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HUMAN RECEPTOR-ASSOCIATED PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human
receptor-
associated proteins and to the use of these sequences in the diagnosis,
treatment, and prevention of
cell proliferative, autoimmune/inflammatory, reproductive, cardiovascular, and
gastrointestinal
disorders.
BACKGROUND OF THE INVENTION
The term receptor describes proteins that specifically recognize other
molecules. The
category is broad and includes proteins with a variety of functions. The bulk
of the proteins
termed receptors are cell surface proteins which, when they bind extracellular
ligands, produce
cellular responses in the areas of growth, differentiation, endocytosis, and
immune response.
Other receptors facilitate the specific transport of proteins out of the
endoplasmic reticulum and
localize enzymes to a particular compartment of the cell. The term may also be
applied to proteins
which act as receptors for ligands (with known or unknown chemical
composition) which interact
with other cellular components. For example, the steroid hormone receptors
bind to and regulate
transcription of genomic DNA.
Regulation of cell proliferation, differentiation, and migration is important
for the
formation and function of tissues. Secreted regulatory proteins such as growth
factors
coordinately control these cellular processes and act as mediators in cell-
cell signaling pathways.
Growth factors are secreted from the cell, and bind to specific cell-surface
receptors on target
cells. The bound receptors trigger intracellular signal transduction pathways
which activate
various downstream effectors. Such processes regulate many cell functions
including cell
proliferation, differentiation, gene transcription, cell motility, and
oncogenic transformation.
Cell. surface receptors are typically integral membrane proteins of the plasma
membrane.
These receptors recognize hormones including the catecholamines, such as
epinephrine,
norepinephrine, and histamine; peptide hormones such as glucagon, insulin,
gastrin, secretin,
cholecystokinin, adrenocorticotropic hormone, follicle stimulating hormone,
luteinizing hormone,
thyroid stimulating hormone, parathyroid hormone, and vasopressin; growth and
differentiation
factors such as epidermal growth factor, fibroblast growth factor,
transforming growth factor,
insulin-like growth factor, platelet-derived growth factor, nerve growth
factor, colony-stimulating
factors, and erythropoietin; cytokines such as chemokines, interleukins,
interferons, and tumor
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necrosis factor; small peptide factors such as bombesin, oxytocin, endothelin,
angiotensin II,
vasoactive intestinal peptide, and bradykinin; neurotransmitters such as
neuropeptide Y,
neurotensin, neuromedin N, melanocortins, somatostatin, galanin, tachykinins;
opioids such as
enkephalins, endorphins and dynorphins; and circulatory system-borne signaling
molecules such
as angiotensin, complement, calcitonin, endothelins, and formyl-methionyl
peptides. Cell surface
receptors on immune system cells recognize antigens, antibodies, and major
histocompatibility
complex (MHC)-bound peptide. Other cell surface receptors bind ligands to be
internalized by the
cell. This receptor-mediated endocytosis functions in the uptake of low
density lipoproteins
(LDL), transfenrin, glucose- or mannose-terminal glycoproteins, galactose-
terminal glycoproteins,
immunoglobulins, phosphovitellogenins, fibrin, proteinase-inhibitor complexes,
plasminogen
activators, and thrombospondin (Lodish, H. et al. (1995) Molecular Cell
Biolo~v, Scientific
American Books, New York, NY, p. ?23; Mikhailenko, I. et al. (1997) J. Biol.
Chem. 272:6784-
6791 ).
Tumor necrosis factor (TNF) is a pleiotropic cytokine that mediates immune
regulation
and inflammatory responses. The cellular responses triggered by TNF are
initiated through its
interaction with two distinct cell surface receptors, TNF-R1 and TNF-R2
(Tartaglia, L.A. and
Goeddel, D.V. (1992) Immunol. Today 13:151-153). Both TNF receptors are part
of the TNF
receptor (TNFR) superfamily, whose members include the Fas antigen, the p75
subunit of the
NGF receptor, the TRAIL receptor, TRUNND, Sa1F19R, CD27, CD30, and CD40.
