and similar
procedures which ultimately
require dissociation of YEAS, preferably in active form, from the antibody
(Catty, D. (1988)
Antibodies, Volume I: A Practical Approach, IRL Press, Washington, DC;
Liddell, J.E. and Cryer, A.
( 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 VEAS-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, sera, and
Coligan et al. supra.)
In another embodiment of the invention, the polynucleotides encoding VEAS, or
any fragment
or complement thereof, may be used for therapeutic purposes. In one aspect,
the complement of the
polynucleotide encoding VEAS may be used in situations in which it would be
desirable to block the
transcription of the mRNA. In particular, cells may be transformed with
sequences complementary to
polynucleotides encoding VEAS. Thus, complementary molecules or fragments may
be used to
modulate VEAS activity, or to achieve regulation of gene function. Such
technology is now well known
in the art, and sense or antisense oligonucleotides or larger fragments can be
designed from various
locations along the coding or control regions of sequences encoding VEAS.
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. Methods which are well known to those skilled in
the art can be used to
construct vectors to express nucleic acid sequences complementary to the
polynucleotides encoding
VEAS. (See, e.g., Sambrook, supra; Ausubel, 1995, s_upra.)
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WO 00/60082 PCT/US00/09353
Genes encoding YEAS can be turned off by transforming a cell or tissue with
expression
vectors which express high levels of a polynucleotide, or fragment thereof,
encoding YEAS. Such
constructs may be used to introduce untranslatable sense or antisense
sequences into a cell. Even in the
absence of integration into the DNA, such vectors may continue to transcribe
RNA molecules until they
are disabled by endogenous nucleases. Transient expression may last for a
month or more with a
non-replicating vector, and may last even longer if appropriate replication
elements are part of the
vector system.
As mentioned above, modifications of gene expression can be obtained by
designing
complementary sequences or antisense molecules (DNA, RNA, or PNA) to the
control, 5', or regulatory
regions of the gene encoding YEAS. Oligonucleotides derived from the
transcription initiation site, e.g.,
between about positions -10 and +10 from the start site, may be employed.
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 ImmunoloQic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-
177.) A
complementary sequence or antisense molecule may also be designed to block
translation of mRNA by
preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding YEAS.
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
oligonucleoddes 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
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WO 00/60082 PCT/US00/09353
encoding YEAS. 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.
Many methods for introducing vectors into cells or tissues are available and
equally suitable for
use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells taken
from the patient and clonally propagated for autologous transplant back into
that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
Biotechnol. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of such
therapy, including, for example, mammals such as humans, dogs, cats, cows,
horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
pharmaceutical or
sterile composition, in conjunction with a pharmaceutically acceptable
carrier, for any of the therapeutic
effects discussed above. Such pharmaceutical compositions may consist of YEAS,
antibodies to
YEAS, and mimetics, agonists, antagonists, or inhibitors of YEAS. The
compositions may be
administered alone or in combination with at least one other agent, such as a
stabilizing compound,
which may be administered in any sterile, biocompatible pharmaceutical carrier
including, but not
limited to, saline, buffered saline, dextrose, and water. The compositions may
be administered to a
patient alone, or in combination with other agents, drugs, or hormones.
The pharmaceutical 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, transdermal, subcutaneous, intraperitoneal,
intranasal, enteral, topical,
sublingual, or rectal means.
In addition to the active ingredients, these pharmaceutical compositions may
contain suitable
pharmaceutically-acceptable carriers comprising excipients and auxiliaries
which facilitate processing
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WO 00/60082 PCT/US00/09353
of the active compounds into preparations which can be used pharmaceutically.
Further details on
techniques for formulation and administration may be found in the latest
edition of Remington's
Pharmaceutical Sciences (Maack Publishing, Easton PA).
Pharmaceutical compositions for oral administration can be formulated using
pharmaceutically
acceptable carriers well known in the art in dosages suitable for oral
administration. Such carriers
enable the pharmaceutical compositions to be formulated as tablets, pills,
dragees, capsules, liquids,
gels, syrups, slurries, suspensions, and the like, for ingestion by the
patient.
Pharmaceutical preparations for oral use can be obtained through combining
active compounds
with solid excipient and processing the resultant mixture of granules
(optionally, after grinding) to
obtain tablets or dragee cores. Suitable auxiliaries can be added, if desired.
Suitable excipients include
carbohydrate or protein fillers, such as sugars, including lactose, sucrose,
mannitol, and sorbitol; starch
from corn, wheat, rice, potato, or other plants; cellulose, such as methyl
cellulose,
hydroxypropylmethyl-cellulose. or sodium carboxymethylcellulose; gums,
including arabic and
tragacanth; and proteins, such as gelatin and collagen. If desired,
disintegrating or solubilizing agents
may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and
alginic acid or a salt thereof,
such as sodium alginate.
Dragee cores may be used in conjunction with suitable coatings, such as
concentrated sugar
solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone,
carbopol gel, polyethylene
glycol, and/or titanium dioxide> lacquer solutions, and suitable organic
solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee coatings for
product identification or to
characterize the quantity of active compound, i.e., dosage.
Pharmaceutical preparations which can be used orally include push-fit capsules
made of
gelatin, as well as soft, sealed capsules made of gelatin and a coating, such
as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers or
binders, such as lactose or
starches, lubricants, such as talc or magnesium stearate, and, optionally,
stabilizers. In soft capsules,
the active compounds may be dissolved or suspended in suitable liquids, such
as fatty oils, liquid, or
liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration may be
formulated in
aqueous solutions, preferably in physiologically compatible buffers such as
Hanks' solution, Ringer's
solution, or physiologically buffered saline. Aqueous injection suspensions
may contain substances
which increase the viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or
dextran. Additionally, suspensions of the active compounds may be prepared as
appropriate oily
injection suspensions. Suitable lipophilic solvents or vehicles include fatty
oils, such as sesame oil, or
synthetic fatty acid esters, such as ethyl oleate, triglycerides, or
liposomes. Non-lipid polycationic
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WO 00/60082 PCT/US00/09353
amino polymers may also be used for delivery. Optionally. the suspension may
also contain suitable
stabilizers or agents to increase the solubility of the compounds and allow
for the preparation of highly
concentrated solutions.
For topical or nasal administration, penetrants appropriate to the particular
barrier to be
permeated are used in the formulation. Such penetrants are generally known in
the art.
The pharmaceutical compositions of the present invention may be manufactured
in a manner
that is known in the art, e.g., by means of conventional mixing, dissolving,
granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
The pharmaceutical composition may be provided as a salt and can be formed
with many acids,
including but not limited to, hydrochloric, sulfuric, acetic, lactic,
tartaric, malic, and succinic acids.
Salts tend to be more soluble in aqueous or other protonic solvents than are
the corresponding free base
forms. In other cases, the preparation may be a lyophilized powder which may
contain any or all of the
following: 1 mM to 50 mM histidine, 0.1 % to 2% sucrose, and 2% to 7%
mannitol, at a pH range of
4.5 to 5.5, that is combined with buffer prior to use.
After pharmaceutical compositions have been prepared, they can be placed in an
appropriate
container and labeled for treatment of an indicated condition. For
administration of VEAS, such
labeling would include amount, frequency, and method of administration.
Pharmaceutical compositions suitable for use in the invention include
compositions wherein the
active ingredients are contained in an effective amount to achieve the
intended purpose. The
determination of an effective dose is well within the capability of those
skilled in the art.
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, 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 YEAS
or fragments thereof, antibodies of YEAS, and agonists, antagonists or
inhibitors of YEAS, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by
standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDSO (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDSO/EDSO ratio.
Pharmaceutical compositions which
exhibit large therapeutic indices are preferred. The data obtained from cell
culture assays and animal
studies are used to formulate a ranee of dosaee for human use. The dosaee
contained in such
CA 02365421 2001-09-26
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compositions is preferably within a range of circulating concentrations that
includes the ED;o with little
or no toxicity. The dosage varies within this range depending upon the dosage
form employed, the
sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the subject
requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the active
moiety or to maintain the desired effect. Factors which may be taken into
account include the severity
of the disease state, the general health of the subject, the age, weight, and
gender of the subject, time
and frequency of administration, drug combination(s), reaction sensitivities,
and response to therapy.
Long-acting pharmaceutical compositions may be administered every 3 to 4 days,
every week, or
biweekly depending on the half-life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about 0.1 ~g to 100,000 fig, 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 VEAS may be used for
the diagnosis
of disorders characterized by expression of VEAS, or in assays to monitor
patients being treated with
YEAS or agonists, antagonists, or inhibitors of YEAS. Antibodies useful for
diagnostic purposes may
be prepared in the same manner as described above for therapeutics. Diagnostic
assays for YEAS
include methods which utilize the antibody and a label to detect VEAS 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 YEAS, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
VEAS expression. Normal or
standard values for YEAS expression are established by combining body fluids
or cell extracts taken
from normal mammalian subjects, for example, human subjects, with antibody to
VEAS under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of VEAS
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 YEAS may
be used for
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diagnostic purposes. The polynucleotides which may be used include
oiigonucleotide 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 YEAS may
be correlated with
disease. The diagnostic assay may be used to determine absence, presence, and
excess expression of
VEAS, and to monitor regulation of YEAS levels during therapeutic
intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding YEAS or closely related
molecules may be used to
identify nucleic acid sequences which encode YEAS. 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 YEAS, allelic
variants, or related
sequences.
Probes may also be used for the detection of related sequences, and may have
at least 5090
sequence identity to any of the VEAS encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:20-38 or from
genomic sequences including promoters, enhancers, and introns of the VEAS
gene.
Means for producing specific hybridization probes for DNAs encoding VEAS
include the
cloning of polynucleodde sequences encoding VEAS or VEAS 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 35S, or by
enzymatic labels, such as
alkaline phosphatase coupled to the probe via avidin/biotin coupling systems,
and the like.
Polynucleotide sequences encoding YEAS may be used for the diagnosis of
disorders associated
with expression of VEAS. Examples of such disorders include, but are not
limited to, a transport
disorder, such as akinesia, amyotrophic lateral sclerosis, ataxia
telangiectasia, cystic fibrosis, Becker's
muscular dystrophy, Bell's palsy, Charcot-Marie Tooth disease, diabetes
mellitus, diabetes insipidus,
diabetic neuropathy, Duchenne muscular dystrophy, hyperkalemic periodic
paralysis, normokalemic
periodic paralysis, Parkinson's disease, malignant hyperthermia, multidrug
resistance, myasthenia
gravis, myotonic dystrophy, catatonia, tardive dyskinesia, dystonias,
peripheral neuropathy, cerebral
neoplasms, prostate cancer, cardiac disorders associated with transport, e.g.,
angina, bradyarrythmia,
tachyarrythmia, hypertension, Long QT syndrome, myocarditis, cardiomyopathy,
nemaline myopathy,
centronuclear myopathy, lipid myopathy, mitochondria) myopathy. thyrotoxic
myopathy, ethanol
myopathy, dermatomyositis, inclusion body myositis, infectious myositis,
polymyositis, neurological
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disorders associated with transport, e.g., Alzheimer's disease, amnesia,
bipolar disorder, dementia,
depression, epilepsy, Tourette's disorder, paranoid psychoses, and
schizophrenia, and other disorders
associated with transport, e.g., neurofibromatosis, postherpetic neuralgia,
trigeminal neuropathy,
sarcoidosis, sickle cell anemia, cataracts, infertility, pulmonary artery
stenosis, sensorineural autosomal
deafness, hyperglycemia, hypoglycemia, Grave's disease, goiter, glucose-
galactose malabsorption
syndrome, and hypercholesterolemia, Cushing's disease, and Addison's disease,
gastrointestinal
disorders including ulcerative colitis, gastric and duodenal ulcers, other
conditions associated with
abnormal vesicle trafficking, including acquired immunodeficiency syndrome
(AIDS), allergies
including hay fever, asthma, and urticaria (hives), autoimmune hemolytic
anemia, proliferative
glomerulonephritis, inflammatory bowel disease, multiple sclerosis, rheumatoid
and osteoarthritis,
scleroderma, Chediak-Higashi and Sjogren's syndromes, systemic lupus
erythematosus, toxic shock
syndrome, traumatic tissue damage, viral, bacterial, fungal, helminthic, and
protozoal infections,
cysdnuria, dibasicaminoaciduria, hypercystinuria, lysinuria, hartnup disease,
tryptophan malabsorption,
methionine malabsorption, histidinuria, iminoglycinuria,
dicarboxylicaminoaciduria, cystinosis, remal
glycosuria, hypouricemia, familial hypophophatemic rickets, congenital
chloridorrhea, distal renal
tubular acidosis, Menkes' disease, Wilson's disease, lethal diarrhea, juvenile
pernicious anemia, folate
malabsorption, adrenoleukodystrophy, hereditary myoglobinuria, and Zellweger
syndrome; an
autoimmune/inflammatory disorder, such as acquired immunodeficiency syndrome
(AIDS),
Addison's disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis,
anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune
thyroiditis,
autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED),
bronchitis,
cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis,
dermatomyositis, diabetes
mellitus, emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis fetalis,
erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's
syndrome, gout, Graves'
disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome,
multiple sclerosis,
myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis,
osteoporosis, pancreatids,
polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma,
Sjogren's syndrome,
systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura,
ulcerative colitis, uveitis, Werner syndrome, complications of cancer,
hemodialysis, and
extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal,
and helminthic infections, and
trauma; and a cancer, such as 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.
3~ The polynucleotide sequences encoding YEAS may be used in Southern or
northern analysis, dot blot,
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WO 00/60082 PCT/US00/09353
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 VEAS
expression. Such qualitative or quantitative methods are well known in the
art.
In a particular aspect, the nucleotide sequences encoding YEAS may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding VEAS 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
VEAS 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 YEAS,
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 YEAS, under conditions suitable for hybridization
or amplification.
Standard hybridization may be quantified by comparing the values obtained from
normal subjects with
values from an experiment in which a known amount of a substantially purified
polynucleotide is used.
Standard values obtained in this manner may be compared with values obtained
from samples from
patients who are symptomatic for a disorder. Deviation from standard values is
used to establish the
presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the development
of the disease, or may provide a means for detecting the disease prior to the
appearance of actual
clinical symptoms. A more definitive diagnosis of this type may allow health
professionals to employ
preventative measures or aggressive treatment earlier thereby preventing the
development or further
progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding YEAS
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WO 00/60082 PCT/US00/09353
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 VEAS,
or a fragment of a polynucleotide complementary to the polynucleotide encoding
YEAS, 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.
Methods which may also be used to quantify the expression of VEAS 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 of interest is presented
in various dilutions and a spectrophotometric or colorimetric response gives
rapid quantitation.
In further embodiments, oligonucleoddes or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as targets in a
microarray. The microarray can
be used to monitor the expression level of large numbers of genes
simultaneously and 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, and to
develop and monitor the
activities of therapeutic agents.
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;
Shalon, 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.)
In another embodiment of the invention, nucleic acid sequences encoding YEAS
may be used to
generate hybridization probes useful in mapping the naturally occurring
genomic sequence. 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.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
chromosome
mapping techniques 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
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
gene encoding YEAS on a physical chromosomal map and a specific disorder, or a
predisposition to a
specific disorder, may help define the region of DNA associated with that
disorder. The nucleotide
sequences of the invention may be used to detect differences in gene sequences
among normal. carrier,
and affected individuals.
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 number or arm of a particular human
chromosome is not known.
New sequences can be assigned to chromosomal arms by physical mapping. This
provides valuable
information to investigators searching for disease genes using positional
cloning or other gene discovery
techniques. Once the disease or syndrome has been crudely localized by genetic
linkage to a particular
genomic region, e.g., ataxia-telangiectasia to l 1q22-23, any sequences
mapping to that area may
represent associated or regulatory genes for further investigation. (See,
e.g., Gatti, R.A. et al. (1988)
Nature 336:577-580.) The nucleotide sequence of the subject 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, VEAS, 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 YEAS 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 YEAS, or
fragments thereof, and
washed. Bound YEAS is then detected by methods well known in the art. Purified
YEAS 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 VEAS specifically compete with a test compound
for binding VEAS. In
this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with YEAS.
In additional embodiments, the nucleotide sequences which encode VEAS may be
used in any
molecular biology techniques that have yet to be developed, provided the new
techniques rely on
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WO 00/60082 PCT/US00/09353
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following preferred specific
embodiments are, therefore, to be construed as merely illustrative, and not
limitative of the remainder of
the disclosure in any way whatsoever.
The disclosures of all patents, applications and publications, mentioned above
and below, in
particular U.S. Ser. No. 60/128,193 and U.S. Ser. No. 60/144,701, 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, supra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleodde 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 S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose eel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e. g.,
PBLUESCRIPT plasmid (Stratagene), PSPORTl plasmid (Life Technologies),
pcDNA2.1 plasmid
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WO 00/60082 PCT/US00/09353
(Invitrogen, Carlsbad CA), or pINCY plasmid (Incyte Pharmaceuticals, Palo Alto
CA). Recombinant
plasmids were transformed into competent E. coli cells including XL1-Blue, XL1-
BlueMRF, or SOLR
from Stratagene or DHSa, DHlOB, or ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones
Plasmids 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
cDNA sequencing reactions were processed using standard methods or high-
throughput
instrumentation such as the ABI CATALYST 800 (Perkin-Elmer) 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 (Perkin-
Elmer).
Electrophoredc 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 (Perkin-Elmer) 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,
references, and threshold parameters. The first column of Table 5 shows the
tools, programs, and
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WO 00/60082 PCT/US00/09353
algorithms used, the second column provides brief descriptions thereof, the
third column presents
appropriate references, all of which are incorporated by reference herein in
their entirety, and the fourth
column presents, where applicable, the scores, probability values, and other
parameters used to evaluate
the strength of a match between two sequences (the higher the score, the
greater the homology between
two sequences). Sequences were analyzed using MACDNASIS PRO software (Hitachi
Software
Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
Polynucleotide and
polypeptide sequence alignments were generated using the default parameters
specified by the clustal
algorithm as incorporated into the MEGALIGN multisequence alignment program
(DNASTAR), which
also calculates the percent identity between aligned sequences.
The polynucleotide sequences were validated by removing vector, linker, and
polyA sequences
and by masking ambiguous bases, using algorithms and programs based on BLAST,
dynamic
programing, and dinucleotide nearest neighbor analysis. The sequences were
then queried against a
selection of public databases such as the GenBank primate, rodent, mammalian,
vertebrate, and
eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM to acquire
annotation
using programs based on BLAST, FASTA, and BLIMPS. The sequences were assembled
into full
length polynucleotide sequences using programs based on Phred, Phrap, and
Consed, and were screened
for open reading frames using programs based on GeneMark, BLAST, and FASTA.
The full length
polynucleotide sequences were translated to derive the corresponding full
length amino acid sequences,
and these full length sequences were subsequently analyzed by querying against
databases such as the
GenBank databases (described above), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM,
Prosite,
and Hidden Markov Model (HMM)-based protein family databases such as PFAM. HMM
is a
probabilistic approach which analyzes consensus primary structures of gene
families. (See, e.g., Eddy,
S.R. (1996) 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
N0:20-38. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
amplification technologies were described in The Invention section above.
IV. Northern Analysis
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
molecules in nucleotide databases such as GenBank or LIFESEQ (Incyte
Pharmaceuticals). This
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WO 00/60082 PCT/US00/09353
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:
sequence identity x % maximum BLAST score
100
The product score takes into account both the degree of similarity between two
sequences and the length
of the sequence match. For example, with a product score of 40, the match will
be exact within a 1 % to
2% error, and, with a product score of 70, the match will be exact. Similar
molecules are usually
identified by selecting those which show product scores between 15 and 40,
although lower scores may
identify related molecules.