Members of
the TNFR superfamily share the TNFR/NGFR family cysteine-rich region
signature, which
consists of cysteine-rich pseudo-repeats in the extracellular domains (ExPASy
PROSITE
document PDOC00561; Pan, G. et al. (1998) FEBS Lett 424:41-45; Bairoch, A. et
al. (1997) Nucl.
Acids. Res. 25:217-221; and Smith, C.A. et al. (1994) Cell 76:959-962).
Many growth factor receptors, including epidermal growth factor, platelet-
derived growth
factor, and fibroblast growth factor, contain intrinsic protein kinase
activities. When the
polypeptide growth factor binds to the receptor, it triggers the
autophosphorylation of a tyrosine
residue on the receptor. It is believed that these phosphorylated sites are
recognition sites for the
binding of other cytoplasmic signaling proteins in the signaling pathway that
eventually links the
initial receptor activation at the cell surface to the activation of a
specific intracellular target
molecule. These signaling proteins contain a common domain referred to as a
SRC homology 2
(SH2) domain. SH2 domains are found in a variety of signaling molecules and
oncogenic proteins
such as phospholipase C-y, Ras GTPase activating protein, and pp60''sxc
(Lowenstein, E.J. et al.
(1992) Cell 70:431-42).
G protein coupled receptors (GPCR) are integral membrane proteins containing
seven
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transmembrane regions, an extracellular N-terminus that binds ligand, and a
cytoplasmic C-
terminus that interacts with G proteins (Strosberg, A.D. (1991) Eur. J.
Biochem. 196:1-10). The
seven hydrophobic transmembrane domains form a bundle of antiparallel alpha
helices which
account for structural and functional features of the receptor. In most cases,
the bundle of helices
forms a binding pocket; however, when the binding site must accommodate bulky
molecules, the
extracellular N-terminal segment or one or more of the three extracellular
loops participate in
ligand binding which induces a conformational change in intracellular portions
of the receptor.
The activated receptor interacts with an intracellular heterotrimeric G-
protein complex which
triggers further intracellular signaling activities. These activities include
interactions with guanine
nucleotide binding (G) proteins and the production of second messengers such
as cyclic AMP
(cAMP), phospholipase C, inositol triphosphate, or interactions with ion
channel proteins
(Baldwin, J.M. (1994) Curr. Opin. Cell Biol. 6:180-190).
The amino-terminus of the GPCR is extracellular, of variable length, and often
glycosylated; the carboxy-terminus is cytoplasmic and generally
phosphorylated. Extracellular
IS loops of the GPCR alternate with intracellular loops and link the
transmembrane domains. The
most conserved domains of GPCRs are the transmembrane domains and the first
two cytoplasmic
loops. GPCRs range in size from under 400 to over 1000 amino acids (Coughlin,
S.R. ( 1994)
Curr. Opin. Cell Biol. 6:191-197).
GPCRs respond to a diverse array of ligands including lipid analogs, amino
acids and their
derivatives, peptides, cytokines, and specialized stimuli such as light,
taste, and odor. GPCRs
function in physiological processes including vision (the rhodopsins), smell
(the olfactory
receptors), neurotransmission (muscarinic acetylcholine, dopamine, and
adrenergic receptors),
and hormonal response (luteinizing hormone and thyroid-stimulating hormone
receptors).
GPCR mutations, which may cause loss of function or constitutive activation,
have been
associated with numerous human diseases (Coughlin, supra). For instance,
retinitis pigmentosa
may arise from mutations in the rhodopsin gene. Parma, J. et al. ( 1993,
Nature 365:649-65 I )
report that somatic activating mutations in the thyrotropin receptor cause
hyperfunctioning thyroid
adenomas and suggest that certain G-protein-coupled receptors susceptible to
constitutive
activation may behave as proto-oncogenes.