The results of northern analyses are reported as a percentage distribution of
libraries in which
the transcript encoding VEAS 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 VEAS Encoding Polynucleotides
The cDNA sequences which were used to assemble SEQ ID N0:35-38 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 N0:35-38 were assembled into clusters of contiguous and overlapping
sequences using assembly
algorithms such as Phrap (Table 5). Radiation hybrid and genetic mapping data
available from public
resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute
for Genome
Research (WIGR), and Genethon were used to determine if any of the clustered
sequences had been
previously mapped. Inclusion of a mapped sequence in a cluster resulted in the
assignment of all
sequences of that cluster, including its particular SEQ ID NO:, to that map
location.
The genetic map locations of SEQ ID N0:38 are described in The Invention as
ranges, or
intervals, of human chromosomes. More than one map location is reported for
SEQ ID N0:38,
indicating that previously mapped sequences having similarity, but not
complete identity, to SEQ ID
N0:38 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
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
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 Genethon
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://www.ncbi.nlm.nih.gov/genemap/), can
be employed to
determine if previously identified disease genes map within or in proximity to
the intervals indicated
above.
VI. Extension of VEAS Encoding Polynucleotides
The full length nucleic acid sequences of SEQ ID N0:20-38 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,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+, (NH4)2S04,
and ~i-mercaptoethanol, Taq DNA polymerise (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerise (Stratagene), with the following
parameters for primer
pair PCI A and PCI B: Step l: 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:
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 pl
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 pl of undiluted PCR product into each well of an opaque tluorimeter
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
51
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
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, 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 (Perkin-Elmer).
In like manner, the nucleotide sequences of SEQ ID N0:20-38 are used to obtain
5' regulatory
sequences using the procedure above, oligonucleotides designed for such
extension, and an appropriate
genomic library.
VII. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:20-38 are employed to screen
cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide fragments.
Oligonucleotides are designed using state-of the-art software such as OLIGO
4.06 software (National
Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ~Ci of ~~y-
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
52
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
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
A chemical coupling procedure and an ink jet device can be used to synthesize
array elements
on the surface of a substrate. (See, e.g., Baldeschweiler, supra.) An array
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 by
hand or using
available methods and machines and contain any appropriate number of elements.
After hybridization,
nonhybridized probes are removed and a scanner used to determine the levels
and patterns of
fluorescence. The degree of complementarity and the relative abundance of each
probe which
hybridizes to an element on the microarray may be assessed through analysis of
the scanned images.
Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may
comprise the
elements of the microarray. Fragments suitable for hybridization can be
selected using software well
known in the art such as LASERGENE software (DNASTAR). Full-length cDNAs,
ESTs, or
fragments thereof corresponding to one of the nucleotide sequences of the
present invention, or selected
at random from a cDNA library relevant to the present invention, are arranged
on an appropriate
substrate, e.g., a glass slide. The cDNA is fixed to the slide using, e.g., UV
cross-linking followed by
thermal and chemical treatments and subsequent drying. (See, e.g., Schena, M.
et al. (1995) Science
270:467-470; Shalom D. et al. (1996) Genome Res. 6:639-645.) Fluorescent
probes are prepared and
used for hybridization to the elements on the substrate. The substrate is
analyzed by procedures
described above.
IX. Complementary Polynucleotides
Sequences complementary to the VEAS-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring VEAS. 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 YEAS. To
inhibit transcription, a
complementary oligonucleotide is designed from the most unique 5' sequence and
used to prevent
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CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
promoter binding to the coding sequence. To inhibit translation, a
complementary oligonucleotide is
designed to prevent ribosomal binding to the YEAS-encoding transcript.
X. Expression of VEAS
Expression and purification of VEAS is achieved using bacterial or virus-based
expression
systems. For expression of VEAS 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 YEAS upon induction with isopropyl beta-
D-thiogalactopyranoside
(IPTG). Expression of YEAS in eukaryotic cells is achieved by infecting insect
or mammalian cell
lines with recombinant Auto~ranhica californica nuclear polyhedrosis virus
(AcMNPV), commonly
known as baculovirus. The nonessential polyhedrin gene of baculovirus is
replaced with cDNA
encoding VEAS 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 Spodontera
fruQiperda (Sf9) insect cells in most cases, or human hepatocytes, in some
cases. Infection of the latter
requires additional genetic modifications to baculovirus. (See Engelhard, E.K.
et al. (1994) Proc. Natl.
Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-
1945.)
In most expression systems, VEAS 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 Schistosomalaponicum, 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 VEAS 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 VEAS obtained by these methods can be used directly
in the following activity
assay.
XI. Demonstration of VEAS Activity
VEAS activity is measured by its inclusion in coated vesicles. VEAS can be
expressed by
transforming a mammalina cell line such as COS7, HeLa, or CHO with an
eukaryotic expression vector
54
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
encoding YEAS. Eukaryotic expression vectors are commercially available, and
the techniques to
introduce them into cells are well known to those skilled in the art. A small
amount of a second
plasmid, which expresses any one of a number of marker genes, such as ~-
galactosidase, is co-
transformed into the cells in order to allow rapid identification of those
cells which have taken up and
expressed the foreign DNA. The cells are incubated for 48-72 hours after
transformation under
conditions appropriate for the cell line to allow expression and accumulation
of VEAS and /3-
ealactosidase.
Transformed cells are collected and cell lysates are assayed for vesicle
formation. A non-
hydrolyzable form of GTP, GTPyS, and an ATP regenerating system are added to
the lysate and the
mixture is incubated at 37 °C for 10 minutes. Under these conditions,
over 90% of the vesicles remain
coated (Orci, L. et al. (1989) Cell 56:357-368). Transport vesicles are salt-
released from the Golgi
membranes, loaded under a sucrose gradient, centrifuged, and fractions are
collected and analyzed by
SDS-PAGE. Co-localization of YEAS with clathrin or COP coatamer is indicative
of VEAS activity in
vesicle formation. The contribution of VEAS in vesicle formation can be
confirmed by incubating
lysates with antibodies specific for VEAS prior to GTPyS addition. The
antibody will bind to VEAS
and interfere with its activity, thus preventing vesicle formation.
In the alternative, VEAS activity is measured by its ability to alter vesicle
trafficking
pathways. Vesicle trafficking in cells transformed with VEAS is examined using
fluorescence
microscopy. Antibodies specific for vesicle coat proteins or typical vesicle
trafficking substrates such as
transferrin or the mannose-6-phosphate receptor are commercially available.
Various cellular
components such as ER, Golgi bodies, peroxisomes, endosomes, lysosomes, and
the plasmalemma are
examined. Alterations in the numbers and locations of vesicles in cells
transformed with YEAS as
compared to control cells are characteristic of VEAS activity.
XII. Functional Assays
VEAS function is assessed by expressing the sequences encoding VEAS 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 ~cg 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;
JJ
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate the
apoptotic state of the cells and other cellular properties. FCM detects and
quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events include
changes in nuclear DNA content as measured by staining of DNA with propidium
iodide; changes in
cell size and granularity as measured by forward light scatter and 90 degree
side light scatter; down-
regulation of DNA synthesis as measured by decrease in bromodeoxyuridine
uptake; alterations in
expression of cell surface and intracellular proteins as measured by
reactivity with specific antibodies;
and alterations in plasma membrane composition as measured by the binding of
fluorescein-conjugated
Annexin V protein to the cell surface. Methods in flow cytometry are discussed
in Ormerod, M.G.
(1994) Flow Cytometry, Oxford, New York NY.
The influence of VEAS on gene expression can be assessed using highly purified
populations
of cells transfected with sequences encoding VEAS 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 VEAS and other genes of interest can be analyzed by northern
analysis or
microarray techniques.
XIII. Production of VEAS Specific Antibodies
VEAS 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 VEAS amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, suvra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to KL,H
(Sigma-Aldrich, St.
Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
to increase
immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with
the oligopepdde-KL,H
complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide and anti-VEAS
activity by, for example, binding the peptide or VEAS to a substrate, blocking
with 1 % BSA, reacting
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CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
with rabbit antisera, washing, and reacting with radio-iodinated goat anti-
rabbit IgG.
XIV. Purification of Naturally Occurring VEAS Using Specific Antibodies
Naturally occurring or recombinant YEAS is substantially purified by
immunoaffinity
chromatography using antibodies specific for VEAS. An immunoaffinity column is
constructed by
covalently coupling anti-VEAS antibody to an activated chromatographic resin,
such as
CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the
resin is
blocked and washed according to the manufacturer's instructions.
Media containing VEAS are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of VEAS (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/VEAS 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 VEAS is collected.
XV. Identification of Molecules Which Interact with VEAS
YEAS, or biologically active fragments thereof, are labeled with'~I 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 mufti-well plate are incubated with the
labeled VEAS, washed, and
any wells with labeled VEAS complex are assayed. Data obtained using different
concentrations of
VEAS are used to calculate values for the number, affinity, and association of
VEAS with the candidate
molecules.
Alternatively, molecules interacting with VEAS 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).
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.
57
CA 02365421 2001-09-26
WO 00/60082 PCT/LTS00/09353
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CA 02365421 2001-09-26
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7''
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
C7 ~o
U
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7;
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
SEQUEP1CE LISTING
<110> INCYTE PHARMACEUTICAL: , iilC.
LnL, Preeti
Y ;JE , henry
F-iILLMAN, Jennifer L.
BAUGHN, Mariah R.
TANG, Y. Tom
LU, Dyung Aina M.
AZIMZAI, Yalda
<120> VESICLE ASSOCIATED PROTEINS
<130> PF-0684 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/128,193; 60/144,701
<151> 1999-04-07; 1999-07-20
<160> 38
<170> PERL Program
<210> 1
<211> 144
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 665637CD1
<400> 1
Met Ile Lys Phe Phe Leu Met Val Asn Lys Gln Gly Gln Thr Arg
1 5 10 15
Leu Ser Lys Tyr Tyr Glu His Val Asp Ile Asn Lys Arg Thr Leu
20 25 30
Leu Glu Thr Glu Val Ile Lys Ser Cys Leu Ser Arg Ser Asn Glu
35 40 45
Gln Cys Ser Phe I12 Glu Tyr Lys Asp Phe Lys Leu Ile Tyr Arg
50 55 60
Gln Tyr Ala Ala Leu Phe Ile Val Val Gly Val ASn Asp Thr Glu
65 70 75
Asn Glu Met Ala Ile Tyr Glu Phe Ile His Asn Phe Val Glu Val
80 85 90
Leu Asp Glu Tyr Phe Ser Arg Val Ser Glu Leu Asp Ile Met Phe
95 100 105
Asn Leu Asp Lys Val His Ile I12 Leu Asp Glu Met Val Leu Asn
110 115 120
Gly Cys Ile Val Glu Thr Asn Arg Ala Arg Ile Leu Ala Pro Leu
125 13 0 13 5
Leu Ile Leu Asp Lys Met Ser Glu Ser
140
<210> 2