The frizzled cell surface receptor, originally identified in Drosophila
melanogaster, is
important for proper bristle and hair polarity on the wing, leg, thorax,
abdomen, and eye of the
developing insect (Wang, Y. et al. (1996) J. Biol. Chem. 271:4468-4476). The
frizzled gene
encodes a 587 amino acid protein which contains an N-terminal signal sequence
and seven
putative transmembrane regions. The cysteine-rich N-terminus is probably
extracellular and the
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C-terminus is probably cytosolic. Multiple frizzled gene homologs have been
found in rat, mouse,
and human. The frizzled receptors are not homologous to other seven-
transmembrane-region
receptors and their ligands are still unknown.
T cells play a dual role in the immune system as effectors and regulators,
coupling antigen
recognition with the transmission of signals that induce cell death in
infected cells and stimulate
proliferation of other immune cells. Although a population of T cells can
recognize a wide range
of different antigens, an individual T cell can only recognize a single
antigen and only when it is
presented to the T cell receptor (TCR) as a peptide complexed with a major
histocompatibility
molecule (MHC) on the surface of an antigen presenting cell. The TCR on most T
cells consists
of immunoglobulin-like integral membrane glycoproteins containing two
polypeptide subunits, a
and ~, of similar molecular weight. The TCR ~ subunit has an extracellular
domain containing
both variable and constant regions, a transmembrane domain that traverses the
membrane once,
and a short intracellular domain (Saito, H. et al. ( 1984) Nature 309:757-
762). The genes for the
TCR subunits are constructed through somatic rearrangement of different gene
segments.
Interaction of antigen in the proper MHC context with the TCR initiates
signaling cascades that
induce the proliferation, maturation, and function of cellular components of
the immune system
(Weiss, A. (1991) Annu. Rev. Genet. 25: 487-510). Rearrangements in TCR genes
and alterations
in TCR expression have been noted in lymphomas, leukemias, autoimmune
disorders, and
immunodeficiency disorders (Aisenberg, A.C. et al. (1985) N. Engl. J. Med.
313:529-533; Weiss,
su ra .
The immunoglobulin E (IgE) receptor is another receptor important in
regulating the
immune response. The IgE receptor is responsible for initiating the allergic
response which begins
with the binding of an allergen to receptor-bound IgE. This binding leads to
cell activation and the
release of mediators such as histamine that are responsible for the
manifestations of allergy
(Kuester, H. et al. (1992) J. Biol. Chern. 2b7:12782-12787). The high affinity
IgE receptor
(FceRI) is a tetrameric hetero-oligomer composed of an a chain, a p chain, and
two disulfide-
linked y chains. The (3 chain contains four transmembrane segments and long
cytoplasmic
domains thought to play an important role in intracellular signaling.
Visual excitation and the phototransmission of light signals is another form
of signaling
cascade in which receptors play an important role. The process begins in
retinal rod cells with the
absorption of light by the photoreceptor rhodopsin, a seven-transmembrane
containing protein
composed of a 40-kDa protein, opsin, and a chromophore, 11-cis-retinal. The
photoisomerization
of the retinal chromophore initiates a biochemical cascade that leads to a
reduction in cyclic-GMP
and closure of cyclic-GMP regulated, Ca2+-specific channels in the plasma
membrane of the rod.
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The resultant membrane hyperpolarization generates a nerve signal. Recovery of
the dark state of
the rod involves another receptor, recoverin. Recoverin is a Ca2'-binding
protein that detects the
lowering of cytosolic Ca2+ and subsequently binds to and activates guanylate
cyclase. Activation
of guanylate cyclase leads to increased cyclic-GMP levels and the reopening of
Caz+-specific
channels (Stryer, L. (1991) J. Biol. Chem. 266:1071 I-10714).
Abnormal receptor activity is associated with a variety of diseases and
disorders.