<211> 177
<212> PRT
<213> Homo Sapiens
<220>
1/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
<221> misc_reature
<223> IncyLe ID No: 745823CD1
<400> 2
Diet Gly Asn Ile Phe Glu Lys Leu Phe Lys Ser Leu Leu Gly Lys
1 5 10 15
Lys Lys Me. Arg Ile Leu I12 Leu Ser Leu Asp Thr Ala Gly Lys
20 25 30
Thr Thr Ile Leu Tyr Lys Leu Lys Leu Gly Glu Thr Val Pro Ala
35 . 40 45
Val Pro Thr Val Gly Phe Cys Val Glu Thr Val Glu Tyr Lys Asn
50 55 60
Asn Thr Phe Ala Val Trp Asp Val Gly Ser His Phe Lys Ile Arg
65 70 75
Pro Leu Trp Gln His Phe Phe Gln Asn Thr Lys Gly Ala Arg Ser
80 85 90
Pro Gly Ser Thr His Gln Gly Ser Leu Ala Ser Gly Val Leu Pro
95 100 105
Ile Lys Cys Ser His Val Glu Phe Gly Met Trp Lys Gly Gly Arg
110 115 120
Ser His Pro Phe Leu Pro His Ser Ser Arg Cys Ala Gly Ser Gly
125 130 135
Gly Gln Leu Asp Ser Ile Leu Pro His Gln Ser Pro Ala Trp Gly
140 145 150
Pro Trp Gly Cys Lys Asp Leu Ser Ser Gly Phe Pro Ser Phe Leu
155 160 165
Thr Ser Ser Ile Leu Trp Lys Ser Ala Val Val Lys
170 175
<210> 3
<211> 408
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 776854CD1
<400> 3
Met Leu Val Asp Gly Pro Ser Glu Arg Pro Ala Leu Cys Phe Leu
1 5 10 15
Leu Leu Ala Val Ala Met Ser Phe Phe Gly Ser Ala Leu Ser Ile
20 25 30
Asp Glu Thr Arg Ala His Leu Leu Leu Lys Glu Lys Met Met Arg
35 40 45
Leu Gly Gly Arg Leu Val Leu Asn Thr Lys Glu Glu Leu Ala Asn
50 55 60
Glu Arg Leu Met Thr Leu Lys Ile Ala Glu Met Lys Glu Ala Met
65 70 75
Arg Thr Leu Ile Phe Pro Pro Ser Met His Phe Phe Gln Ala Lys
80 85 90
His Leu Ile Glu Arg Ser Gln Val Phe Asn Ile Leu Arg Met Met
95 100 105
Pro Lys Gly Ala Ala Leu His Leu His Asp Ile Gly Ile Val Thr
110 115 120
Met Asp Trp Leu Val Arg Asn Val Thr 'I'yr Arg Pro His Cys His
125 130 135
Ile Cys Phe Thr Pro Arg Gly Ile Met Gln Phe Arg Phe Ala His
140 145 150
Pro Thr Pro Arg Pro Ser Glu Lys Cys Ser Lys Trp Ile Leu Leu
155 160 165
Glu Asp Tyr Arg Lys Arg Val Gln Asn Val Thr Glu Phe Asp Asp
170 175 180
Ser Leu Leu Arg Asn Phe Thr Leu Val T~r Gln His Pro Glu Val
185 19G 195
2/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
Ile Tyr Thr Asn Gln Asn Val Val Trp Ser Lys Phe Glu Thr Ile
200 205 210
Phe Phe Thr Ile Ser Gly Leu Ile His Tyr Ala Pro Val Phe Arg
215 220 225
Asp Tyr Val Phe Arg Ser Met Gln Glu Phe 'I"~.~r Glu Asp Asn Val
230 235 240
Leu Tyr Met Giu Ile Arg Ala Arg Leu Leu Pro Val T=rr Glu Leu
245 250 255
Ser Gly Glu His His Asp Glu Glu Trp Ser Val Lys Thr Tyr Gln
260 265 270
Glu Val Ala Gln Lys Phe Val Glu Thr His Pro Glu Phe Ile Gly
275 280 285
Ile Lys Ile Ile Tyr Ser Asp His Arg Ser Lys Asp Val Ala Val
290 295 300
Ile Ala Glu Ser Ile Arg Met Ala Met Gly Leu Arg Ile Lys Phe
305 310 315
Pro Thr Val Val Ala Gly Phe Asp Leu Val Gly His Glu Asp Thr
320 325 330
Gly His Ser Leu Arg Asp Tyr Lys Glu Ala Leu Met I1e Pro Ala
335 340 345
Lys Asp Gly Val Lys Leu Pro Tyr Phe Phe His Ala Gly Glu Thr
350 355 360
Asp Pro Lys Glu Asp Leu Gly Gly Gly Cys Ser His Gly Gly Arg
365 370 375
Asp Gln Glu Gly Ser Ser Leu Gln His Pro Thr Cys His Pro Arg
380 385 390
Lys Thr Lys Ala Gly Trp Arg Glu Trp Trp Ala Val Ser Phe Pro
395 400 405
Thr Gln Ala
<210> 4
<211> 553
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1273556CD1
<400> 4
Met Thr Arg Gly Gly Pro Gly Gly Arg Pro Gly Leu Pro Gln Pro
1 5 10 15
Pro Pro Leu Leu Leu Leu Leu Leu Leu Leu Pro Leu Leu Leu Val
20 25 30
Thr Ala Glu Pro Pro Lys Pro Ala Gly Val Tyr Tyr Ala Thr Ala
35 40 45
Tyr Trp Met Pro Ala Glu Lys Thr Val Gln Val Lys Asn Val Met
50 55 60
Asp Lys Asn Gly Asp Ala Tyr Gly Phe Tyr Asn Asn Ser Val Lys
65 70 75
Thr Thr Gly Trp Gly Ile Leu Glu Ile Arg Ala Gly Tyr Gly Ser
80 85 90
Gln Thr Leu Ser Asn Glu Ile Ile Met Phe Val Ala Gly Phe Leu
95 100 105
Glu Gly Tyr Leu Thr Ala Pro His Met Asn Asp His Tyr Thr Asn
110 115 120
Leu Tyr Pro Gln Leu Ile Thr Lys Pro Ser Ile Met Asp Lys Val
125 130 135
Gln Asp Phe Met Glu Lys Gln Asp Lys Trp Thr Arg Lys Asn Ile
140 145 150
Lys Glu Tyr Lys Thr Asp Ser Phe Trp Arg His Thr Gly Tyr Val
155 160 165
Met Ala Gln Ile Asp Gly Leu Tyr Val Gly Ala Lys Lys Arg Ala
170 175 180
3/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
Ile Leu Glu Gly Thr Lys Pro Met Thr Leu Phe Gln Ile Gln Phe
185 190 195
Leu Asn Ser Val Gly Asp Leu Leu Asp Leu Ile Pro Ser Leu Ser
2GG 205 210
Pro Thr Lys Asn Gly Ser Leu Lys Val Phe Lys Arg Trp Asp Met
215 220 225
Gly His C_,~s Ser Ala Leu Iie Lys Val Leu Pro Gly Phe Glu Asn
230 235 240
Ile Leu Phe Ala His Ser Ser Trp Tyr Thr Tyr Ala Ala Met Leu
245 250 255
Arg Ile Tyr Lys His Trp Asp Phe Asn Ile Ile Asp Lys Asp Thr
260 265 270
Ser Ser Ser Arg Leu Ser Phe Ser Ser 'I'yr Pro Gly Phe Leu Glu
275 280 285
Ser Leu Asp Asp Phe Tyr Ile Leu Ser Ser Gly Leu Ile Leu Leu
290 295 300
Gln Thr Thr Asn Ser Val Phe Asn Lys Thr Leu Leu Lys Gln Val
305 310 315
Ile Pro Glu Thr Leu Leu Ser Trp Gln Arg Val Arg Val Ala Asn
320 325 330
Met Met Ala Asp Ser Gly Lys Arg Trp Ala Asp Ile Phe Ser Lys
335 340 345
Tyr Asn Ser Gly Thr Tyr Asn Asn Gln Tyr Met Val Leu Asp Leu
350 355 360
Lys Lys Va1 Lys Leu Asn His Ser Leu Asp Lys Gly Thr Leu Tyr
365 370 375
Ile Val Glu Gln Ile Pro Thr '1'~lrr Val Glu Tyr Ser Glu Gln Thr
380 385 390
Asp Val Leu Arg Lys Gly Tyr Trp Pro Ser Tyr Asn Val Pro Phe
395 400 405
His Glu Lys Ile Tyr Asn Trp Ser Gly Tyr Pro Leu Leu Val Gln
410 415 420
Lys Leu Gly Leu Asp Tyr Ser Tyr Asp Leu Ala Pro Arg Ala Lys
425 430 435
Ile Phe Arg Arg Asp Gln Gly Lys Val Thr Asp Thr Ala Ser Met
440 445 450
Lys Tyr Ile Met Arg Tyr Asn Asn Tyr Lys Lys Asp Pro Tyr Ser
455 460 465
Arg Gly Asp Pro Cys Asn Thr Ile Cys Cys Arg Glu Asp Leu Asn
470 475 480
Ser Pro Asn Pro Ser Pro Gly Gly Cys 'I',rr Asp Thr Lys Val Ala
485 490 495
Asp Ile Tyr Leu Ala Ser Gln Tyr Thr Ser Tyr Ala Ile Ser Gly
500 505 510
Pro Thr Val Gln Gly Gly Leu Pro Val Phe Arg Trp Asp Arg Phe
515 520 525
Asn Lys Thr Leu His Gln Gly Met Pro Glu Val Tyr Asn Phe Asp
530 535 540
Phe Ile Thr Met Lys Pro Ile Leu Lys Leu Asp Ile Lys
545 550
<210> 5
<211> 179
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: _505808CD1
<400> 5
Met Asp Pro Ala Gly Pro Ser Ala Pro ~.rg Arg Trp Thr Ala Arg
1 5 10 15
Ser Pro Gly Arg Lys ~sn Thr Arg Ser Arg Pro yr Leu Arg Val
4/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
20 25 30
Ser Ser Asp Pro Trp Arg Ser His Cys Gly Ser Arg Ala Iie Ala
35 40 45
Met Glu Phe Val Met Lys Gln Ala Leu Gly Gly Ala Thr Lys Asp
50 55 60
Met Gly Lys Met Leu Gly Gly Asp Glu Glu Lys Asp Pro Asp Ala
65 70 75
Ala Lys Lys Glu Glu Glu Arg Gln Glu Ala Leu Arg Gln Ala Glu
80 85 9G
Glu Glu Arg Lys Ala Lys Tyr Ala Lys Met Glu Ala Glu Arg Glu
95 100 105
Ala Val Arg Gln Gly Ile Arg Asp Lys 'I";rr Gly Ile Lys Lys Lys
110 115 120
Glu Glu Arg Glu Ala Glu Ala Gln Ala Ala Met Glu Ala Asn Ser
125 130 135
Glu Gly Ser Leu Thr Arg Pro Lys Lys Ala Ile Pro Pro Gly Cys
140 145 150
Gly Asp Glu Val Glu Glu Glu Asp Glu Ser Ile Leu Asp Thr Val
155 160 165
Ile Lys Tyr Leu Pro Gly Pro Leu Gln Asp Met Leu Lys Lys
170 175
<210> 6
<211> 336
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1814911CD1
<400> 6
Met Glu Lys Leu Arg Leu Leu Gly Leu Arg Tyr Gln Glu Tyr Val
1 5 10 15
Thr Arg His Pro Ala Ala Thr Ala Gln Leu Glu Thr Ala Val Arg
20 25 30
Gly Phe Ser Tyr Leu Leu Ala Gly Arg Phe Ala Asp Ser His Glu
35 40 45
Leu Ser Glu Leu Val Tyr Ser Ala Ser Asn Leu Leu Val Leu Leu
50 55 60
Asn Asp Gly Ile Leu Arg Lys Glu Leu Arg Lys Lys Leu Pro Val
65 70 75
Ser Leu Ser Gln Gln Lys Leu Leu Thr Trp Leu Ser Val Leu Glu
80 85 90
Cys Val Glu Val Phe Met Glu Met Gly Ala Ala Lys Val Trp Gly
95 100 105
Glu Val Gly Arg Trp Leu Val Ile Ala Leu Ile Gln Leu Ala Lys
110 115 120
Ala Val Leu Arg Met Leu Leu Leu Leu Trp Phe Lys Ala Gly Leu
125 130 135
Gln Thr Ser Pro Pro Ile Val Pro Leu Asp Arg Glu Thr Gln Ala
140 145 150
Gln Pro Pro Asp Gly Asp His Ser Pro Gly Asn His Glu Gln Ser
155 160 165
Tyr Val Gly Lys Arg Ser Asn Arg Val Val Arg Thr Leu Gln Asn
170 175 180
Thr Pro Ser Leu His Ser Arg His Trp Gly Ala Pro Gln Gln Arg
185 190 195
Glu Gly Arg Gln Gln Gln His His Glu Glu Leu Ser Ala Thr Pro
200 205 210
Thr Pro Leu Gly Leu Gln Glu Thr Ile Ala Glu Phe Leu T1~r Ile
215 220 225
Ala Arg Pro Leu Leu His Leu Leu Ser Leu Gly Leu Trp Gly Gln
230 235 24C
S/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
Arg Ser Trp Lys Pro Trp Leu Leu Ala Gly Val Val Asp Val Thr
245 250 255
Ser Leu Ser Leu Leu Ser Asp Arg Lys Gly Leu Thr Arg Arg Glu
260 265 270
Arg Arg Glu Leu Arg Arg Arg Thr Ile Leu Leu Leu 'I'~~r '?'~yr Leu
275 280 285
Leu Arg S2r Pro Phe Tyr Asp Arg Ph2 Ser Glu Ala Arg Ile Leu
290 295 300
Phe Leu Leu Gln Leu Leu Ala Asp His Val Pro Gly Val Gly Leu
305 310 315
Val Thr Arg Pro Leu Met Asp Tyr Leu Pro Thr Trp Gln Lys Ile
320 325 330
Tyr Phe Tyr Ser Trp Gly
335
<210> 7
<211> 240
<212> PRT
<213> Homo Sapiens
<220>
<221> misc ~eature
<223> Incyre ID No: 2087812CD1
<400> 7
Met Ser Prc Leu Leu Phe Gly Ala Gly Leu Val Val Leu Asn Leu
1 5 10 15
Val Thr Ser Ala Arg Ser Gln Lys Thr Glu Pro Leu Ser Gly Ser
20 25 30
Gly Asp Gln Pro Leu Phe Arg Gly Ala Asp Arg Tyr Asp Phe Ala
35 40 45
Ile Met Ile Pro Pro Gly Gly Thr Glu Cys Phe Trp Gln Phe Ala
50 55 60
His Gln Thr Gly Tyr Phe Tyr Phe Ser Tyr Glu Val Gln Arg Thr
65 70 75
Val Gly Met Ser His Asp Arg His Val Ala Ala Thr Ala His Asn
80 85 90
Pro Gln Gly Phe Leu Ile Asp Thr Ser Gln Gly Val Arg Gly Gln
95 100 105
Ile Asn Phe Ser Thr Gln Glu Thr Gly Phe Tyr Gln Leu Cys Leu
110 115 120
Ser Asn Gln His Asn His Phe Gly Ser Val Gln Val Z"yr Leu Asn
125 130 135
Phe Gly Val Phe Tyr Glu Gly Pro Glu Thr Asp His Lys Gln Lys
140 145 150
Glu Arg Lys Gln Leu Asn Asp Thr Leu Asp Ala Ile Glu Asp Gly
155 160 165
Thr Gln Lys Val Gln Asn Asn Ile Phe His Met Trp Arg Tyr Tyr
170 175 180
Asn Phe Ala Arg Met Arg Lys Met Ala Asp Phe Phe Leu Ile Gln
185 190 195
Ser Asn Tyr Asn Tyr Val Asn Trp Trp Ser Thr Ala Gln Ser Leu
200 205 210
Val Ile Ile Leu Ser Gly Ile Leu Gln Leu Tyr Phe Leu Lys Arg
215 220 225
Leu Phe Asn Val Pro Thr Thr Thr Asp Thr Lys Lys Pro Arg Cys
230 235 240
<210> 8
<211> 955
<212> PRT
6/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
<213> Homo Sapiens
<220>
<221> misc_teature
<223> Incyte ID Nc: 2149274CD1
<400> 8
Met Pro Ala Val Ser Lys Gly Asp Gly Met Arg Gly Leu Ala Val
1 5 10 15
Phe Ile Ser Asp Ile Arg Asn Cys Lys Ser Lys Glu Ala Glu Ile
20 25 30
Lys Arg Ile Asn Lys Glu Leu Ala Asn Ile Arg Ser Lys Phe Lys
35 40 45
Gly Asp Lys Ala Leu Asp Gly Tyr Ser Lys Lys Lys Tyr Val Cys
50 55 60
Lys Leu Leu Phe Ile Phe Leu Leu Gly His Asp Ile Asp Phe Gly
65 70 75
His Met Glu Ala Val Asn Leu Leu Ser Ser Asn Lys Tyr Thr Glu
80 85 90
Lys Gln Ile Gly Tyr Leu Phe Ile Ser Val Leu Val Asn Ser Asn
95 100 105
Ser Glu Leu Ile Arg Leu I12 Asn Asn Ala Ile Lys Asn Asp Leu
110 115 120
Ala Ser Arg Asn Pro Thr Phe Met Cys Leu Ala Leu His Cys Ile
125 130 135
Ala Asn Val Gly ser Arg Glu Met Gly Glu Ala Phe Ala Ala Asp
140 145 150
Ile Pro Arg Ile Leu Val Ala Gly Asp Ser Met Asp Ser Val Lys
155 160 165
Gln Ser Ala Ala Leu Cys Leu Leu Arg Leu Tyr Lys Ala Ser Pro
170 175 180
Asp Leu Val Pro Met Gly Glu Trp Thr Ala Arg Val Val His Leu
185 190 195
Leu Asn Asp Gln His Met Gly Val Val Thr Ala Ala Val Ser Leu
200 205 210
Ile Thr Cys Leu Cys Lys Lys Asn Pro Asp Asp Phe Lys Thr Cys
215 220 225
Val Ser Leu Ala Val Ser Arg Leu Ser Arg Ile Val Ser Ser Asp
230 235 240
Ser Thr Glu Leu Gln Asp Tyr Thr Tyr Tyr Phe Val Pro Ala Pro
245 250 255
Trp Leu Ser Val Lys Leu Leu Arg Leu Leu Gln Cys Tyr Pro Pro
260 265 270
Pro Glu Asp Ala Ala Val Lys Gly Arg Leu Val Glu Cys Leu Glu
275 280 285
Thr Val Leu Asn Lys Ala Gln Glu Pro Pro Lys Ser Lys Lys Val
290 295 300
Gln His Ser Asn Ala Lys Asn Ala Ile Leu Phe Glu Thr I1e Ser
305 310 315
Leu Ile Ile His Tyr Asp Ser Glu Pro Asn Leu Leu Val Arg Ala
320 325 330
Cys Asn Gln Leu Gly Gln Phe Leu Gln His Arg Glu Thr Asn Leu
335 340 345
Arg Tyr Leu Ala Leu Glu Ser Met Cys Thr Leu Ala Ser Ser Glu
350 355 360
Phe Ser His Glu Ala Val Lys Thr His Ile Asp Thr Val Ile Asn
365 370 375
Ala Leu Lys Thr Glu Arg Asp Val Ser Val Arg Gln Arg Ala Ala
380 385 390
Asp Leu Leu Tyr Ala Met Cys Asp Arg Ser Asn Ala Lys Gln Ile
395 400 405
Val Ser Glu Met Leu Arg Tyr Leu Glu Thr Ala Asp Tyr Ala Ile
410 415 420
Arg Glu Glu Ile Val Leu Lys Val Ala Ile Leu Ala Glu Lys Tyr
425 430 435
Ala Val Asp Tyr Ser Trp Tyr Val Asp Thr Ile Leu Asn Leu Ile
440 445 450
7/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
Arg Ile Ala Cys Asp Tyr Val Ser Glu Glu Val Trp Tyr Arg Val
455 460 465
Leu Gln Ile Val Thr Asn Arg Asp Asp Val Gln Gly Tyr Ala Ala
470 475 480
Lys Thr Val Phe G1u Ala Leu Gln Ala Pro Ala Cys His Glu Asn
485 490 495
Met Val Lys Val Gly Gly Tyr I12 Leu Gly Glu Phe Gly Asn Leu
500 505 510
Ile Ala Gly Asp Pro Arg Ser Ser Pro Pro Val Gln Phe Ser Leu
515 520 525
Leu His Ser Lys Phe His Leu Cys Ser Val Ala Thr Arg Ala Leu
530 535 540
Leu Leu Ser Thr Tyr Ile Lys Phe Ile Asn Leu Phe Pro Glu Thr
545 550 555
Lys Ala Thr Ile Gln Gly Val Leu Arg Ala Gly Ser Gln Leu Arg
560 565 570
Asn Ala Asp Val Glu Leu Gln Gln Arg Ala Val Glu Tyr Leu Thr
575 580 585
Leu Ser Ser Val Ala Ser Thr Asp Val Leu Ala Thr Val Leu Glu
590 595 600
Glu Met Pro Pro Phe Pro Glu Arg Glu Ser Ser I12 Leu Ala Lys
605 610 615
Leu Lys Arg Lys Lys Gly Pro Gly Ala Gly Ser Ala Leu Asp Asp
620 625 630
Gly Arg Arg Asp Pro Ser Ser Asn Asp Ile Asn Gly Gly Met Glu
635 640 645
Pro Thr Pro Ser Thr Val Ser Thr Pro Ser Pro Ser Ala Asp Leu
650 655 660
Leu Gly Leu Arg Ala Ala Pro Pro Pro Ala Ala Pro Pro Ala Ser
665 670 675
Ala Gly Ala Gly Asn Leu Leu Val Asp Val Phe Asp Gly Pro Ala
680 685 690
Ala Gln Pro Ser Leu Gly Pro Thr Pro Glu Glu Ala Phe Leu Ser
695 700 705
Pro Gly Pro Glu Asp Ile Gly Pro Pro Ile Pro Glu Ala Asp Glu
710 715 720
Leu Leu Asn Lys Phe Val Cys Lys Asn Asn Gly Val Leu Phe Glu
725 730 735
Asn Gln Leu Leu Gln Ile Gly Val Lys Ser Glu Phe Arg Gln Asn
740 745 750
Leu Gly Arg Met Tyr Leu Phe Tyr Gly Asn Lys Thr Ser Val Gln
755 760 765
Phe Gln Asn Phe Ser Pro Thr Val Val His Pro Gly Asp Leu Gln
770 775 780
Thr Gln Leu Ala Val Gln Thr Lys Arg Val Ala Ala Gln Val Asp
785 790 795
Gly Gly Ala Gln Val Gln Gln Val Leu Asn Ile Glu Cys Leu Arg
800 805 810
Asp Phe Leu Thr Pro Pro Leu Leu Ser Val Arg Phe Arg Tyr Gly
815 820 825
Gly Ala Pro Gln Ala Leu Thr Leu Lys Leu Pro Val Thr Ile Asn
830 835 840
Lys Phe Phe Gln Pro Thr Glu Met Ala Ala Gln Asp Phe Phe Gln
845 850 855
Arg Trp Lys Gln Leu Ser Leu Pro Gln Gln Glu Ala Gln Lys Ile
860 865 870
Phe Lys Ala Asn His Pro Met Asp Ala Glu Val Thr Lys Ala Lys
875 880 885
Leu Leu Gly Phe Gly Ser Ala Leu Leu Asp Asn Val Asp Pro Asn
890 895 900
Pro Glu Asn Phe Val Gly Ala Gly Ile Ile Gln Th= Lys Ala Leu
905 910 915
Gln Val Gly Cys Leu Leu Arg Leu Glu Pro Asn Ala Gln Ala Gln
920 925 930
Met 'I'~~rr Arg Leu Thr Leu Arg Thr Ser Lya Glu Pro Val Ser Arg
935 940 945
His Leu Cys Glu Leu Leu Ala Gln Gln Phe
8/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
950 955
<210> 9
<211> 247
<212> PRT
<213> Homo sabiens
<220>
<221> misc_feature
<223> Incyte ID No: 2355124CD1
<400> 9
Met Leu Val Leu Arg Ser Gly Leu Thr Lys Ala Leu Ala Ser Arg
1 5 10 15
Thr Leu Ala Pro Gln Val Cys Ser Ser Phe Ala Thr Gly Pro Arg
20 25 30
Gln Tyr Asp Gly Thr Phe Tyr Glu Phe Arg Thr Tyr Tyr Leu Lys
35 40 45
Pro Ser Asn Met Asn Ala Phe Met Glu Asn Leu Lys Lys Asn Ile
50 55 60
His Leu Arg Thr Ser Tyr Ser Glu Leu Val Gly Phe Trp Ser Val
65 70 75
Glu Phe Gly Gly Arg Thr Asn Lys Val Phe His Ile Trp Lys Tyr
80 85 90
Asp Asn Phe Ala His Arg Thr Glu Val Gln Lys Ala Leu Ala Lys
95 100 105
Asp Lys Glu Trp Gln Glu Gln Phe Leu Ile Pro Asn Leu Ala Leu
110 115 120
Ile Asp Lys Gln Glu Ser Glu Ile Thr '1'~,rr Leu Val Pro Trp Cys
125 130 135
Lys Leu Glu Lys Pro Pro Lys Glu Gly Val Tyr Glu Leu Ala Thr
140 145 150
Phe Gln Met Lys Pro Gly Gly Pro Ala Leu Trp Gly Asp Ala Phe
155 160 165
Lys Arg Ala Val His Ala His Val Asn Leu Gly Tyr Thr Lys Leu
170 175 180
Val Gly Val Phe His Thr Glu Tyr Gly Ala Leu Asn Arg Val His
185 190 195
Val Leu Trp Trp Asn Glu Ser Ala Asp Ser Arg Ala Ala Gly Arg
200 205 210
His Lys Ser His Glu Asp Pro Arg Val Val Ala Ala Val Arg Glu
215 220 225
Ser Val Asn 'I"srr Leu Val Ser Gln Gln Asn Met Leu Leu Ile Pro
230 235 240
Thr Ser Phe Ser Pro Leu Lys
245
<210> 10
<211> 532
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incl~te ID No: 2366939CD1
<400> 10
Met Gly Pro Gln Tyr C;rs Val Cys Lys Val Glu Leu Ser Va1 Ser
1 5 10 15
Gly Gln Asn Leu Leu Asp Arg Asp Val T'_:r Ser Lys Ser Asp Pro
2G 25 30
9/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
Phe Cys Val Leu Phe Thr Glu Asn Asn Gly Arg Trp Ile Glu Tyr
35 40 45
Asp Arg Thr Glu Thr Ala Ile Asn Asn Leu Asn Pro Ala Phe Ser
50 55 60
Lys Lys Phe Val Leu Asp Tyr His Phe Glu Giu Val Gln Lys Leu
65 70 75
Lys Phe Ala Leu Phe Asp Gln Asp L~~s Ser Ser Met Arg Leu Asp
80 85 90
Glu His Asp Phe Leu Gly Gln Phe Ser Cys Ser Leu Gly Thr Ile
95 100 105
Val Ser Ser Lys Lys Ile Thr Arg Pro Leu Leu Leu Leu Asn Asp
110 115 120
Lys Pro Ala Gly Lys Gly Leu Ile Thr Ile Ala Ala Gln Glu Leu
125 130 135
Ser Asp Asr. Arg Val Ile Thr Leu Ser Leu Ala Gly Arg Arg Leu
140 145 150
Asp Lys Lys Asp Leu Phe Gly Lys Ser Asp Pro Phe Leu Glu Phe
155 160 165
Tyr Lys Prc Gly Asp Asp Gly Lys Trp Met Leu Val His Arg Thr
170 175 180
Glu Val Ile Lys Tyr Thr Leu Asp Pro Val Trp Lys Pro Phe Thr
185 190 195
Val Pro Leu Val Ser Leu Cys Asp Gly Asp Met Glu Lys Pro Ile
200 205 210
Gln Val Met Cys Tyr Asp Tyr Asp Asn Asp Gly Gly His Asp Phe
215 220 225
Ile Gly Glu Phe Gln Thr Ser Val Ser Gln Met Cys Glu Ala Arg
230 235 240
Asp Ser Val Pro Leu Glu Phe Glu Cys Ile Asn Pro Lys Lys Gln
245 250 255
Arg Lys Lys Lys Asn Tyr Lys Asn Ser Gly Ile Ile Ile Leu Arg
260 265 270
Ser Cys Lys Ile Asn Arg Asp Tyr Ser Phe Leu Asp Tyr Ile Leu
275 280 285
Gly Gly Cys Gln Leu Met Phe Thr Val Gly Ile Asp Phe Thr Ala
290 295 300
Ser Asn Gly Asn Pro Leu Asp Pro Ser Ser Leu His Tyr Ile Asn
305 310 315
Pro Met Gly Thr Asn Glu Tyr Leu Ser Ala Ile Trp Ala Val Gly
320 325 330
Gln Ile Ile Gln Asp Tyr Asp Ser Asp Lys Met Phe Pro Ala Leu
335 340 345
Gly Phe Gly Ala Gln Leu Pro Pro Asp Trp Lys Val Ser His Glu
350 355 360
Phe Ala Ile Asn Phe Asn Pro Thr Asn Pro Phe Cys Ser Gly Val
365 370 375
Asp Gly I12 Ala Gln Ala Tyr Ser Ala Cys Leu Pro His Ile Arg
380 385 390
Phe Tyr Gly Pro Thr Asn Phe Ser Pro Ile Val Asn His Val Ala
395 400 405
Arg Phe Ala Ala Gln Ala Thr Gln Gln Arg Thr Ala Thr Gln Tyr
410 415 420
Phe Ile Leu Leu Ile Ile Thr Asp Gly Val Ile Ser Asp Met Glu
425 430 435
Glu Thr Arg His Ala Val Val Gln Ala Ser Lys Leu Pro Met Ser
440 445 450
Ile Ile Ile Val Gly Val Gly Asn Ala Asp Phe Ala Ala Met Glu
455 460 465
Phe Leu Asp Gly Asp Ser Arg Met Leu Arg Ser His Thr Gly Glu
470 475 480
Glu Ala Ala rg Asp Ile Val Gln Phe Val Pro Phe Arg Glu Phe
485 490 495
Arg Asn Ala .la Lys Glu Thr Leu Ala Lys Ala Val Leu Ala Glu
500 505 510
Leu Pro Gl.-: ~ln Val Val G1n Tyr Phe Lys His Lys Asn Leu Pro
515 520 525
Pro Thr As_-. Ser Glu Pro Ala
10/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
53 0
<210> 11
<211> 154
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2483906CD1
<400> 11
Met Ile His Phe Ile Leu Leu Phe Ser Arg Gln Gly Lys Leu Arg
1 5 10 15
Leu Gln Lys Trp Tyr Ile Thr Leu Pro Asp Lys Glu Arg Lys Lys
20 25 30
Ile Thr Arg Glu Ile Val Gln I1e Ile Leu Ser Arg Gly His Arg
35 40 45
Thr Ser Ser Phe Val Asp Trp Lys Glu Leu Lys Leu Val Tyr Lys
50 55 60
Arg Tyr Ala Ser Leu Tyr Phe Cys Cys Ala Ile Glu Asn Gln Asp
65 70 75
Asn Glu Leu Leu Thr Leu Glu Ile Val His Arg Tyr Val Glu Leu
80 85 90
Leu Asp Lys Tyr Phe Gly Asn Val Cys Glu Leu Asp Ile Ile Phe
95 100 105
Asn Phe Glu Lys Ala '1'~,~r Phe Ile Leu Asp Glu Phe Ile Ile Gly
110 115 120
Gly Glu Ile Gln Glu Thr Ser Lys Lys Ile Ala Val Lys Ala Ile
125 130 135
Glu Asp Ser Asp Met Leu Gln Glu Thr Met Glu Glu Tyr Met Asn
140 145 150
Lys Pro Thr Phe
<210> 12
<211> 684
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2499488CD1
<400> 12
Met Trp His Glu Thr Ser Asn Arg Ile Pro Ser Lys Arg Thr Val
1 5 10 15
Ser Phe Val Leu Ser Thr Gly Leu Tyr Pro Ile Ser Phe Gln Val
20 25 30
His Leu Val Arg Asp Gly Val Leu Thr Arg Phe Ser Val Ala Thr
35 40 45
Glu Tyr Gln Lys Asn Ala Gln Ile Glu Lys Leu Arg Ser Glu Ile
50 55 60
Val Val Leu Lys Glu Glu Leu Gln Leu Thr Arg Ser Glu Leu Glu
65 70 75
Ala Ala His His Ala Ser Ala Val Arg Phe Ser Lys Glu 'I'~~rr Glu
80 85 90
Met Gln Lys Thr Lys Glu Glu Asp Phe Leu Lys Leu Phe Asp Arg
95 1C0 105
Trp Lys Glu Glu Glu Lys Glu Lys Leu Val Asp Glu Met Glu Lys
110 115 120
Val Lys Glu Met Phe Met Lys Glu Phe Lys Glu Leu T:r Ser Lys
11; 33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
125 130 135
Asn Ser Ala Leu Glu Tyr Glr. Leu Ser Glu Ile Gln Lys Ser Asn
140 145 150
Met G1n Ile Lys Ser Asn Ile Gly Thr Leu Lys Asp Ala His Glu
155 160 165
Phe Lys Glu Asp Arg Ser Pro T"_~r Pro Gln Asp Phe His Asn Val
170 175 180
Met Gln Leu Leu Asp Ser Gln Glu Ser Lys Trp Thr Ala Arg Val
185 190 195
Gln Ala Ile His Gln Glu His Lys Lys Glu Lys Gly Arg Leu Leu
200 205 210
Ser His Ile Glu Lys Leu Arg Thr Ser Met Ile Asp Asp Leu Asn
215 220 225
Ala Ser Asn Val Phe Tyr Lys Lys Arg Ile Glu Glu Leu Gly Gln
230 235 240
Arg Leu Gln Glu Gln Asn Glu Leu Ile Ile Thr Gln Arg Gln Gln
245 250 255
Ile Lys Asp Phe Thr Cys Asn Pro Leu Asn Ser Ile Ser Glu Pro
260 265 270
Lys Gly Asn Pro Leu Ala Trp Gln Ala Phe Glu Ser Gln Pro Ala
275 280 285
Ala Pro Ala Val Pro Met Asn Ala Pro Ala Leu His Thr Leu Glu
290 295 300
Thr Lys Ser Ser Leu Pro Met Val His Glu Gln Ala Phe Ser Ser
305 310 315
His Ile Leu Glu Pro Ile Glu Glu Leu Ser Glu Glu Glu Lys Gly
320 325 330
Arg Glu Asn Glu Gln Lys Leu Asn Asn Asn Lys Met His Leu Arg
335 340 345
Lys Ala Leu Lys Ser Asn Ser Ser Leu Thr Lys Gly Leu Arg Thr
350 355 360
Met Val Glu Gln Asn Leu Met Glu Lys Leu Glu Thr Leu Gly Ile
365 370 375
Asn Ala Asp Ile Arg Gly Ile Ser Ser Asp Gln Leu His Arg Val
380 385 390
Leu Lys Ser Val Glu Ser Glu Arg His Lys Gln Glu Arg Glu Ile
395 400 405
Pro Asn Phe His Gln Ile Arg Glu Phe Leu Glu His Gln Val Ser
410 415 420
Cys Lys Ile Glu Glu Lys Ala Leu Leu Ser Ser Asp Gln Cys Ser
425 430 435
Val Ser Gln Met Asp Thr Leu Ser Thr Gly Glu Val Pro Lys Met
440 445 450
Ile Gln Leu Pro Ser Lys Asn Arg Gln Leu Ile Arg Gln Lys Ala
455 460 465
Val Ser Thr Asp Arg Thr Ser Val Pro Lys Ile Lys Lys Asn Val
470 475 480
Met Glu Asp Pro Phe Pro Arg Lys Ser Ser Thr Ile Thr Thr Pro
485 490 495
Pro Phe Ser Ser Glu Glu Glu Gln Glu Asp Asp Asp Leu Ile Arg
500 505 510
Ala Tyr Ala Ser Pro Gly Pro Leu Pro Val Pro Pro Pro Gln Asn
515 520 525
Lys Gly Ser Phe Gly Lys Asn Thr Val Lys Ser Asp Ala Asp Gly
530 535 540
Thr Glu Gly Ser Glu Ile Glu Asp Thr Asp Asp Ser Pro Lys Pro
545 550 555
Ala Gly Val Ala Val Lys Thr Pro Thr Glu Lys Val Glu Lys Met
560 565 570
Phe Pro His Arg Lys Asn Val Asn Lys Pro Val Gly Gly Thr Asn
575 580 585
Val Pro Glu Met Phe Ile Lys Lys Glu Glu Leu Gln Glu Leu Lys
590 595 600
Cys Ala Asp Val Glu Asp Glu Asp Trp Asp Ile Ser Ser Leu Glu
605 610 615
Glu Glu Ile Ser Leu Gly Lys Lys Ser Gly Lys Glu Gln Lys Glu
620 625 63G
12/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
Pro Pro Pro Ala Lys Asn Glu Pro His Phe Aia His Val Leu Asn
635 64U 645
Ala Trp Gly Ala Phe Asn Pro Lys Gly Pro Lys Gly Glu Gly Leu
650 655 660
Gln Glu Asn Glu Ser Ser Thr Leu Lys Ser Ser Leu Val T::r Val
665 670 675
Thr Asp Trp Ser Asp Thr Ser Asp Val
680
<210> 13
<211> 576
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2559148CD1
<400> 13
Met Ser Thr Ser Ser Leu Arg Arg Gln Met Lys Asn Ile Val His
1 5 10 15
Asn Tyr Ser Glu Ala Glu Ile Lys Val Arg G1u Ala Thr Ser Asn
20 25 30
Asp Pro Trp Gly Pro Ser Ser Ser Leu Met Ser Glu Ile Ala Asp
35 40 45
Leu Thr T~,rr Asn Val Val Ala Phe Ser Glu Ile Met Ser Met Ile
50 55 60
Trp Lys Arg Leu Asn Asp His Gly Lys Asn Trp Arg His Val Tyr
65 70 75
Lys Ala Met Thr Leu Met Glu '1'yr Leu Ile Lys Thr Gly Ser Glu
80 85 90
Arg Val Ser Gln Gln Cys Lys Glu Asn Met Tyr Ala Val Gln Thr
95 100 105
Leu Lys Asp Phe Gln Tyr Val Asp Arg Asp Gly Lys Asp Gln Gly
110 115 120
Val Asn Val Arg Glu Lys Ala Lys Gln Leu Val Ala Leu Leu Arg
125 130 135
Asp Glu Asp Arg Leu Arg Glu Glu Arg Ala His Ala Leu Lys Thr
140 145 150
Lys Glu Lys Leu Ala G1n Thr Ala Thr Ala Ser Ser Ala Ala Val
155 160 165
Gly Ser Gly Pro Pro Pro Glu Ala Glu Gln Ala Trp Pro Gln Ser
170 175 180
Ser Gly Glu Glu Glu Leu Gln Leu Gln Leu Ala Leu Ala Met Ser
185 190 195
Lys Glu Glu Ala Asp Gln Pro Pro Ser Cys Gly Pro Glu Asp Asp
200 205 210
Ala Gln Leu Gln Leu Ala Leu Ser Leu Ser Arg Glu Glu His Asp
215 220 225
Lys Glu Glu Arg Ile Arg Arg Gly Asp Asp Leu Arg Leu G1n Met
230 235 240
Ala Ile Glu Glu Ser Lys Arg Glu Thr Gly Gly Lys Glu Glu Ser
245 250 255
Ser Leu Met Asp Leu Ala Asp Val Phe Thr Ala Pro Ala Pro Ala
260 265 270
Pro Thr Thr Asp Pro Trp Gly Gly Pro Ala Pro Met Ala Ala Ala
275 280 285
Val Pro Thr Ala A1a Pro Thr Ser Asp Pro Trp Gly Gly Pro Pro
290 295 300
Val Pro Pro Ala Ala Asp Pro Trp Gly Gly Pro Ala Pro Thr Pro
305 310 315
Ala Ser Gly Asp Pro Trp Arg Pro Ala Ala Pro Ala Gly Pro Ser
320 325 330
Val Asp Pro Trp Gly Gly Thr Pro Ala Pro Ala Ala Gly Glu Gly
13/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
335 340 345
Pro Thr Pro Asp Pro Trp Gly Ser Ser Asp Gly Gly Val Pro Val
350 355 360
Ser Gly Pro Ser Ala Ser Asp Pro Trp Thr Pro Ala Pro Ala Phe
365 370 375
Ser Asp Pro Trp Gly Gly Ser Pro Ala Lys Pro Ser Thr Asn Gly
380 385 390
Thr Thr Ala Ala Gly Gly Phe Asp Thr Glu Pro Asp Glu Phe Ser
395 400 405
Asp Phe Asp Arg Leu Arg Thr Ala Leu Pro Thr Ser Gly Ser Ser
410 415 420
Ala Gly Glu Leu Glu Leu Leu Ala Gly Glu Val Pro Ala Arg Ser
425 430 435
Pro Gly Ala Phe Asp Met Ser Gly Val Arg Gly Ser Leu Ala Glu
440 445 450
Ala Val Gly Ser Pro Pro Pro Ala Ala Thr Pro Thr Pro Thr Pro
455 460 465
Pro Thr Arg Lys Thr Pro Glu Ser Phe Leu Gly Pro Asn Ala Ala
470 475 480
Leu Val Asp Leu Asp Ser Leu Val Ser Arg Pro Gly Pro Thr Pro
485 490 495
Pro Gly Ala Lys Ala Ser Asn Pro Phe Leu Pro Gly Gly Gly Pro
500 505 510
Ala Thr Gly Pro Ser Val Thr Asn Pro Phe Gln Pro Ala Pro Pro
515 520 525
Ala Thr Leu Thr Leu Asn Gln Leu Arg Leu Ser Pro Val Pro Pro
530 535 540
Val Pro Gly Ala Pro Pro Thr Tyr Ile Ser Pro Leu Gly Gly Gly
545 550 555
Pro Gly Leu Pro Pro Met Met Pro Pro Gly Pro Pro Ala Pro Asn
560 565 570
Thr Asn Pro Phe Leu Leu
575
<210> 14
<211> 425
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_~eature
<223> Incyte ID No: 3321481CD1
<400> 14
Met Ser Ala Ser Ala Val Phe Ile Leu Asp Val Lys Gly Lys Pro
1 5 10 15
Leu Ile Ser Arg Asn Tyr Lys Gly Asp Val Ala Met Ser Lys Ile
20 25 30
Glu His Phe Met Pro Leu Leu Val Gln Arg Glu Glu Glu Gly Ala
35 40 45
Leu Ala Pro Leu Leu Ser His Gly Gln Val His Phe Leu Trp Ile
50 55 60
Lys His Ser Asn Leu Tyr Leu Val Ala Thr Thr Ser Lys Asn Ala
65 70 75
Asn Ala Ser Leu Val Tyr Ser Phe Leu Tyr Lys Thr Ile Glu Val
80 85 90
Phe Cys Glu Tyr Phe Lys Glu Leu Glu Glu Glu Ser Ile Arg Asp
95 100 105
Asn Phe Val Ile Val Tyr Glu Leu Leu Asp Glu Leu Met Asp Phe
110 115 120
Gly Phe Pro Gln Thr Thr Asp Ser Lys Ile Leu Gln Glu Tyr Ile
125 130 135
Thr Gln Gln Ser Asn Lys Leu Glu Thr Gly Lys Ser Ara Val Prc
140 145 150
14/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
Pro Thr Val Thr Asn Ala Val Ser Trp Arg Ser Glu Gly Ile Lys
155 160 165
Tyr Lys Lys Asn Glu Val Phe Ile Asp Val Ile Glu Ser Val Asn
170 175 180
Leu Leu Val Asn Ala Asn Gly Ser Val Leu Leu Ser Glu Ile Val
185 190 195
Gly Thr Ile Lys Leu Lys Val Phe Leu Ser Gly Met Pro Glu Leu
200 205 210
Arg Leu Gly Leu Asn Asp Arg Val Leu Phe Glu Leu Thr Gly Leu
215 220 225
Ser Gly Ser Lys Asn Lys Ser Val Glu Leu Glu Asp Val Lys Phe
230 235 240
His Gln Cys Val Arg Leu Ser Arg Phe Asp Asn Asp Arg Thr Ile
245 250 255