Abnormal hormonal secretion is linked to disorders including diabetes
insipidus, hyper- and
hypoglycemia, Grave's disease and goiter, and Cushing's and Addison's
diseases. 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 secreted from
IS intestinal tumors. Tumors may exhibit ectopic synthesis and secretion of
biologically active
peptides, including 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.
Inflammation is a molecular, cellular, and tissue program during which foreign
substances
and pathogens are destroyed, and injured tissue is repaired through a variety
of biochemical,
biophysical, and cellular mechanisms. The principal cellular mediators of
inflammation are
leukocytes, particularly granulocytes and the monocytes/macrophages.
Macrophages recognize,
internalize, and destroy a variety of foreign (non-self) and endogenous
substances and pathogens,
including bacteria, parasites, and viruses. The exact recognition mechanism
for non-self
pathogens is unknown, but it has been proposed that receptors with broad
binding specificity are
used to discriminate between self and non-self antigens. Macrophages are also
thought to play an
important role in the immune response by presenting foreign antigens to
lymphocytes.
The discovery of new human receptor-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 cell proliferative,
autoimmune/inflammatory, reproductive,
cardiovascular, and gastrointestinal disorders.
SUMMARY OF THE INVENTION
The invention features substantially purified polypeptides, human receptor-
associated
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proteins, referred to collectively as "HRAP". In one aspect, the invention
provides a substantially
purified polypeptide comprising an amino acid sequence selected from the group
consisting of
SEQ ID NO:1-16, and fragments thereof.
The invention further provides a substantially purified variant having at
least 90% amino
acid identity to at least one of the atriino acid sequences selected from the
group consisting of SEQ
ID NO:1-16, and fragments thereof. The invention also provides an isolated and
purified
polynucleotide encoding the polypeptide comprising an amino acid sequence
selected from the
group consisting of SEQ ID NO:I-16, and fragments thereof. The invention also
includes an
isolated and purified polynucleotide variant having at least 70%
polynucleotide sequence identity
to the polynucleotide encoding the polypeptide comprising an amino acid
sequence selected from
the group consisting of SEQ ID NO:1-16, and fragments thereof.
Additionally, the invention provides an isolated and purified polynucleotide
which
hybridizes under stringent conditions to the polynucleotide encoding the
polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID NO:1-16,
and fragments
thereof. The invention also provides an isolated and purified polynucleotide
having a sequence
which is complementary to the polynucleotide encoding the polypeptide
comprising the amino
acid sequence selected from the group consisting of SEQ ID NO:1-16, and
fragments thereof.
The invention also provides an isolated and purified poiynucleotide comprising
a
polynucleotide sequence selected from the group consisting of SEQ ID N0:17-32,
and fragments
thereof. The invention further provides an isolated and purified
polynucleotide variant having at
least 90% polynucleotide sequence identity to the polynucieotide sequence
selected from the
group consisting of SEQ ID N0:17-32, and fragments thereof. The invention also
provides an
isolated and purified polynucleotide having a sequence which is complementary
to the
polynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ
ID N0:17-32, and fragments thereof.
The invention also provides a method for detecting a polynucleotide in a
sample
containing nucleic acids, the method comprising the steps of (a) hybridizing
the complement of the
polynucleotide sequence to at least one of the polynucleotides of the sample,
thereby forming a
hybridization complex; and (b) detecting the hybridization complex, wherein
the presence of the
hybridization complex correlates with the presence of a polynucleotide in the
sample. In one
aspect, the method further comprises amplifying the polynucleotide prior to
hybridization.
The invention further provides an expression vector containing at least a
fragment of the
polynucleotide encoding the polypeptide comprising an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-16, and fragments thereof. In another aspect,
the expression
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vector is contained within a host cell.
The invention also provides a method for producing a polypeptide, the method
comprising
the steps of (a) culturing the host cell containing an expression vector
containing at least a
fragment of a polynucleotide under conditions suitable for the expression of
the polypeptide; and
(b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a
substantially
purified polypeptide having the amino acid sequence selected from the group
consisting of SEQ
ID NO:I-16, and fragments thereof, in conjunction with a suitable
pharmaceutical carrier.
The invention further includes a purified antibody which binds to a
polypeptide selected
from the group consisting of SEQ ID NO:1-16, and fragments thereof. The
invention also
provides a purified agonist and a purified antagonist to the polypeptide.
The invention also provides a method for treating or preventing a disorder
associated with
decreased expression or activity of HRAP, the method comprising administering
to a subject in
need of such treatment an effective amount of a phanmaceutical composition
comprising a
substantially purified polypeptide having the amino acid sequence selected
from the group
consisting of SEQ ID NO:1-16, and fragments thereof, in conjunction with a
suitable
pharmaceutical carrier.
The invention also provides a method for treating or preventing a disorder
associated with
increased expression or activity of HRAP, the method comprising administering
to a subject in
need of such treatment an effective amount of an antagonist of a polypeptide
having an amino acid
sequence selected from the group consisting of SEQ ID NO:1-16, and fragments
thereof.
BRIEF DESCRIPTION OF THE TABLES
Table 1 shows nucleotide and polypeptide sequence identification numbers (SEQ
ID NO),
clone identification numbers (clone ID), cDNA libraries, and cDNA fragments
used to assemble
full-length sequences encoding HRAP.
Table 2 shows features of each polypeptide sequence including potential
motifs,
homologous sequences, and methods and algorithms used for identification of
HRAP.
Table 3 shows the tissue-specific expression patterns of each nucleic acid
sequence as
determined by northern analysis, diseases or disorders associated with these
tissues, selected
fragments of the nucleotide sequences encoding HItAP which are useful as
hybridization probes,
and the vector into which each cDNA was cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which
Incyte
cDNA clones encoding HRAP were isolated.
Table 5 shows the programs, their descriptions, references, and threshold
parameters used
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to analyze HRAP.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is
understood that this invention is not limited to the particular machines,
materials and methods
described, as these may vary. It is also to be understood that the terminology
used herein is for the
purpose of describing particular embodiments only, and is not intended to
limit the scope of the
present invention which will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a,"
"an," and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for
example, a reference to "a host cell" includes a plurality of such host cells,
and a reference to "an
antibody" is a reference to one or more antibodies and equivalents thereof
known to those skilled
in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention
belongs. Although any machines, materials, and methods similar or equivalent
to those described
herein can be used to practice or test the present invention, the preferred
machines, materials and
methods are now described. All publications mentioned herein are cited for the
purpose of
describing and disclosing the cell lines, protocols, reagents and vectors
which are reported in the
publications and which might be used in connection with the invention. Nothing
herein is to be
construed as an admission that the invention is not entitled to antedate such
disclosure by virtue of
prior invention.
DEFINITIONS
"HRAP" refers to the amino acid sequences of substantially purified HRAP
obtained from
any species, particularly a mammalian species, including bovine, ovine,
porcine, murine, equine,
and preferably the human species, from any source, whether natural, synthetic,
semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which, when bound to HR.AP, increases
or
prolongs the duration of the effect of HRAP. Agonists may include proteins,
nucleic acids,
carbohydrates, or any other molecules which bind to and modulate the effect of
HRAP.
An "allelic variant" is an alternative form of the gene encoding HRAP. Allelic
variants
may result from at least one mutation in the nucleic acid sequence and may
result in altered
mltNAs or in polypeptides whose structure or function may or may not be
altered. Any given
natural or recombinant gene may have none, one, or many allelic forms. Common
mutational
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changes which give rise to allelic variants are generally ascribed to natural
deletions, additions, or
substitutions of nucleotides. Each of these types of changes may occur alone,
or in combination
with the others, one or more times in a given sequence.
"Altered" nucleic acid sequences encoding I-IRAP include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polynucleotide the same as
HRAP or a polypeptide with at least one functional characteristic of HRAP.