Ser Phe Ile Pro Pro Asp Gly Asp Phe Glu Leu Met Ser Tyr Arg
260 265 270
Leu Ser Thr Gln Val Lys Pro Leu Ile Trp Ile Glu Ser Val Ile
275 280 285
Glu Lys Phe Ser His Ser Arg Val Glu Ile Met Val Lys Ala Lys
290 295 300
Gly Gln Phe Lys Lys Gln Ser Val Ala Asn Gly Val Glu Ile Ser
305 310 315
Val Pro Val Pro Ser Asp Ala Asp Ser Pro Arg Phe Lys Thr Ser
320 325 330
Val Gly Ser Ala Lys Tyr Val Pro Glu Arg Asn Val Val Ile Trp
335 340 345
Ser Ile Lys Ser Phe Pro Gly Gly Lys Glu Tyr Leu Met Arg Ala
350 355 360
His Phe Gly Leu Pro Ser Val Glu Lys Glu Glu Val Glu Gly Arg
365 370 375
Pro Pro Ile Gly Val Lys Phe Glu Ile Pro Tyr Phe Thr Val Ser
380 385 390
Gly Ile Gln 'Jal Arg 1'yr Met Lys Ile Ile Glu Lys Ser Gly Tyr
395 400 405
Gln Ala Leu Pro Trp Val Arg Tyr Ile Thr Gln Ser Gly Asp Tyr
410 415 420
Gln Leu Arg Thr Ser
425
<210> 15
<211> 167
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3367918CD1
<400> 15
Met Leu Ser Trp Ala Trp Trp Gln Gly Ala Gly Asn Pro Val Leu
1 5 10 15
Gly Arg Leu Arg Gln Glu Asn His Leu Asn Pro Gly Gly Arg Gly
20 25 30
Cys Ser Glu Gln Lys Lys Lys Lys Lys Lys Leu Thr Ser Leu Leu
35 40 45
Pro Thr Ile Leu Leu Ser Gln Glu Ile Tyr Lys Asp Arg Gln Lys
50 55 60
Phe Ser Glu Gln Val Phe Lys Val Ala Ser Ser Asp Leu Val Asn
65 70 75
Met Gly Ile Ser Val Val Ser Tyr Thr Leu Lys Asp Ile His Asp
80 85 90
Asp Gln Asp Tyr Leu His Ser Leu Gly Lys Ala Arg Thr Ala Gln
95 100 105
Val Gln Lys Asp Ala =rg Ile Gly Glu Ala Glu Ala Lys Arg Asp
15/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
110 115 120
Ala Gly Ile Arg Glu Ala Lys Ala Lys Gln Glu Lys Val 5er Aia
125 130 135
Gln Tyr Leu Ser Glu Ile Glu Met Ala Lys Ala Gln Arg Asp Tyr
140 145 15C
Glu Leu Lys Lys Ala Ala Tyr Asp Ile Glu Val Asn Thr Pro Ser
155 160 lo'~
Thr Gly
<210> 16
<211> 739
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte ID No: 1227327CD1
<400> 16
Met Pro Tyr Leu Gly Ser Glu Asp Val Val Lys Glu Leu Lys Lys
1 5 10 15
Ala Leu Cys Asn Pro His Ile Gln Ala Asp Arg Leu Arg Tyr Arg
20 25 30
Asn Val Ile Gln Arg Val Ile Arg 'I"yr Met Thr Gln Gly Leu Asp
35 40 45
Met Ser Gly Val Phe Met Glu Met Val Lys Ala Ser Ala Thr Val
50 55 60
Asp Ile Val Gln Lys Lys Leu Val Tyr Leu Tyr Met Cys Thr Tyr
65 70 75
Ala Pro Leu Lys Pro Asp Leu Ala Leu Leu Ala Ile Asn Thr Leu
80 85 90
Cys Lys Asp Cys Ser Asp Pro Asn Pro Met Val Arg Gly Leu Ala
95 100 105
Leu Arg Ser Met Cys Ser Leu Arg Met Pro Gly Val Gln Glu Tyr
110 115 120
Ile Gln Gln Pro Ile Leu Asn Gly Leu Arg Asp Lys Ala Ser Tyr
125 130 135
Val Arg Arg Val Ala Val Leu Gly Cys Ala Lys Met His Asn Leu
140 145 150
His Gly Asp Ser Glu Val Asp Gly Ala Leu Val Asn Glu Leu Tyr
155 160 165
Ser Leu Leu Arg Asp Gln Asp Pro Ile Val Val Val Asn Cys Leu
170 175 180
Arg Ser Leu Glu Glu Ile Leu Lys Gln Glu Gly Gly Val Val Ile
185 190 195
Asn Lys Pro Ile Ala His His Leu Leu Asn Arg Met Ser Lys Leu
200 205 210
Asp Gln Trp Gly Gln Ala Glu Val Leu Asn Phe Leu Leu Arg Tyr
215 220 225
Gln Pro Arg Ser Glu Glu Glu Leu Phe Asp Ile Leu Asn Leu Leu
230 235 240
Asp Ser Phe Leu Lys Ser Ser Ser Pro Gly Val Val Met Gly Ala
245 250 255
Thr Lys Leu Phe Leu Ile Leu Ala Lys Met Phe Pro His Val Gln
260 265 270
Thr Asp Val Leu Val Arg Val Lys Gly Pro Leu Leu Ala Ala Cys
275 280 285
Ser Ser Glu Ser Arg Glu Leu Cys Phe Val Ala Leu Cys His Val
290 295 300
Arg Gln Ile Leu His Ser Leu Pro Gly His Phe Ser Ser His Tyr
305 310 315
Lys Lys Phe Phe Cys Ser Tyr Ser Glu Pro His Tyr Ile Lys Leu
320 325 330
Gln Lys Val Glu Val Leu Cys Glu Leu Val Asn Asp Glu Asn Val
16/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
335 340 345
Gln Gln Val Leu Glu Glu Leu Arg Gly T"yr C,,rs ~1'hr Asp Val Ser
350 355 360
Ala Asp Phe Ala Gln Ala Ala Ile Phe Ala Ile Gly Gly Ile Ala
365 370 375
Arg Thr '=';-r Thr Asp Gln Cys Val Gln Ile Leu Thr Glu Leu Leu
380 385 390
Gly Leu Arg Gln Glu His Ile Thr Thr Val Val Val Gln Thr Phe
395 400 405
Arg Asp Leu Val Trp Leu Cys Pro Gln Cys Thr Glu Ala Val Cys
410 415 420
Gln Ala Leu Pro Gly Cys Glu Glu Asn Ile Gln Asp Ser Glu Gly
425 430 435
Lys Gln Ala Leu Ile Trp Leu Leu Gly Val His Gly Glu Arg Ile
440 445 450
Pro Asn Ala Pro Tyr Val Leu Glu Asp Phe Val Glu Asn Val Lys
455 460 465
Ser Glu Thr Phe Pro Ala Val Lys Met Glu Leu Leu Thr Ala Leu
470 475 480
Leu Arg Leu Phe Leu Ser Arg Pro Ala Glu Cys Gln Asp Met Leu
485 490 495
Gly Arg Leu Leu Tyr Tyr Cys Ile Glu Glu Glu Lys Asp Met Ala
500 505 510
Val Arg Asp Arg Gly Leu Phe Tyr Tyr Arg Leu Leu Leu Val Gly
515 520 525
Ile Asp Glu Val Lys Arg Ile Leu Cys Ser Pro Lys Ser Asp Pro
530 535 540
Thr Leu Gly Leu Leu Glu Asp Pro Ala Glu Arg Pro Val Asn Ser
545 550 555
Trp Ala Ser Asp Phe Asn Thr Leu Val Pro Val Tyr Gly Lys Ala
560 565 570
His Trp Ala Thr Ile Ser Lys Cys Gln Gly Ala Glu Arg Cys Asp
575 580 585
Pro Glu Leu Pro Lys Thr Ser Ser Phe Ala Ala Ser Gly Pro Leu
590 595 600
Ile Pro Glu Glu Asn Lys Glu Arg Val Gln Glu Leu Pro Asp Ser
605 610 615
Gly Ala Leu Met Leu Val Pro Asn Arg Gln Leu Thr Ala Asp Tyr
620 625 630
Phe Glu Lys Thr Trp Leu Ser Leu Lys Val Ala His Gln Gln Val
635 640 645
Leu Pro Trp Arg Gly Glu Phe His Pro Asp Thr Leu Gln Met Ala
650 655 660
Leu Gln Val Val Asn Ile Gln Thr Ile Ala Met Ser Arg Ala Gly
665 670 675
Ser Arg Pro Trp Lys Ala Tyr Leu Ser Ala Gln Asp Asp Thr Gly
680 685 690
Cys Leu Phe Leu Thr Glu Leu Leu Leu Glu Pro Gly Asn Ser Glu
695 700 705
Met Gln Ile Ser Val Lys Gln Asn Glu Ala Arg Thr Glu Thr Leu
710 715 720
Asn Ser Phe Ile Ser Val Leu Glu Thr Val Ile Gly Thr Ile Glu
725 730 735
Glu Ile Lys Ser
<210> 17
<211> 742
<212> PRT
<213> Homo sapiens
<220>
<221> mist feature
<223> Incyte ID No: 1416292CD1
17/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
<400> 17
Met Ala Asp Ile Leu Ser Gln Ser Glu Thr Leu Ala Ser Gln Asp
1 5 10 15
Leu Ser Gly Asp Phe Lys Lys Pro Ala Leu Pro Val Ser Pro Ala
2G 25 30
Ala Arg Ser Lys Ala Pro Ala Ser Ser Ser Ser Asn Pro Glu Glu
35 40 45
Val Gln Lys Glu Gly Pro Thr Ala Leu Gln Asp Ser Asn Ser Gly
50 55 60
Glu Pro Asp Ile Pro Pro Pro Gln Pro Asp Cys Gly Asp Phe Arg
65 70 75
Ser Leu Gln Glu Glu Gln Ser Arg Pro Thr Thr Ala Val Ser Ser
80 85 90
Pro Gly Gly Pro Ala Arg Ala Pro Pro Tyr Gln Glu Pro Pro Trp
95 100 105
Gly Gly Pro Ala Thr Ala Pro Tyr Ser Leu Glu Thr Leu Lys Gly
110 115 120
Gly Thr Ile Leu Gly Thr Arg Ser Leu Lys Gly Thr Ser Tyr Cys
125 130 135
Leu Phe Gly Arg Leu Ser Gly Cys Asp Val Cys Leu Glu His Pro
140 145 150
Ser Val Ser Arg Tyr His Ala Val Leu Gln His Arg Ala Ser Gly
155 160 165
Pro Asp Gly Glu Cys Asp Ser Asn Gly Pro Gly Phe Tyr Leu Tyr
170 175 180
Asp Leu Gly Ser Thr His Gly Thr Phe Leu Asn Lys Thr Arg Ile
185 190 195
Pro Pro Arg Thr Tyr Cys Arg Val His Val Gly His Val Val Arg
200 205 210
Phe Gly Gly Ser Thr Arg Leu Phe Ile Leu Gln Gly Pro Glu Glu
215 220 225
Asp Arg Glu Ala Glu Ser Glu Leu Thr Val Thr Gln Leu Lys Glu
230 235 240
Leu Arg Lys Gln Gln Gln Ile Leu Leu Glu Lys Lys Met Leu Gly
245 250 255
Glu Asp Ser Asp Glu Glu Glu Glu Met Asp Thr Ser Glu Arg Lys
260 265 270
Ile Asn Ala Gly Ser Gln Asp Asp Glu Met Gly Cys Thr Trp Gly
275 280 285
Met Gly Glu Asp Ala Val Glu Asp Asp Ala Glu Glu Asn Pro Ile
290 295 300
Val Leu Glu Phe Gln Gln Glu Arg Glu Ala Phe Tyr Ile Lys Asp
305 310 315
Pro Lys Lys Ala Leu Gln Gly Phe Phe Asp Arg Glu Gly Glu Glu
320 325 330
Leu Glu Tyr Glu Phe Asp Glu Gln Gly His Ser Thr Trp Leu Cys
335 340 345
Arg Val Arg Leu Pro Val Asp Asp Ser Thr Gly Lys Gln Leu Val
350 355 360
Ala Glu Ala Ile His Ser Gly Lys Lys Lys Glu Ala Met Ile Gln
365 370 375
Cys Ser Leu Glu Ala Cys Arg Ile Leu Asp Thr Leu Gly Leu Leu
380 385 390
Arg Gln Glu Ala Val Ser Arg Lys Arg Lys Ala Lys Asn Trp Glu
395 400 405
Asp Glu Asp Phe Tyr Asp Ser Asp Asp Asp Thr Phe Leu Asp Arg
410 415 420
Thr Gly Leu Ile Glu Lys Lys Arg Leu Asn Arg Met Lys Lys Ala
425 430 435
Gly Lys Ile Asp Glu Lys Pro Glu Thr Phe Glu Ser Leu Val Ala
440 445 450
Lys Leu Asn Asp Ala Glu Arg Glu Leu Ser Glu Ile Ser Glu Arg
455 460 465
Leu Lys Ala Ser Ser Gln Val Leu Ser Glu Ser Pro Ser Gln Asp
470 475 480
Ser Leu Asp Ala Phe Met Ser Glu Met Lys Ser Gly Ser Thr Leu
485 490 495
18/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
Asp Gly Val Ser Arg Lys Lys Leu His Leu Arg Thr Phe Glu Leu
500 505 5i0
Arg Lys Glu Gln Gln Arg Leu Lys Gly Leu I1e Lys Ile Val Lys
515 520 525
Pro Ala Glu Ile Pro Glu Leu Lys Lys Thr Glu Thr Gln Thr Thr
530 535 540
Gly Ala Glu Asn Lys Ala Lys Lys Leu Thr Leu P=o Leu Phe Gly
545 550 555
Ala Met Lys Gly Gly Ser Lys Phe Lys Leu Lys Thr Gly Thr Val
560 565 570
Gly Lys Leu Pro Pro Lys Arg Pro Glu Leu Pro Pro Thr Leu Met
575 580 585
Arg Met Lys Asp Glu Pro Glu Val Glu Glu Glu Glu Glu Glu Glu
590 595 600
Glu Glu Glu Glu Lys Glu Lys Glu Glu His Glu Lys Lys Lys Leu
605 610 615
Glu Asp Gly Ser Leu Ser Arg Pro Gln Pro Glu Ile Glu Pro Glu
620 625 630
Ala Ala Val Gln Glu Met Arg Pro Pro Thr Asp Leu Thr His Phe
635 640 645
Lys Glu Thr Gln Thr His Glu Asn Met Ser Gln Leu Ser Glu Glu
650 655 660
Glu Gln Asn Lys Asp Tyr Gln Asp Cys Ser Lys Thr Thr Ser Leu
665 670 675
Cys Ala Gly Pro Ser Ala Ser Lys Asn Glu Tyr Glu Lys Ser Arg
680 685 690
Gly Glu Leu Lys Lys Lys Lys Thr Pro Gly Pro Gly Lys Leu Pro
695 700 705
Pro Thr Leu Ser Ser Lys Tyr Pro Glu Asp Asp Pro Asp Tyr Cys
710 715 720
Val Trp Val Pro Pro Glu Gly Gln Ser Gly Asp Gly Arg Thr His
725 730 735
Leu Asn Asp Lys Tyr Gly Tyr
740
<210> 18
<211> 325
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2611704CD1
<400> 18
Met Ala His Phe Val Gln Gly Thr Ser Arg Met Ile Ala Ala Glu
1 S 10 15
Ser Ser Thr Glu His Lys Glu Cys Ala Glu Pro Ser Thr Arg Lys
20 25 30
Asn Leu Met Asn Ser Leu Glu Gln Lys Ile Arg Cys Leu Glu Lys
35 40 45
Gln Arg Lys Glu Leu Leu Glu Val Asn Gln Gln Trp Asp Gln Gln
50 55 60
Phe Arg Ser Met Lys Glu Leu Tyr Glu Arg Lys Val Ala Glu Leu
65 70 75
Lys Thr Lys Leu Asp Ala Ala Glu Arg Phe Leu Ser Thr Arg Glu
80 85 90
Lys Asp Pro His Gln Arg Gln Arg Lys Asp Asp Arg Gln Arg Glu
95 100 105
Asp Asp Arg Gln Arg Asp Leu Thr Arg Asp Arg Leu Gln Arg Glu
110 115 120
Glu Lys Glu Lys Glu Arg Leu Asn Glu Glu Leu His Glu Leu Lys
125 13 0 135
Glu Glu Asn Lys Leu Leu Lys Gly Lys Asn Thr Leu Ala Asn Lys
19/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
140 145 150
Glu Lys Glu His Tyr Glu Cys Glu Ile Lys Arg Leu Asn Lys Ala
155 160 165
Leu Gln Asp Ala Leu Asn Ile Lys Cys Ser Phe Ser Glu Asp Cys
170 175 180
Leu Arg Lys Ser Arg Val Glu Phe Cys His Glu Glu Met Arg Thr
185 190 195
Glu Met Glu Val Leu Lys Gln Gln Val G1n Ile Tyr Glu Glu Asp
200 205 210
Phe Lys Lys Glu Arg Ser Asp Arg Glu Arg Leu Asn Gln Glu Lys
215 220 225
Glu Glu Leu Gln Gln Ile Asn Glu Thr Ser Gln Ser Gln Leu Asn
230 235 240
Arg Leu Asn Ser Gln Ile Lys Ala Cys Gln Met Glu Lys Glu Lys
245 250 255
Leu Glu Lys Gln Leu Lys Gln Met Tyr Cys Pro Pro Cys Asn Cys
260 265 270
Gly Leu Val Phe His Leu Gln Asp Pro Trp Val Pro Thr Gly Pro
275 280 285
Gly Ala Val Gln Lys Gln Arg Glu His Pro Pro Asp Tyr Gln Trp
290 295 300
Tyr Ala Leu Asp Gln Leu Pro Pro Asp Val Gln His Lys Ala Asn
305 310 315
Gly Leu Ser Ser Val Lys Lys Val His Pro
320 325
<210> 19
<211> 299
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5136672CD1
<400> 19
Met Glu His Phe Asp Ala Ser Leu Ser Thr Tyr Phe Lys Ala Leu
1 5 10 15
Leu Gly Pro Arg Asp Thr Arg Val Lys Gly Trp Phe Leu Leu Asp
20 25 30
Asn Tyr Ile Pro Thr Phe Ile Cys Ser Val Ile Tyr Leu Leu Ile
35 40 45
Val Trp Leu Gly Pro Lys Tyr Met Arg Asn Lys Gln Pro Phe Ser
50 55 60
Cys Arg Gly Ile Leu Val Val Tyr Asn Leu Gly Leu Thr Leu Leu
65 70 75
Ser Leu Tyr Met Phe Cys Glu Leu Val Thr Gly Val Trp Glu Gly
80 85 90
Lys Tyr Asn Phe Phe Cys Gln Gly Thr Arg Thr Ala Gly Glu Ser
95 100 105
Asp Met Lys Ile Ile Arg Val Leu Trp Trp Tyr Tyr Phe Ser Lys
110 115 120
Leu Ile Glu Phe Met Asp Thr Phe Phe Phe Ile Leu Arg Lys Asn
125 13 0 135
Asn His Gln Ile Thr Val Leu His Val 'I'yr His His Ala Ser Met
140 145 150
Leu Asn Ile Trp Trp Phe Val Met Asn Trp Val Pro Cys Gly His
155 160 165
Ser Tyr Phe Gly Ala Thr Leu Asn Ser Phe Ile His Val Leu Met
170 175 180
Tyr Ser Tyr Tyr Gly Leu Ser Ser Val Pro Ser Met Arg Pro Tyr
185 190 195
Leu Trp Trp Lys Lys Tyr Ile Thr Gln Gly Gln Leu Leu Gln Phe
200 2G5 210
2U/33
CA 02365421 2001-09-26
WO 00/60082 PCT/LJS00/09353
Val Leu Thr Ile Ile Gln Thr Ser Cys Gly Va1 Ile Trp Pro Cys
215 220 225
Thr Phe Pro Leu Gly Trp Leu Tyr Phe Gln Ile Gly '1'~lrr Met Ile
230 235 240
Ser Leu Ile Ala Leu Phe Thr Asn Phe Tyr Iie Gln Thr '1'~~rr Asn
245 250 255
Lys Lys Gly Ala Ser Arg Arg Lys Asp His Leu Lys Asp His Gln
260 265 270
Asn Gly Ser Met Ala Ala Val Asn Gly His Thr Asn Ser Phe Ser
275 280 285
Pro Leu Glu Asn Asn Val Lys Pro Arg Lys Leu Arg Lys Asp
290 295
<210> 20
<211> 1050
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 665637CB1
<400> 20
aagccaaaat ggctgccccg aggaggcccg caccgcgtag ccagtgaagg ttggggagca 60
agcttatgcg ggaaagaggg agggggactc caggaaaagc cgttgagagg accatcacaa 120
cctgagcagc acaggttcca gttacagcca tcccttgtca taacttttga actgtatttg 180
gaaaaatact ggccaggaaa gaaaaatgat aaaatttttc ctcatggtga ataaacaagg 240
gcagactcga ctttctaagt actatgaaca tgtggatatt aataagcgta cacttctgga 300
aacagaagtc ataaagagct gtctctctcg atccaatgaa caatgctctt tcattgaata 360
taaggatttt aagctgatat atcggcagta tgcagctctc ttcattgtgg ttggagttaa 420
tgacactgag aacgagatgg ctatttatga attcattcat aactttgtgg aagttttaga 480
tgagtatttc agccgagtga gtgaattaga tataatgttt aatttggata aagtacacat 540
cattttggat gagatggtgt taaatggctg cattgtggaa actaacaggg caagaattct 600
tgcccctcta ctaattcttg ataagatgtc agaaagctga aaggaagtct cttcgagaca 660
atatggattt atcagaaatg cgagtaccgt ggaatacatc tcaacatgtt aacccagaag 720
aatctggaag accacaatta caaaatgggg tatccttcca aagacattat aaataggcat 780
tttccacagt tcctaaaaag aaaacacaac tgtactttaa aatatgtaca aagaaaaaaa 840
tttctttaaa ctgagagaga agttttattt tctaattgta aacatatctg tcgcacttta 900
aattctgttg agcacctaag gaacccttct tggtctatgc ttcttttgca aattgaattc 960