Included within this
definition are polymorphisms which may or may not be readily detectable using
a particular
oligonucleotide probe of the polynucleotide encoding HRAP, and improper or
unexpected
hybridization to allelic variants, with a locus other than the normal
chromosomal locus for the
polynucleotide sequence encoding HItAP. 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 HRAP. Deliberate amino acid
substitutions may be made
on the basis of similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity, and/or the
amphipathic nature of the residues, as long as the biological or immunological
activity of I~iRAP is
retained. For example, negatively charged amino acids may include aspartic
acid and glutamic
acid, positively charged amino acids may include lysine and arginine, and
amino acids with
uncharged polar head groups having similar hydrophilicity values may include
leucine, isoleucine,
and valine; glycine and alanine; asparagine and glutamine; serine and
threonine; and
phenylalanine and tyrosine.
The terms "amino acid" or "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. In this context, "fragments," "immunogenic fragments," or
"antigenic
fragments" refer to fragments of HRAP which are preferably at least 5 to about
I S amino acids in
length, most preferably at least 14 amino acids, and which retain some
biological activity or
immunological activity of HRAP. Where "amino acid sequence" is recited to
refer to an amino
acid sequence of a naturally occurring protein molecule, "amino acid sequence"
and like tenors 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 team "antagonist" refers to a molecule which, when bound to HRAP,
decreases the
amount or the duration of the effect of the biological or immunological
activity of I-IRAP.
Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or
any other molecules
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which decrease the effect of HRAP.
The term "antibody" refers to intact molecules as well as to fragments
thereof, such as
Fab, F(ab'~, and Fv fragments, which are capable of binding the epitopic
determinant. Antibodies
that bind HRAP 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 (ICLH). The coupled peptide is then used to
immunize the animal.
The term "antigenic determinant" refers to that fragment 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 (given regions or three-
dimensional structures on
the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
I S used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition containing a nucleic acid
sequence which
is complementary to the "sense" strand of a specific nucleic acid sequence.
Antisense molecules
may be produced by any method including synthesis or transcription. Once
introduced into a cell,
the complementary nucleotides combine with natural sequences produced by the
cell to form
duplexes and to block either transcription or translation. The designation
"negative" can refer to
the antisense strand, and the designation "positive" can refer to the sense
strand.
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 HRAP, 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" or "complementarily" refer to the natural binding of
polynucleotides by base pairing. For example, the sequence "5' A-G-T 3"' bonds
to the
complementary sequence "3' T-C-A 5'." Complementarily 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 complementarily exists between the single stranded molecules. The degree
of
complementarily 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 acids
strands, and in the
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design and use of peptide nucleic acid (PNA) molecules.
A "composition comprising a given polynucleotide sequence" or 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.
HRAP or
fragments of HRAP may be employed as hybridization probes. The probes may be
stored in
freeze-dried form and may be associated with a stabilizing agent such as a
carbohydrate. In
hybridizations, the probe may be deployed in an aqueous solution containing
salts (e.g., NaCI),
detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g.,
Denhardt's solution,
dry milk, salmon sperm DNA, etc.).
"Consensus sequence"refers to a nucleic acid sequence which has been
resequenced to
resolve uncalled bases, extended using 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
more than one Incyte Clone using a computer program for fragment assembly,
such as the
GELVIEW Fragment Assembly system (GCG, Madison WI). Some sequences have been
both
extended and assembled to produce the consensus sequence.
The term "correlates with expression of a polynucleotide" indicates that the
detection of
the presence of nucleic acids, the same or related to a nucleic acid sequence
encoding HRAP, by
northern analysis is indicative of the presence of nucleic acids encoding HRAP
in a sample, and
thereby correlates with expression of the transcript from the polynucleotide
encoding HRAP.
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, or amino group. A
derivative polynucleotide
encodes a polypeptide which retains at least one biological or immunological
function of the
natural molecule. A derivative polypeptide is one modified by glycosylation,
pegylation, or any
similar process that retains at least one biological or immunological function
of the polypeptide
from which it was derived.