aggaatagca ggatggtagt gggaagaaag tatggcagtt ttccgtcagc caataaagtt 1020
ttaaaattta aacacaaaaa aaaaaaaaaa 1050
<210> 21
<211> 916
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 745823CB1
<400> 21
gaacatggta ttgccagagc ttcatggcgt cagctacggc gctccctccc tcccaggccc 60
ttactcttct ttgcgttggc cagatatgtc gtcactcaca aaacttcgag ctcattggtg 120
caaaagctcc aggaggcgga gggtgattgg ttggttgctg ttcctgcccc cacgcatgtc 180
gtggtttagc tgaactgagc tgaaatccta aaggccgcgg agtcggcggt gttgtaggta 240
gcggtacct~ gagtggcaac agaattcgat taaattacaa tgggaaacat ttttgaaaag 300
ctctttaaaa gtctacttgg gaaaaaaaag atgcggattc ttatattgag tttggataca 360
gctggaaaaa ccaccatctt gtataaattg aagctggggg agactgtgcc tgccgtccct 420
acagtaggtt ~ctgtgtgga gacagtagaa tataaaaata acaccttcgc tgtctgggat 480
gttggcagcc acttcaaaat cagacctctg tggcagcatt ttttccagaa cacaaaaggt 540
gccagaagcc caggaagcac acatcaaggc tcacttgcca gcggggtgct gccaataaaa 600
tgtagtcacg tggaatttgg aatgtggaaa ggaggtagaa gtcatccttt cctcccccat 660
21 /33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
agcagcaggt gtgcaggctc tggtggtcag ctggactcca tactccccca ccagtcacca 720
gcctggggac cgtggggctg caaggacctc agcagcggtt tcccaagttL cctgacttct 780
tccatcctct ggaaatcagc tgtggtaaag tagcctgaaa gccagtggtg caaccccatc 840
cccacaacct tcaccacctc tagcacctcc agtgataagc actaattgcc tatatacaac 900
ccttttttgt ttgaaa 916
<210> 22
<211> 1930
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 776854CB1
<400> 22
cttttccggt gctctgcaca gatgctgggg cactgagcaa acagccctca gtttctggag 60
ctgttccgag tcccgtggag tctccatctg agccctttcc tagtccaggc atcccgatgt 120
tggtggatgg cccatctgag cggccagccc tgtgcttctt gctgttggct gtggcaatgt 180
ctttcttcgg ctcagctcta tccatagatg aaacacgggc gcatctgttg ttgaaagaaa 240
agatgatgcg gctcgggggg cggctggtgc tgaacaccaa ggaggagctg gccaatgaga 300
ggctcatgac gctcaaaatc gctgagatga aggaggccat gaggaccctg atattcccac 360
ccagcatgca ctttttccag gccaagcatc tcattgagag aagtcaagtg tttaatattc 420
taaggatgat gccaaaaggg gctgccttgc acctccatga cattggcatc gtgactatgg 480
actggctggt gaggaatgtc acctacaggc ctcactgcca catctgttt,c accccaaggg 540
ggatcatgca gttcagattt gctcacccaa ctccccgtcc atcagaaaaa tgttccaagt 600
ggattctgct ggaggattat cggaagcggg tgcagaacgt cactgagttt gatgacagct 660
tgctgaggaa tttcactctg gtgacccagc acccggaggt gatttacaca aaccaaaatg 720
ttgtctggtc gaaatttgaa accatcttct tcaccatctc tggtctcatc cattacgcac 780
cagtgttcag agactatgtc ttccggagca tgcaggagtt ctacgaggac aacgtgctct 840
acatggagat cagagccagg ctgctgccgg tgtatgagct cagtggagag caccatgacg 900
aagagtggtc agtgaagact taccaggaag tagctcagaa gtttgtggaa actcaccctg 960
agtttattgg aatcaaaatc atttattcgg atcacagatc caaagatgtg gctgtcatcg 1020
cagaatccat ccgaatggcc atggggctcc gaatcaagtt ccccacggtg gtggcagggt 1080
ttgacctggt ggggcatgag gacactggcc actccttgcg tgactacaag gaagctctga 1140
tgatccccgc caaggatggc gttaagctgc cttacttctt ccacgccgga gaaacagatc 1200
caaaggaaga tttgggagga ggctgttcac atggaggccg ggaccaggag ggcagctctc 1260
ttcagcatcc cacctgtcac cctcggaaga ccaaggcggg gtggagagag tggtgggcag 1320
tttcatttcc cacccaggct taagctctgc tctatcacac cttctctcag agaaagggac 1380
cttgtgcatt aatctctttt tcctgacatt tcgagtcaat ttagatccaa tttagatcaa 1440
agtctgctct aggagagcag aagaattgtt tgagggggag ggggttgggc gagggtgttg 1500
ttaaccttga agagcttgtg gtctggttgg ggaaaatcat acttatgtgt gtgaaataat 1560
tctggcccta caaaacattg cagcacagga cctaacaggt tccaagatgt atggtccttg 1620
gagatgattt tgccaggact tgggtatcta atacctaaaa gtaatgtcct tatttttatt 1680
tttttatttt attttatgtt attttttgag acggagtctc gctctgtact ccagcctggc 1740
gacagagcaa gactgtgtct caaaaaacaa caacaacaac aacaaaaaac tcggttttgc 1800
tacttactgt gtgaacatcc ttggtcaatt tatagaagct attaagtttc aactttctca 1860
ttagaatgat aaattaataa attagatgat taatttaata aagccatatc atacctctct 1920
ttagcattga 1930
<210> 23
<211> 1809
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1273556CB1
<400> 23
cgcagaggaa ggtcgcggcc gtggagcgat gacccgcggc ggtccgggcg ggcgcccggg 60
gctgccacag ccgccgccgc ttctgctgct gctgctgctg ctgccgctgt tgttagtcac 120
cgcggagccg ccgaaacctg caggagtcta ctatgcaact gcatactgga tgcctgctga 180
aaagacagta caagtcaaaa atgtaatgga caagaatggg gacgcctatg gcttttacaa 240
taactctgtg aaaaccacag gctggggcat cctggagatc agagctggcc atggctctca 30G
22/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
aaccctgagc aatgagatca tcatgtttgt ggctggcttt ttggagggtt acctcactgc 360
cccacacatg aatgaccact acacaaacct ctacccacag ctgatcacga aaccttccat 420
catggataaa gtgcaggatt ttatggagaa gcaagataag tggacccgga aaaatatcaa 480
agaatacaag actgattcat tttggagaca tacaggctat gtgatggcac aaatagatgg 540
cctctatgta ggagcaaaga agagggctat tttagaaggg acaaagccaa tgaccctgtt 600
ccagattcag ttcctgaata gtgttggaga tctattggat ctgattccct cactctctcc 660
cacaaaaaac ggcagcctaa aggtttttaa gagatgggac atgggacatt gctccgctct 720
tatcaaggtt cttcctggat ttgagaacat cctttttgct cactcaagct ggtacacgta 780
tgcagccatg ctcaggatat ataaacactg ggacttcaac atcatagata aagataccag 840
cagtagtcgc ctctctttca gcagttaccc agggtttttg gagtctctgg atgattttta 900
cattcttagc agtggattga tattgctgca gaccacaaac agtgtgttta ataaaaccct 960
gctaaagcag gtaatacccg agactctcct gtcctggcaa agagtccgtg tggccaatat 1020
gatggcagat agtggcaaga ggtgggcaga catcttttca aaatacaact ctggcaccta 1080
taacaatcaa tacatggttc tggacctgaa gaaagtaaag ctgaaccaca gtcttgacaa 1140
aggcactctg tacattgtgg agcaaattcc tacatatgta gaatattctg aacaaactga 1200
tgttctacgg aaaggatatt ggccctccta caatgttcct ttccatgaaa aaatctacaa 1260
ctggagtggc tatccactgt tagttcagaa gctgggcttg gactactctt atgatttagc 1320
tccacgagcc aaaattttcc ggcgtgacca agggaaagtg actgatacgg catccatgaa 1380
atatatcatg cgatacaaca attataagaa ggatccttac agtagaggtg acccctgtaa 1440
taccatctgc tgccgtgagg acctgaactc acctaaccca agtcctggag gttgttatga 1500
cacaaaggtg gcagatatct acctagcatc tcagtacaca tcctatgcca taagtggtcc 1560
cacagtacaa ggtggcctcc ctgtttttcg ctgggaccgt ttcaacaaaa ctctacatca 1620
gggcatgcca gaggtctaca actttgattt tattaccatg aaaccaattt tgaaacttga 1680
tataaaatga aggagggaga tgacggacta gaagactgta aataagatac caaaggcact 1740
attttagcta tgtttttccc atcagaatta tgcaataaaa tatattaatt tgtcaaaaaa 1800
aaaaaaaaa 1809
<210> 24
<211> 638
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1505808CB1
<400> 24
tcaccgggcg ccacatggac ccagccggcc cgagcgcgcc ccgccgctgg accgcccgct 60
ccccgggaag gaagaacact cgctcccggc catacttgcg tgtgagttct gacccctgga 120
ggagccactg tggaagcaga gcaatcgcca tggagtttgt gatgaagcag gctctaggag 180
gggccaccaa ggacatgggg aagatgctgg ggggtgacga ggagaaggac ccagacgccg 240
ccaagaagga ggaggagcgg caggaggcgc tgcgccaggc ggaggaggag cgcaaggcca 300
agtacgccaa gatggaggcg gagcgcgagg ccgtgcgcca gggcatccga gacaagtacg 360
gcatcaagaa gaaggaggag cgcgaggccg aggcccaggc cgccatggag gccaactccg 420
aggggagctt gacgcggccc aagaaggcca tcccgccggg ctgcggggac gaggtggagg 480
aggaggacga gagcatcctg gacaccgtca tcaagtacct gcccgggccg ctgcaggaca 540
tgctcaagaa gtagccccgc gcgggacagc ggccccgcgg agcccccgcc cctcccccta 600
cagatcctcc gcggaggccc ctgagggacg agcagagc 638
<210> 25
<211> 1477
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1814911CB1
<400> 25
gcggttcaag agatggggtg aagggtggtt caccggttct tcaagtcctc agccttctgg 60
cccgcggaag ttaagcaacc aagaggcggg cctaagaccg gaagcaggaa ggagggcgca 120
ggaagcaggg cgccgcagcc tgtcgtacgg tccttctgtg ggtctgtcgg tgccgagggc 180
aggatggaga agctgcggct cctgggcctc cgctaccagg agtacgtgac tcgtcacccg 240
gccgccacgg cccagctgga gacagcagtg cggggcttca gttacctgct ggcaggtcga 300
ttcgccgatt cgcacgagct gtcagagctg gtgtactctg cctctaacct gcttgtgctg 360
3/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
ctcaatgacg ggatcctacg gaaggagctt cggaaaaagt tgcctgtgtc gctgtcccag 42G
cagaagctgc tgacatggct gagcgtgctg gagtgcgtgg aggtgttcat ggagatggga 48G
gctgccaagg tgtggggtga agtgggccgc tggcttgtca tcgccctcat ccagctggcc 540
aaggctgtac tgcggatgct cctgctgctc tggttcaagg ctggcctcca gacttcaccc 600
cctatcgttc cactggacag agagacccag gcacagcccc cggatggtga ccacagccct 660
ggcaaccatg agcagtccta cgtggggaag cggtcaaacc gggtggtgcg aaccctccag 72G
aacacgccgt ccctgcactc caggcactgg ggagctcccc agcagcggga gggacggcag 78G
cagcagcatc acgaggagct gagtgcgacc cccacccccc tggggctgca ggagaccatc 840
gcagagtttt tgtacattgc ccggccgctg ctgcacttgc tcagcctggg cctgtggggt 900
cagaggtcgt ggaaaccctg gctcttggct ggtgttgtgg acgtgaccag cctgagcctc 96G
ctgagtgaca gaaagggcct gacccggagg gagcggcggg agctgcggcg ccggaccatc 1020
ctgctgctct actacctgct gcgctctcct ttctacgacc gcttctccga ggccaggatc 1080
ctcttcctgc tccagttgct ggccgaccac gtccctggcg ttggcctggt cacaaggccg 1140
ctcatggatt acttgcccac ctggcagaaa atctacttct acagttgggg ctgacagacc 1200
tcccggaagg agggtgtggg gaggggtggg gcagggagcc cctcttccct aataaaactg 1260
actccggcag cgtcccagcg tgcggcctct ccgtgcctac cggccaggcc ccacacacag 1320
ccctggtcgc caccagcgtt cctcccagga cacccttgac tgcgctctcc tgtgacgatg 1380
ccactgcagc ccgcaccttg tcactgctgg gccaagaagc cttcactagg agtgggatcc 1440
aggctcctct cccacagaaa gcggtgactt cacctca 1477
<210> 26
<211> 1137
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2087812CB1
<400> 26
ctccagaaag cggcatgCcc cctttgctct ttggggctgg gctggtcgtt ctgaatctag 60
tgacgtctgc caggagccag aagacagaac ctctaagtgg ctctggggac cagccactct 120
tccgtggagc tgatcgatat gactttgcca tcatgatacc tccaggaggc acggaatgct 180
tttggcaatt tgcccaccag actggatact tctatttcag ttacgaggtt cagcggacag 240
tggggatgtc acatgaccgg catgttgctg ccacggcaca taacccacag ggatttctca 300
tagacacctc ccagggtgtt cggggccaga ttaacttctc tacccaagag acaggttttt 360
atcagctttg tctaagtaat cagcataatc acttcggttc tgtgcaagtg tacctcaact 420
ttggggtctt ctatgagggg cctgagactg atcacaaaca gaaggaaaga aaacaactga 480
atgatactct ggatgcaatt gaggacggca cacaaaaggt gcagaacaat atctttcaca 540
tgtggcgata ctacaacttt gcccggatga ggaaaatggc tgactttttc cttatccaat 600
caaactataa ctacgtgaac tggtggtcga cagcccagag ccttgttatt attctttctg 660
ggatcctgca actgtatttc ttgaagcgtc tcttcaatgt tccaacaact acagatacaa 72G
agaagccaag atgctaagct aaggtgacta tagcaccctg gctgttttct tctggggctt 780
agtcgaatca gctttgtaat gttatgggac aaaaatcaat tatctcatta atgttttagt 840
ctgctgcaca catctaaaaa agcaaaatgg caataaaatc ataacagtga aaaagttctg 9GC
aagaagtatg tattaaatgt ataaaggaaa atactgttca tcatatagca gtcccttagc 960
cttcactgag tctacttaga ggctaacaga aaaccttaga aggaagatga gttgaaaaag 1020
gccacaatca agtttgctta catataaaag caggagttcg taatgtttta ttcttaaaga 1080
cttcacctct ggaacttaaa ataagcaata tattcagtaa agaaatagtt gaattta 1137
<210> 27
<211> 3343
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2149274CB1
<400> 27
cggctcggct ccttggcgct gcctggggtc ctttccgccc ggtccccgct tgccagcccc
cgctgctctg tgccctgtcc ggccaggcct ggagccgaca ccaccgccat catgccggcc 12G
gtgtccaagg gcgatgggat gcgggggctc gcggtgttca tctccgacat ccggaactgt 18~
aagagcaaag aggcggaaat taagagaatc aacaaggaac tggccaacat ccgctccaag 24G
ttcaaaggag acaaagcctt ggatggctac agtaagaaaa aatatgtgtg taaactgctt 30G
24/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
ttcatcttcc tgcttggcca tgacattgac tttgggcaca tggaggctgt gaatctgttg 360
agttccaata aatacacaga gaagcaaata ggttacctgt tcatttctgt gctggtgaac 420
tcgaactcgg agctgatccg cctcatcaac aacgccatca agaatgacct ggccagccgc 480
aaccccacct tcatgtgcct ggccctgcac tgcatcgcca acgtgggcag ccgggagatg 54C
ggcgaggcct ttgccgctga catcccccgc atcctggtgg ccggggacag catggacagt 600
gtcaagcaga gtgcggccct gtgcctcctt cgactgtaca aggcctcgcc tgacctggtg 660
cccatgggcg agtggacggc gcgtgtggta cacctgctca atgaccagca catgggtgtg 720
gtcacggccg ccgtcagcct catcacctgt ctctgcaaga agaacccaga tgacttcaag 780
acgtgcgtct ctctggctgt gtcgcgcctg agccggatcg tctcctctga ctccaccgaa 840
ctccaggact acacctacta cttcgtccca gcaccctggc tctcggtgaa gctcctgcgg 900
ctgctgcagt gctacccgcc tccagaggat gcggctgtaa aggggcggct ggtggaatgt 960
ctggagactg tgctcaacaa ggcccaggag ccccccaaat ccaagaaggt gcagcattcc 1020
aacgccaaga acgccatcct cttcgagacc atcagcctca tcatccacta tgacagtgag 1080
cccaacctcc tggttcgggc ctgcaaccag ctgggccagt tcctgcagca ccgggagacc 1140
aacctgcgct acctggccct ggagagcatg tgcacgctgg ccagctccga gttctcccat 1200
gaagccgtca agacgcacat tgacaccgtc atcaatgccc tcaagacgga gcgggacgtc 1260
agcgtgcggc agcgggcggc tgacctcctc tacgccatgt gtgaccggag caatgccaag 1320
cagatcgtgt cggagatgct gcggtacctg gagacggcag actacgccat ccgcgaggag 1380
atcgtcctga aggtggccat cctggccgag aagtacgccg tggactacag ctggtacgtg 1440
gacaccatcc tcaacctcat ccgcattgcg tgcgactacg tgagtgagga ggtgtggtac 1500
cgtgtgctac agatcgtcac caaccgtgat gacgtccagg gctatgccgc caagaccgtc 1560
tttgaggcgc tccaggcccc tgcctgtcac gagaacatgg tgaaggttgg cggctacatc 1620
cttggggagt ttgggaacct gattgctggg gacccccgct ccagcccccc agtgcagttc 1680
tccctgctcc actccaagtt ccatctgtgc agcgtggcca cgcgggcgct gctgctgtcc 1740
acctacatca agttcatcaa cctcttcccc gagaccaagg ccaccatcca gggcgtcctg 1800
cgggccggct cccagctgcg caatgctgac gtggagctgc agcagcgagc cgtggagtac 1860
ctcaccctca gctcagtggc cagcaccgac gtcctggcca cggtgctgga ggagatgccg 1920
cccttccccg agcgcgagtc gtccatcctg gccaagctga aacgcaagaa ggggccaggg 1980
gccggcagcg ccctggacga tggccggagg gaccccagca gcaacgacat caacgggggc 2040
atggagccca cccccagcac tgtgtcgacg ccctcgccct ccgccgacct cctggggctg 2100
cgggcagccc ctcccccggc agcacccccg gcttctgcag gagcagggaa ccttctggtg 2160
gacgtcttcg atggcccggc cgcccagccc agcctggggc ccacccccga ggaggccttc 2220
ctcagcccag gtcctgagga catcggccct cccattccgg aagccgatga gttgctgaat 2280
aagtttgtgt gtaagaacaa cggggtcctg ttcgagaacc agctgctgca gatcggagtc 2340
aagtcagagt tccgacagaa cctgggccgc atgtatctct tctatggcaa caagacctcg 2400
gtgcagttcc agaatttctc acccactgtg gttcacccgg gagacctcca gactcagctg 2460
gctgtgcaga ccaagcgcgt ggcggcgcag gtggacggcg gcgcgcaggt gcagcaggtg 2520
ctcaatatcg agtgcctgcg ggacttcctg acgcccccgc tgctgtccgt gcgcttccgg 2580
tacggtggcg ccccccaggc cctcaccctg aagctcccag tgaccatcaa caagttcttc 2640
cagcccaccg agatggcggc ccaggatttc ttccagcgct ggaagcagct gagcctccct 2700
caacaggagg cgcagaaaat cttcaaagcc aaccacccca tggacgcaga agttactaag 2760
gccaagcttc tggggtttgg ctctgctctc ctggacaatg tggaccccaa ccctgagaac 2820
ttcgtggggg cggggatcat ccagactaaa gccctgcagg tgggctgtct gcttcggctg 2880