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
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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 stringency. This is not to say that
conditions of reduced
stringency are such that 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" or "% identity" refer to the percentage of
sequence
similarity found in a comparison of two or more amino acid or nucleic acid
sequences. Percent
identity can be determined electronically, e.g., by using the MEGALIGN program
(DNASTAR,
Madison WI). The MEGALIGN program can create alignments between two or more
sequences
IS according to different methods, e.g., the clustal method. (See, e.g.,
Higgins, D.G. and P.M. Sharp
(1988) Gene 73:237-244.) The clustal algorithm groups sequences into clusters
by examining the
distances between all pairs. The clusters are aligned pairwise and then in
groups. The percentage
similarity between two amino acid sequences, e.g., sequence A and sequence B,
is calculated by
dividing the length of sequence A, minus the number of gap residues in
sequence A, minus the
number of gap residues in sequence B, into the sum of the residue matches
between sequence A
and sequence B, times one hundred. Gaps of low or of no similarity between the
two amino acid
sequences are not included in determining percentage similarity. Percent
identity between nucleic
acid sequences can also be counted or calculated by other methods known in the
art, e.g., the Jotun
Hein method. (See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.)
Identity between
sequences can also be determined by other methods known in the art, e.g., by
varying
hybridization conditions.
"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 any process by which a strand of nucleic acid binds
with a
complementary strand through base pairing.
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The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a
solid support (e.g., paper, membranes, filters, chips, pins or glass slides,
or any other appropriate
substrate to which cells or their nucleic acids have been fixed).
The words "insertion" or "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,
to the sequence found in the naturally occurring molecule.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by
expression of various factors, e.g., cytokines, chemokines, and other
signaling molecules, which
may affect cellular and systemic defense systems.
The term "microarray" refers to an arrangement of distinct polynucleotides on
a substrate.
The terms "element" or "array element" in a microarray context, refer to
hybridizable
polynucleotides arranged on the surface of a substrate.
The term "modulate" refers to a change in the activity of HRAP. For example,
modulation may cause an increase or a decrease in protein activity, binding
characteristics, or any
other biological, functional, or immunological properties of HRAP.
The phrases "nucleic acid" or "nucleic acid sequence," as used herein, 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. In this context, "fragments" refers to those
nucleic acid
sequences which, comprise a region of unique polynucleotide sequence that
specifically identifies
SEQ ID N0:17-32, for example, as distinct from any other sequence in the same
genome. For
example, a fragment of SEQ ID N0:17-32 is useful in hybridization and
amplification
technologies and in analogous methods that distinguish SEQ ID N0:17-32 from
related
polynucleotide sequences. A fragment of SEQ ID N0:17-32 is at least about 15-
20 nucleotides in
length. The precise length of the fragment of SEQ ID N0:17-32 and the region
of SEQ ID
N0:17-32 to which the fragment corresponds are routinely determinable by one
of ordinary skill
in the art based on the intended purpose for the fragment. In some cases, a
fragment, when
translated, would produce polypeptides retaining some functional
characteristic, e.g., antigenicity,
or structural domain characteristic, e.g., ATP-binding site, of the full-
length polypeptide.
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The terms "operably associated" or "operably linked" refer to functionally
related nucleic
acid sequences. A promoter is operably associated or operably linked with a
coding sequence if
the promoter controls the translation of the encoded polypeptide. While
operably associated or
operably linked nucleic acid sequences can be contiguous and in the same
reading frame, certain
genetic elements, e.g., repressor genes, are not contiguously linked to the
sequence encoding the
polypeptide but still bind to operator sequences that control expression of
the polypeptide.