gagcccaatg cccaggccca gatgtaccgg ctgaccctgc gcaccagcaa ggagcccgtc 2940
tcccgtcacc tgtgtgagct gctggcacag cagttctgag ccctggactc tgccccgggg 3000
gatgtggccg gcactgggca gccccttgga ctgaggcagt tttggtggat gggggacctc 3060
cactggtgac agagaagaca ccagggtttg ggggatgcct gggactttcc tccggccttt 3120
tgtattttta tttttgttca tctgctgctg tttacattct ggggggttag ggggagtccc 3180
cctccctccc tttccccccc aagcacagag gggagagggg ccagggaagt ggatgtctcc 3240
tcccctccca ccccaccctg ttgtagcccc tcctaccccc tccccatcca ggggctgtgt 3300
attattgtga gcgaataaac agagagacgc taaaaaaaaa aaa 3343
<210> 28
<211> 1639
<212> DNA
<213> Homo sapiens
<220>
<221> misc_~eature
<223> Incyte ID No: 2355124CB1
<400> 28
cgccagtggt ccaggagccg cttttttcca ctcgggaaga cttcagagaa gtctcacaaa 60
ggactcggct ggctgctttt ctcagtgccg aagccgcgcc atgctcgttc tcagaagcgg 120
cctgaccaag gcgcttgcct cacggacgct cgcgcctcag gtgtgttcat cttttgctac 180
gggccctaga caatacgatg gaacgttcta tgaatttcgt acttattacc ttaaaccttc 24G
25/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
aaatatgaat gcgttcatgg aaaatcttaa gaaaaacatt catcttcgga cctcttactc 300
tgaattggtt ggattctgga gtgtagaatt tggaggcaga acgaataaag tgtttcatat 360
ttggaagtat gataattttg ctcatcgaac tgaagttcag aaagccttgg ccaaagataa 420
ggaatggcaa gaacaattcc tcattccaaa tttggctctc attgataaac aagagagtga 480
gattacttat ctggtaccat ggtgcaaatt agaaaaacct ccaaaagaag gagtctatga 540
actggccact tttcagatga aacctggtgg gccagctctg tggggtgatg catttaaaag 600
ggcagttcat gctcatgtca atctaggcta cacaaaacta gttggagtgt tccacacaga 660
gtacggagca ctcaacagag ttcatgttct ttggtggaat gagagtgcag atagtcgtgc 720
agctgggaga cataagtccc atgaggatcc cagagttgtg gcagctgttc gggaaagtgt 780
caactaccta gtatctcagc agaatatgct tctgattcct acatcgtttt caccactgaa 840
atagttttct actgaaatac aaaacatttc attaactgct ataggatctg tctgctaatg 900
gtgcttaaat tctcccaaga ggttctcact tttatttgaa ggaggtgtta agttaatttg 960
ctatgtttct tgcattatga aggctacatc tgtgctttgt aagtaccact tcaaaaaata 1020
gttctgttta ctttctgcat ggtatttcag tgtctgtcat acattaaaaa tacttgtcac 1080
tgttttaaga tcttgactct tcatttgttt cagaatagct cttctactgt attctgacaa 1140
ctctttgctt tatagcattt tgttgtattc aaatgataat ggtagcattt ccatgcttgt 1200
gacagcattt ttaagttatt aatatatttt atcaaccttt ccatcatgtc tgttttcctg 1260
gttttttttg gttgtttttt gaccagtaaa atttattttg taataccaaa taggatttaa 1320
gaaaattaac gtatttcttt actatggaaa accacattgt catttgtgac atcatctata 1380
ttaaatatgg ttttcacatt agttatttgt cacttacttg gaaaatgatg ctgttaggtc 1440
ctggtattaa aaatctagaa aagacttgtt ggtttatgtg ctgaaatgtc tttatttata 1500
attaatttta actactattt acttcatttc ggatcctgtt taacaaagat acttgagaca 1560
tccatttgtt ttaatgaaat ctgtatggat atggaaatgc ttgccctaat aaaagcctac 1620
atataaaaaa aaaaaaaaa 1639
<210> 29
<211> 2074
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2366939CB1
<400> 29
ccccgcctgc cccaactcca gacatcatgt gctccccgtt tcggggatcc agaagcttcc 60
agccagagga ctgccaggaa cgtcctgcca ccgccaccgg ctcccatggc ccacataccc 120
agctgggggt gccccagcag cgggggcagc ccccatgggc ccccagtatt gcgtgtgcaa 180
ggtggagctg tcagtgagtg gccagaacct actggaccgg gatgttacct ccaagtccga 240
ccccttctgt gtcctcttta cagagaacaa tggcagatgg atcgagtacg acaggacaga 300
aaccgcgatc aacaacctca accccgcctt ctccaagaag ttcgtgcttg actaccactt 360
cgaggaggta cagaagctca agttcgcgct ctttgaccag gacaagtcca gtatgcggct 420
ggacgagcat gacttcctgg gccagttctc ctgcagcctg ggcacgatcg tctccagcaa 480
gaagatcact aggcctctgc tgctgctgaa tgacaagcct gcggggaagg gcttgattac 540
gatcgctgcc caggagctgt ccaacaaccg cgtcatcaca ctaagcctgg cgggcaggag 600
gctggacaag aaggacctct ttgggaagtc agaccccttt ctggagtttt ataagccagg 660
agacgatggc aagtggatgc tggtccacag gactgaggtg atcaagtaca cactggaccc 720
tgtgtggaag ccattcacag tgcccttggt gtccctgtgt gatggggaca tggagaagcc 780
catccaggtc atgtgctacg actatgacaa tgacgggggc catgacttca tcggcgagtt 840
ccagacctca gtgtcacaga tgtgtgaggc tcgagacagc gtcccgctgg agttcgagtg 900
catcaacccc aagaagcaga ggaagaagaa gaactataaa aactcgggca tcatcatcct 960
gcgatcctgc aagataaacc gagactactc cttccttgac tacatcctgg gaggctgcca 1020
gctcatgttc accgttggaa tagactttac agcctccaac gggaatcccc tcgacccttc 1080
ctctttgcac tatatcaacc ctatgggcac caacgaatat ctgtcggcca tctgggctgt 1140
tgggcagatc attcaggact acgacagtga taagatgttt ccagctctgg gattcggggc 1200
ccagttaccc ccagactgga aggtctccca tgagtttgcc atcaacttca accccaccaa 1260
ccccttctgc tcaggtgtgg atggtattgc ccaggcgtac tcagcttgcc tgccccacat 1320
ccgcttctac ggtcctacca atttctcccc catcgtcaac cacgtggccc ggtttgcggc 1380
ccaggccaca caacagcgga cggccacgca gtacttcatc ctcctcatca tcacggacgg 1440
ggtcatcagt gacatggagg agacacggca tgccgtggtg caggcttcca agctgcccat 1500
gtccatcatc atcgtgggcg tgggcaatgc ggacttcgct gccatggagt tcctggatgg 1560
ggacagccgc atgctgcgct cccacacggg ggaggaggca gcccgcgata ttgtgcagtt 1620
cgttcccttt cgagagttcc gcaacgcagc aaaagagacc ttggccaaag ctgtgctggc 1680
ggagctgccc caacaagttg tgcagtattt caagcataaa aacctgcccc ccaccaactc 1740
ggagcccgcc tgagctccag tgcccagcag cagcatgtca gctgagcctc ctgccctccc 1800
ccaggaacat gcacgctcac tctgcttcct tgtgggtggc ctttttttac cgatcccctt 1860
26/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
ttttattttt tacaaccgga cctccacccc caacttcctc cagcccagct gggcttcctt 1920
tgttggagtc aactgttgat gcttccaggc caaactggct tcctctcctc ctctccccac 1980
ctttgccatt cttaagtatt gaatgtactt tgtataattt tagtggaatt gttattgaga 2040
ataaaatttt tacaatcata aaaaaaaaaa aaaa 2074
<210> 30
<211> 611
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2483906CB1
<400> 30
atggtctaag ccgggcgcct cacctgtcag ccgcaccggc tccagcgctc gcctctcgcc 60
ctcgcttctc cagcgctcct tgctcgcaag gcgggggagg cggcggccca gccacgatga 120
tacatttcat attgctcttc agtcgacaag ggaaattacg gctacagaaa tggtacatca 180
ctctccctga taaagagagg aagaagatca cccgggaaat tgttcagatt attctctccc 240
gtggtcacag gacaagcagt tttgttgact ggaaggagct aaaacttgtt tataaaaggt 300
atgctagttt atatttttgc tgtgcaatag aaaatcagga caatgagctc ttgacgctag 360
agattgtgca tcgttacgtg gagctgctgg acaaatattt tggaaatgtc tgtgagctgg 420
atattatctt taattttgaa aaggcttatt tcatcctgga cgagtttata ataggtgggg 480
aaattcagga aacatccaag aaaattgctg tcaaagccat tgaagactct gatatgttac 540
aggagacaat ggaagaatac atgaacaagc ctacatttta actggaaatc tacttgaaga 600
ctccagcact a 611
<210> 31
<211> 2871
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2499488CB1
<400> 31
cactgggtca aagcaccctg tattattcct tgttaccatt attgctccct gaagagggct 60
ctcatgtatc tatgtaggat aatccaacct caacacagcg gctgaaagaa tctgctaaaa 120
aataattcag tgccccaaaa tacactgggc tccaaataaa ctaaagagca aaaattggct 180
catgtggcat gaaacaagta atcgtatccc aagtaagagg acagtttcct tcgtgttatc 240
aacaggactg taccccataa gtttccaggt acatttggta agagatggtg ttttaacacg 300
attttctgtt gcaacagagt atcagaaaaa tgcacagatt gagaagctcc ggagtgagat 360
cgtcgtattg aaggaagagc tgcagctcac caggtctgag ctagaggctg cacaccatgc 420
cagtgcagtc agattctcca aggaatatga aatgcagaaa acaaaagagg aagacttttt 480
gaagttattt gacaggtgga aagaagaaga aaaggagaaa ctagttgatg aaatggaaaa 540
agtcaaggag atgtttatga aggaatttaa agaattaact tcgaagaatt cagcattaga 600
atatcaactg tcagaaatcc agaagtccaa tatgcagatc aagtccaaca taggcacatt 660
aaaagatgca cacgagttta aagaagaccg ttctccatat ccccaggatt tccataatgt 720
catgcagctt cttgatagtc aggaaagcaa atggacagct cgagttcaag ctattcatca 780
agaacacaag aaagagaagg gtcggctcct gtcacatata gagaaacttc gaacctcaat 840
gatagatgat ctaaatgcaa gcaatgtttt ctataagaaa aggatagaag agctagggca 900
gagactccag gagcagaatg agctgattat aactcagaga cagcagatta aagactttac 960
ctgtaatcca ttaaacagta tcagtgaacc caaaggaaat cctttagcct ggcaggcttt 1020
tgaatctcag ccagctgctc cagctgtgcc tatgaatgcc ccagccctgc acactttgga 1080
aactaaatca agtctgccaa tggtgcatga acaggcattc tcgtcgcaca tactggaacc 1140
aatagaagaa ctttcagagg aagaaaaagg aagggaaaat gaacagaaat taaataacaa 1200
caaaatgcat ttaaggaaag ctttgaagag taactcctcc ctcactaagg gactaagaac 1260
aatggtggag cagaacttga tggagaaact ggaaaccttg gggattaatg cagatatacg 1320
tggcatttca agtgatcagt tgcatagagt actaaaaagt gtggaatcag aaagacataa 1380
gcaagaaaga gaaataccta actttcatca aattcgagaa ttccttgaac atcaagtcag 1440
ctgtaaaatt gaggagaaag cactactctc ttcagatcag tgcagtgttt ctcaaatgga 1500
taccctttca actggagaag tacccaaaat gatacaactt ccttccaaaa acagacaact 1560
gattagacaa aaagctgttt ctactgatag gacatctgtt ccaaaaatta agaaaaatgt 1620
catggaagat ccttttccca gaaagtcttc aactattacg acccctcctt ttagttcaga 1680
27/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
ggaggagcag gaggacgacg acctcatccg ggcatacgca tccccaggcc cacttcctgt 1740
gccgccacca caaaacaagg gcagcttcgg gaagaacaca gtgaaaagtg acgcggacgg 1800
gaccgaggga agcgaaatcg aggacactga tgattctccc aagcccgcag gagtcgccgt 1860
taaaacacct actgaaaaag ttgaaaagat gtttccacat cgcaaaaatg tgaacaaacc 1920
agtcggtgga actaatgtcc ctgagatgtt tatcaaaaaa gaagaattac aagaactaaa 1980
gtgtgcggat gtggaggatg aagactggga catatcatcc ctagaggaag agatatcttt 2040
gggaaaaaaa tctgggaaag aacagaagga acctccacct gcgaaaaatg aaccacattt 2100
tgctcatgtg ctaaatgcct ggggcgcatt taatcctaag gggccaaagg gagaaggact 2160
tcaagaaaat gaatcaagca cattaaaaag cagcttagta actgtgactg attggagcga 2220
cacttcagat gtctaattcc acatgtcaga agattattcc agaagccagc agtatttcag 2280
tatcacagtg tttcagtaat ttgcctccat gattctagtg cttctgcctt accgtgtttc 2340
ccacagcaac acagagactg attcaaagaa caatggtctc tttaatggca cccaatacag 2400
tattgaaaat cagatcatca acagtatttc gaagcatgta aaggtgttta agacttccgc 2460
tgctgcttaa aaataacatg tcattgaagt cataaaaagt tttttcttca gaaaggtact 2520
ctagtgttaa gtgtattttt ttcaactaat tttttagtga atttttttta aacttacagc 2580
atgttttggt ttgaattact aaaactttaa aaaatatttt tcttatgtat gctgtcgtat 2640
cgtaggcgtt tatattataa aattctgtta gtagtcttaa aattgaattg gtggaaccac 2700
taatccttaa aagttagtct ggttattttt catatagaag taagtttaat ccgagtgtgg 2760
tggtgttcac ctttaatccc agctacttgg gaggctgagg tgggaggata acttgagcac 2820
aggagttcaa gaccagcgtg ggcaatatag caagactcca cccctccaca c 2871
<210> 32
<211> 2123
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2559148CB1
<400> 32
cggggccgag atgcggtgac ctgccagcac ctgccgcagc cttcgtccgg gagtcgcccc 60
atctctccac gcatcggggc cctgtgcccc ttgctgctgc agccgggcac catgtcgacc 120
tcgtccttga ggcgccagat gaagaacatc gtccacaact actcagaggc ggagatcaag 180
gttcgagagg ccacgagcaa tgacccctgg ggcccatcca gctccctcat gtcagagatt 240
gccgacctca cctacaacgt tgtcgccttc tcggagatca tgagcatgat ctggaagcgg 300
ctcaatgacc atggcaagaa ctggcgtcac gtttacaagg ccatgacgct gatggagtac 360
ctcatcaaga ccggctcgga gcgcgtgtcg cagcagtgca aggagaacat gtacgccgtg 420
cagacgctga aggacttcca gtacgtggac cgcgacggca aggaccaggg cgtgaacgtg 480
cgtgagaaag ctaagcagct ggtggccctg ctgcgcgacg aggaccggct gcgggaagag 540
cgggcgcacg cgctcaagac caaggaaaag ctggcacaga ccgccacggc ctcatcagca 600
gctgtgggct caggcccccc tcccgaggcg gagcaggcgt ggccgcagag cagcggggag 660
gaggagctgc agctccagct ggccctggcc atgagcaagg aggaggccga ccagcccccg 720
tcctgcggcc ccgaggacga cgcccagctc cagctggccc ttagtttgag ccgagaagag 780
catgataagg aggagcggat ccgtcgcggg gatgacctgc ggctgcagat ggcaatcgag 840
gagagcaaga gggagactgg gggcaaggag gagtcgtccc tcatggacct tgctgacgtc 900
ttcacggccc cagctcctgc cccgaccaca gacccctggg ggggcccagc acccatggct 960
gctgccgtcc ccacggctgc ccccacctcg gacccctggg gcggcccccc tgtccctcca 1020
gctgctgatc cctggggagg tccagccccc acgccggcct ctggggaccc ctggaggcct 1080
gctgcccctg caggaccctc agttgaccct tggggtggga ccccagcccc tgcagctggg 1140
gaggggccca cgcctgatcc atggggaagt tccgatggtg gggtcccggt cagtgggccc 1200
tcagcctccg atccctggac accggccccg gccttctcag atccctgggg agggtcacct 1260
gccaagccca gcaccaatgg cacaacagca gccgggggat tcgacacgga gcccgacgag 1320
ttctctgact ttgaccgact ccgcacggca ctgccgacct ccgggagcag cgcaggagag 1380
ctggagctgc tggcaggaga ggtgccggcc cgaagccctg gggcgtttga catgagtggg 1440
gtcaggggat ctctggctga ggctgtgggc agccccccac ctgcagccac accaactccc 1500
acgcccccca cccggaagac gccggagtca ttcctggggc ccaatgcagc cctcgtcgac 1560
ctggactcgc tggtgagccg gccgggcccc acgccgcctg gagccaaggc ctccaacccc 1620
ttcctgccag gcggaggccc agccactggc ccttccgtca ccaacccctt ccagcccgcg 1680
cctcccgcga cgctcaccct gaaccagctc cgtctcagtc ctgtgcctcc cgtccctgga 1740
gcgccaccca cgtacatctc tccccttggc gggggccctg gcctgccccc catgatgccc 1800
ccgggccccc cggcccccaa cactaatccc ttcctcctat aatccagggc ggaagggggc 1860
ctggctccat ccggctgccc cattccggct ccctgggaga tcagtgttgt gagtgcatgt 1920
gaaatggggg atccccaccc ccagtgccct tccccttcct ggggcccact cacactacac 1980
cctcttcctt tcccacccca cctccccgga gagaaactgg acatggggcc tggggagggg 2040
agctggccag aggaggaccc ctttcccgtg gcattagaag ggggaggggt ggctggggcc 2100
28/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
ctcacccatt ccccctccct ccc 2123
<210> 33
<211> 1973
<212> DNA
<213> Homc Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3321481CB1
<400> 33
agccgacacc gccggtcgcc acgctggagt gcagtggtgc gagctcggct cactgaaacc 60
tccttctctt gggttcaagt gatttgtgtg cctcagcctc tggagtagct gggatttcag 120
ccatcttcaa gttggccgac agcgctgctc acctggccgc ctccccttta agacgcacac 180
acctgggctc tcacctgggc gcaggcgctt ccgcagaaga aggaagcggc gccgccatcg 240
cctcccggcg ctccctcccc gactcctaag tccttcggcc gccaccatgt ccgcctcggc 300
tgtcttcatt ctggacgtta agggcaagcc attgatcagc cgcaactaca agggcgatgt 360
ggccatgagc aagattgagc acttcatgcc tttgctggta cagcgggagg aggaaggcgc 420
cctggccccg ctgctgagcc acggccaggt ccacttccta tggatcaaac acagcaacct 480
ctacttggtg gccaccacat cgaagaatgc caatgcctcc ctggtgtact ccttcctgta 540
taagacaata gaggtattct gcgaatactt caaggagctg gaggaggaga gcatccggga 600
caactttgtc atcgtctacg agttgctgga cgagctcatg gactttggct tcccgcagac 660
caccgacagc aagatcctgc aggagtacat cactcagcag agcaacaagc tggagacggg 720
caagtcacgg gtgccaccca ctgtcaccaa cgctgtgtcc tggcgctccg agggtatcaa 780
gtataagaag aacgaggtct tcattgatgt catagagtct gtcaacctgc tggtcaatgc 840
caacggcagc gtccttctga gcgaaatcgt cggtaccatc aagctcaagg tgtttctgtc 900
aggaatgcca gagctgcggc tgggcctcaa tgaccgcgtg ctcttcgagc tcactggcct 960
ttcaggcagc aagaacaaat cagtagagct ggaggatgta aaattccacc agtgcgtgcg 1020
gctctctcgc tttgacaacg accgcaccat ctccttcatc ccgcctgatg gtgactttga 1080
gctcatgtca taccgcctca gcacccaggt caagccactg atctggattg agtctgtcat 1140
tgagaagttc tcccacagcc gcgtggagat catggtcaag gccaaggggc agtttaagaa 1200
acagtcagtg gccaacggtg tggagatatc tgtgcctgta cccagcgatg ccgactcccc 1260
cagattcaag accagtgtgg gcagcgccaa gtatgtgccg gagagaaacg tcgtgatttg 1320
gagtattaag tctttcccgg ggggcaagga gtaettgatg cgagcccact ttggcctccc 1380
cagtgtggaa aaggaagagg tggagggccg gccccccatc ggggtcaagt ttgagatccc 1440
ctacttcacc gtctctggga tccaggtccg atacatgaag atcattgaga aaagtggtta 1500
ccaggccctg ccctgggttc gctacatcac ccagagtggc gattaccaac ttcgtaccag 1560
ctagaaggga gaagagatgg gggcttgaac acggggcttc cttacagccc cggatgcaga 1620
ttttagaggg agggcaggtg cgggctgtgt gtgtctgtgt gagggcaggt cctggacttg 1680
gcagtttctt gctcccagca cccgcccctt cctcacctct tccttattcc ataggctggg 1740
agagaaactc tctctgcttc cctcgccctt ggagctttcc ccatccccct gattttatat 1800
gaagaaatag aagaggggct tgaagtcccc ctcgcgagtg ccttcttgca attacctgcc 1860
ttagcgggtg ttgcgggtcc ctccttcaca gccgctgagc ccagaggtcc cgctggcccc 1920
tcctctgaat tttaggatgt cattaaaaag atgaatctaa aaaaaaaaaa aaa 1973
<210> 34
<211> 1535
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyt2 ID No: 3367918CB1
<400> 34
gagagacgaa aggagggggg gggtggaagg gagaggaggg cgagcaaggg gaggagagga 60
gtgacggggg cgggggggga gtagagaatg gggaagggga ggagggaacg ggaaggcgca 120
gaggtgagga agaagggtgg ggggagggga aggggggagg aggcgcggaa ggagagggag 180
tgaggatgag acgagaagag cgggggtaga ggggagtgaa aggaatagtg ggcaagcagt 240
gagaagaaga gggaagggag ccacgaatgc aaggaggcga agggggggtg tgccagagga 300
aggaaaacgt ggttgaagaa gagaaaacca ggtggggggt gtgttgcagg acagcgaagg 360
ggggggagga gagggggggg aagaagggga tttgtataag gagggggtag acagaggata 420
gataagagag ccggccatag gaccccaggc aatggtaggt ttgggcgatg aggaacaggg 48G
gtcaaggtgg ggggaagaag aattggcaga ggcgtaggga ataataggcc cgaaggagta 540
29/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
atttcgggta ctggggaggg agagacagga gaatggcttg cgccttggag gcagaggttg 600
cagtgtaccg agatagcgcc actgcactca gtttggggga cagagtgaga acctatttat 660
taaaaagaaa aacaggccag gggggtggct gacatatgta atcccagcac tttgggaggc 720
cgaggcaggt ggatcaccag aggtcgggag ttcaagatca gcctggccaa catggtgaaa 780
cctcattttt attaaaaata caaatgctta gctgggcgtg gtggcagggc gctggtaatc 840
ctgtacttgg gaggctgagg caggagaatc acttgaaccc aggaggcaga ggttgcagtg 900
agcaaaaaaa aaaaaaaaaa aaacttacat cccttctccc caccatcctg ttatcacagg 960
agatctataa ggacaggcag aaattctcag aacaggtttt caaagtggcc tcctcagacc 1020
tggtcaacat gggcatcagt gtggttagct acactctgaa ggacattcac gatgaccagg 1080
actatttgca ctctttgggg aaggctcgaa cagctcaagt ccaaaaagat gcacggattg 1140
gagaagcaga ggccaagaga gatgctggga tccgggaagc taaagccaag caggaaaagg 1200
tgtctgctca gtacctgagt gagatcgaga tggccaaggc acagagagat tacgaactga 1260
agaaggccgc ctatgacatc gaggtcaaca ccccgagcac aggctgacct ggcctatcag 1320
cttcaggctg gagtgcaatg gcgtgatctc agctactgca acctctgcct cctgggttca 1380
agcgattctc ctacttggga ggctgaggca ggagaatcac ttgaacccag gaggcagagg 1440
ttgcagtgag ccaagatcac accactgcac tctagcctgg gcgacagtga gactccacct 1500
caaaaaaaag gaaaaaaaaa aaaaaaaaag tcccg 1535
<210> 35
<211> 2453
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1227327CB1
<400> 35
tgaagagcct agggtggggc tcgcggtgtc gcggagggcg tggaagtgtg tcagggccag 60
cgtgccggcc ctacggaagc cgagcctgag gcgagatctc gccttttgta ttttcactga 120
ctcatatttc ctttactcag gctcccacat caggacgaga aggagccctc gagttaccgt 180
gggagctgtg ggagctgccc tgtgactctt aggaagatgc cgtaccttgg ctccgaggac 240
gtggtgaagg agctgaagaa ggctctgtgc aatcctcaca ttcaagctga taggctgcgc 300
taccggaatg tcatccagcg agtgattagg tacatgactc aaggcttgga catgtctggt 360
gtttttatgg aaatggtgaa ggccagtgcc actgtagata ttgtccagaa gaagttggtt 420
tatctgtaca tgtgcacata tgctcccctg aaaccagatc tggctctcct ggccatcaat 480
acgctgtgca aagactgctc agaccccaat ccaatggtgc gagggctggc gttacggagc 540
atgtgtagcc tcaggatgcc tggtgtgcag gagtatatac aacagcctat tctcaatggt 600
ctgcgggata aggcttcata tgtcaggaga gtggcagtcc ttggatgtgc caagatgcat 660
aatcttcatg gagactctga agtagatggt gccctggtaa atgaattata cagtttgctg 720
cgtgaccagg atccaattgt agttgtgaac tgcttgaggt ctctagagga aattctgaaa 780
caggaaggag gcgttgtcat caataagccc attgctcacc atctcttaaa tcgaatgtca 840
aaactggacc aatggggcca ggctgaagta ttgaactttc tgctacgcta ccaaccccgc 900
agtgaggaag aactatttga cattctcaat ctgttggata gtttcctcaa gagcagtagc 960
ccaggtgtgg tgatgggagc taccaaactt tttctgatct tggcaaaaat gtttccccac 1020
gtacaaactg atgtccttgt gcgggtcaag ggacctttgc tagctgcctg ttcttcagag 1080
agccgtgagc tctgttttgt tgctctttgt catgtacgcc agatcttgca tagtttacca 1140
ggtcacttta gcagccacta caaaaagttt ttttgctcct actcggagcc ccactacatc 1200
aaactacaga aagtggaggt gctgtgtgaa ctggtgaacg atgagaatgt gcagcaggtg 1260
ctagaggagc ttcgagggta ctgcacggat gtgtctgcgg actttgcaca ggctgccatc 1320
tttgccatag gtggcattgc caggacttac acagatcaat gtgttcagat tttaacagag 1380
ttgctgggtc ttcgacaaga gcacattacc acagtggtgg tgcagacttt ccgagacctg 1440
gtttggttgt gtcctcagtg tactgaagct gtatgtcagg ccctgcccgg ctgtgaagag 1500
aacattcaag atagtgaggg gaagcaagca cttatttggc tacttggtgt ccatggggaa 1560
agaattccta atgctcctta tgtgttagag gactttgttg agaatgtgaa gtcggaaaca 1620
tttccagctg ttaagatgga gctgctcact gctttgctgc gccttttcct ctcccgacct 1680
gctgagtgcc aggacatgct aggacgtttg ttgtattact gcatagagga agaaaaagat 1740
atggctgtac gggaccgagg tctcttctat tatcgcctcc tcttagttgg cattgatgaa 1800
gttaagcgga ttctgtgtag ccctaaatct gaccctactc ttggactttt ggaggatccg 1860
gcagaaagac ctgtgaatag ctgggcctca gacttcaaca cactggtgcc agtgtatggc 1920
aaagcccact gggcaactat ctctaaatgc cagggggcag agcgttgtga cccagagctt 1980
cctaaaactt catcctttgc cgcatcagga cccttgattc ctgaagagaa caaggagagg 2040
gtacaagaac tccctgattc tggagccctc atgctagtcc ccaatcgcca gcttactgct 2100
gattattttg agaaaacttg gcttagcctt aaagttgctc atcagcaagt gttgccttgg 2160
cggggagaat tccatcctga caccctccag atggctcttc aagtagtgaa catccagacc 2220
30/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00109353
atcgcaatga gtagggctgg gtctcggcca tggaaagcat acctcagtgc tcaggatgat 2280
actggctgtc tgttcttaac agaactgcta ttggagcctg gaaactcaga aatgcagatc 2340
tctgtgaaac aaaatgaagc aagaacggag acgctgaata gttttatttc tgtattagaa 2400
actgtgattg gaacaattga agaaataaaa tcataacaga gaaaaaaaaa aaa 2453
<210> 36
<211> 2599
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1416292CB1
<400> 36
cgaagttgca ccggttgagg atggctgaca ttctctctca gtcagagacc ctggcgtcgc 60
aagacctcag tggggacttc aagaagccag ctctgccggt gtccccagcg gcgcggagta 120
aggccccggc cagcagttct tcaaaccctg aggaggtaca gaaggaaggg cccactgcgt 180
tgcaggactc caattctggg gagcccgaca tccctcctcc tcagccggac tgcggtgatt 240
ttaggagtct acaggaggag cagtcgcgcc ccacgacagc ggtttcttcc cctggcggtc 300
cagcccgggc tcccccctac caagagcctc catggggtgg ccctgccaca gccccctaca 360
gcttagagac cctgaagggc ggcactatcc ttggcacccg tagcttgaaa gggacgagtt 420
actgcctttt cgggaggctg tctggctgcg acgtgtgcct ggagcaccct tcggtgtctc 480
ggtaccacgc agtgctgcag cacagggcgt ccggccctga cggagaatgc gacagcaacg 540
ggccgggctt ctacctctac gatctgggaa gcacccatgg cacttttctc aacaaaactc 600
gcatcccacc tcgcacctac tgtcgagtcc acgttgggca tgttgttcgc tttggaggca 660
gcacccggct ctttatcctg cagggaccag aggaagaccg agaggcagaa tccgagttaa 720
cagtaacaca gttgaaggaa ttgcgcaagc agcagcaaat attgttggag aagaagatgc 780
taggagaaga ctcagatgaa gaagaggaaa tggatacctc tgaaaggaag ataaatgctg 840
gtagccaaga tgatgagatg ggttgcacct ggggaatggg agaagatgca gtagaggatg 900
atgctgaaga gaaccctatt gtcttagagt ttcagcagga aagggaggcc ttttatataa 960
aggatcccaa aaaggctctc caaggctttt ttgaccgaga aggagaagaa ttagaatatg 1020
aatttgatga acagggacat agcacttggc tctgcagggt gagattacct gtggacgatt 1080
caactggaaa acaactggtg gctgaggcca ttcactcagg aaagaaaaaa gaagcaatga 1140
tccagtgctc attggaagct tgtcggattc ttgacacttt gggattgctt cggcaggaag 1200
cagtatctcg gaaaaggaaa gccaagaact gggaagatga agacttttat gatagtgatg 1260
atgacacatt tcttgatagg actggcctga ttgagaagaa gcgtctgaac agaatgaaga 1320
aggctggcaa gattgatgag aagccagaga cctttgaatc attggttgca aaattaaatg 1380
atgctgaaag ggaactttct gaaatttctg agagattgaa agcctcaagc caagttctat 1440
cagagtctcc atctcaggat tctttagatg cgttcatgtc agaaatgaaa tcaggcagta 1500
cattagatgg tgtgtcccgg aagaaacttc acctgagaac ttttgaactg aggaaagaac 1560
aacagagact taaagggtta ataaaaattg taaagccagc agagattcca gaactaaaaa 1620
agactgaaac tcagactaca ggtgcagaaa acaaagctaa aaagcttaca ttgcctctat 1680
ttggtgccat gaaaggagga agcaaattca aattaaaaac tggaacagta gggaagttac 1740
cccccaagcg tccagaactc cctccaactc taatgagaat gaaagatgag cctgaagtag 1800
aagaggagga ggaagaggaa gaggaagaag agaaagaaaa ggaggagcat gaaaagaaaa 1860
aactggagga tggaagcctc agtaggccac agccagagat agagccagaa gcagcagtgc 1920
aggaaatgag gcctcccaca gatctcacac attttaaaga aacccaaacc catgaaaaca 1980
tgtctcaact tagcgaggaa gaacagaata aagattatca agactgtagc aaaaccactt 2040
cattgtgcgc aggaccctca gcatcaaaga atgaatatga gaaaagcaga ggtgaattga 2100
agaaaaagaa aacacctggt ccaggcaaac ttccaccaac actttcttcc aaatatcctg 2160
aagatgaccc agactactgt gtgtgggtcc cacctgaagg tcaaagtgga gatggcagaa 2220
cccatcttaa tgacaagtat ggctattgat tgcttcagaa tcccaaaaga aaaccttgtg 2280
gaccatgtga catggaatat ttgggataat gtatcaaatt gaatggccag agaagtttag 2340
atgattattt gtaagatctg gtgactggct tttcgttctg tgttcttggc ttcctaaatt 2400
tatctgccca tatgattctc atgcatttga tatttatgtt taaaagtgtt tatatatgta 2460
tgtaaaaagg gaaccatatg ttttgagaat ttgtaaagtg agagacatga tcctattaaa 2520
ataagaaggc aaaaatgttc ctaatatttt attttatttt atttttttta agagacaggg 2580
tcttactctg tcacctagg 2599
<210> 37
<211> 1294
<212> DNA
<213> Homo sapiens
31/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
<220>
<221> misc_reature
<223> Incyte ID No: 2611704CB1
<400> 37
ttcctatcca cagtcacttc tgttcatttt taatgaaaac ctattgctgt tattttctat 60
tatgagttaa cggtgatgtg tgttttttaa attttgtata cttgctctca ccctaaaata 120
taccttaaga gacttttccc ctgtttttct ttctgctttc caagactcca ggaaaaacag 180
cttccatggc acattttgta cagggcacat ctagaatgat tgccgcagaa agttctacgg 240
agcataaaga gtgtgctgaa ccatcaacaa gaaagaactt gatgaattct cttgaacaaa 300
agataaggtg tttggaaaaa caaagaaaag agctcctgga agttaaccag caatgggatc 360
agcaatttag aagtatgaaa gagttatatg aaagaaaggt agcagagctg aagacgaaac 420
tggacgccgc ggaaagattc ctcagcacgc gggagaagga tccgcatcag aggcagagaa 480
aggacgacag gcaaagagag gacgacaggc agcgcgacct gacccgggac cggctgcagc 540
gggaggagaa ggaaaaggaa cgcctaaatg aagaattaca tgaattgaaa gaagagaata 600
aacttttaaa gggaaaaaat actcttgcga acaaggaaaa ggaacattac gaatgtgaaa 660
taaaacgcct caataaggct cttcaggatg ccttgaatat caagtgttca ttttccgagg 720
actgtttgag gaagtctcga gtggaattct gccatgagga gatgagaaca gaaatggaag 780
ttctgaagca gcaggtgcaa atatacgaag aagacttcaa aaaggaacga tcggatcgag 840
agagacttaa tcaagagaaa gaggagctac agcaaattaa tgaaacttcc caatcccagt 900
tgaacaggct gaattcccag ataaaagctt gtcagatgga gaaagaaaaa ctagaaaagc 960
aattaaaaca gatgtattgc ccaccctgta actgcggctt ggttttccac ctgcaagatc 1020
catgggtacc aacaggccct ggagctgtgc agaagcaacg ggagcaccca ccagactatc 1080
agtggtatgc tcttgaccag cttccgccag atgtacaaca caaggcaaat ggtttatcct 1140
cagtaaagaa agtccatccg tagaagtaca cacacacaca catatatagt agtatatgta 1200
taatagaata tatattctaa aatgtagaat gggatgaaaa ctgacctgtc aaaattttgt 1260
aatcagagtc ccagaaccga aagagaaaat gate 1294
<210> 38
<211> 2313
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5136672CB1
<400> 38
gactacgtag ccggcgcctt cctcttccca tcgcgcgggt cctagccacc ggtgtctcct 60
tctacatccg cctctgcgcc ggctgccacc cgcgctccct ccgccgccgc cgccttgctg 120
ctgctcaaag ctgctgccgc cccttgggct aaaaggtttt caaatggaac attttgatgc 180
atcacttagt acctatttca aggcattgct aggccctcga gatactagag taaaaggatg 240
gtttcttctg gacaattata tacccacatt tatctgctct gtcatatatt tactaattgt 300
atggctggga ccaaaataca tgaggaataa acagccattc tcttgccggg ggattttagt 360
ggtgtataac cttggactca cactgctgtc tctgtatatg ttctgtgagt tagtaacagg 420
agtatgggaa ggcaaataca acttcttctg tcagggcaca cgcaccgcag gagaatcaga 480
tatgaagatt atccgtgtcc tctggtggta ctacttctcc aaactcatag aatttatgga 540
cactttcttc ttcatcctgc gcaagaacaa ccaccagatc acggtcctgc acgtctacca 600
ccatgcctcg atgctgaaca tctggtggtt tgtgatgaac tgggtcccct gcggccactc 660
ttattttggt gccacactta atagcttcat ccacgtcctc atgtactctt actatggttt 720
gtcgtcagtc ccttccatgc gtccatacct ctggtggaag aagtacatca ctcaggggca 780
gctgcttcag tttgtgctga caatcatcca gaccagctgc ggggtcatct ggccgtgcac 840
attccctctt ggttggttgt atttccagat tggatacatg atttccctga ttgctctctt 900
cacaaacttc tacattcaga cctacaacaa gaaaggggcc tcccgaagga aagaccacct 960
gaaggaccac cagaatgggt ccatggctgc tgtgaatgga cacaccaaca gcttttcacc 1020
cctggaaaac aatgtgaagc caaggaagct gcggaaggat tgaagtcaaa gaattgaaac 1080
cctccaaacc acgtcatctg attgtaagca caatatgagt tgtgccccaa tgctcgttaa 1140
cagctgctgt aactagtctg gcctacaata gtgtgattca tgtaggactt ctttcatcaa 1200
ttcaaaaccc ctagaaaacg tatacagatt atataagtag ggataagatt tctaacattt 1260
ctgggctctc tgacccctgc gctagactgt ggaaagggag tattattata gtatacaaca 1320
ctgctgttgc cttattagtt ataacatgat aggtgctgaa ttgtgattca caatttaaaa 1380
acactgtaat ccaaactttt ttttttaact gtagatcatg catgtgattg taaatgtaaa 1440
tttgtacaat gttgttatgg tagagaaaca cacatgcctt aaaatttaaa aagcagggcc 1500
caaagcttat tagtttaaat tagggtatgt ttcaagtttg tattaatttg taatagctct 1560
gtttagaaaa aatcaaagac catgatttat gaaactaatg tgacataatt tccagtgact 1620
tgttgatgtg aaatcagaca cggcaccttc agttttgtac tattggcttt gaatcaagca 1680
32/33
CA 02365421 2001-09-26
WO 00/60082 PCT/US00/09353
ggctcaaatc tagtggaaca gtcagtttaa ctttttaaca gatcttattt ttttattttg 1740
agtgccacta ttaatgtaaa aagggggggg ctctacagca gtcgtgatga aacttaaata 1800
tatattcttt gtcctcgaga ttttaggaag ggtgtagggt gagtaggcca tttttaattt 1860
ctgaagtgct aagtgttttt atacagcaaa caaaaagtca attttgcttt ccaccagtgc 1920
gagagaggat gtatactttt caagagagat gattgcctat ttaccgtttg acagagtccc 1980
gtagatgagc aatggggaac tggttgccag ggtctaaatt tggattgatt tatgcactgt 2040
tatctgtttt gacacagatt tccttgtaaa atgtgcctag tttaccaaaa,ttaacaaagg 2100
gggggaaagg accttagaac tttttaaggt aaaatcaaat atagctacag cataagagaa 2160
tcgagaaatt tgatagaggt aacttgttta atgtaaatct aatagtactt gtaatttctt 2220
tctgcttaga atctaaagat gtgtttagaa cctccttgtt aaaataatag actgctattc 2280
ataaatcaca tctcacacat ttggggcagt ggt 2313
33/33