The tenor "oligonucleotide" refers to a nucleic acid sequence of at least
about 6
nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most
preferably about 20
to 25 nucleotides, which can be used in PCR amplification or in a
hybridization assay or
microarray. "Oligonucleotide" is substantially equivalent to the terms
"amplimer," "primer,"
"oligomer," and "probe," as these terms are commonly defined in the art.
"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.
The term "sample" is used in its broadest sense. A sample suspected of
containing nucleic
acids encoding I-IRAP, or fragments thereof, or HRAP 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" or "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, or an antagonist. 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 "stringent conditions" refers to conditions which permit
hybridization between
polynucleotides and the claimed polynucleotides. Stringent conditions can be
defined by salt
concentration, the concentration of organic solvent, e.g., formamide,
temperature, and other
conditions well known in the art. In particular, stringency can be increased
by reducing the
concentration of salt, increasing the concentration of fonmamide, or raising
the hybridization
temperature.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
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removed from their natural environment and are isolated or separated, and are
at least about 60%
free, preferably about 75% free, and most preferably about 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, 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 "variant" of I-IRAP polypeptides refers to an amino acid sequence that is
altered by one
or more amino acid residues. The variant may have "conservative" changes,
wherein a substituted
amino acid has similar structural or chemical properties (e.g., replacement of
leucine with
isoleucine). More rarely, a variant may have "nonconservative" changes (e.g.,
replacement of
glycine with tryptophan). Analogous minor variations may also include amino
acid deletions or
insertions, or both. Guidance in determining which amino acid residues may be
substituted,
inserted, or deleted without abolishing biological or immunological activity
may be found using
computer programs well known in the art, for example, LASERGENE software
(DNASTAR).
The term "variant," when used in the context of a polynucleotide sequence, may
encompass a polynucleotide sequence related to HRAP. This definition may also
include, for
example, "allelic" (as defined above), "splice," "species," or "polymorphic"
variants. 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 an
absence of domains.
Species variants are poIynucleotide sequences that vary from one species to
another. The resulting
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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 base.
The presence
of SNPs may be indicative of, for example, a certain population, a disease
state, or a propensity for
a disease state.
THE INVENTION
The invention is based on the discovery of new human receptor-associated
proteins
(HRAP), the polynucleotides encoding HRAP, and the use of these compositions
for the diagnosis,
treatment, or prevention of cell proliferative, autoimmune/inflammatory,
reproductive,
cardiovascular, and gastrointestinal disorders.
Table I lists the Incyte Clones used to derive full length nucleotide
sequences encoding
HRAP. Columns 1 and 2 show the sequence identification numbers (SEQ ID NO) of
the amino
acid and nucleic acid sequences, respectively. Column 3 shows the Clone ID of
the Incyte Clone
in which nucleic acids encoding each HRAP were identified, and column 4, the
cDNA libraries
from which these clones were isolated. Column 5 shows Incyte clones, their
corresponding cDNA
libraries, and shotgun sequences useful as fragments in hybridization
technologies, and which are
part of the consensus nucleotide sequence of each HRAP.
The columns of Table 2 show various properties 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, potential phosphorylation sites; column 4, potential
glycosylation sites;
column 5, the amino acid residues comprising signature sequences and motifs;
column 6, the
identity of each protein; and column 7, analytical methods used to identify
each protein through
sequence homology and protein motifs.
The columns of Table 3 show the tissue-specificity and disease-association of
nucleotide
sequences encoding HItAP. The first column of Table 3 lists the polynucleotide
sequence
identifiers. The second column lists unique fragments of the nucleotide
sequences encoding HRAP
which are useful as hybridization probes. The third column lists tissue
categories which express
HRAP as a fraction of total tissue categories expressing HRAP. The fourth
column lists the
disease classes associated with those tissues expressing HRAP. The fifth
column lists the vectors
used to subclone the cDNA library.
The invention also encompasses HRAP variants. A preferred HRAP variant is one
which
has at least about 80~/0, more preferably at least about 90%, and most
preferably at least about 95%
amino acid sequence identity to the HRAP amino acid sequence, and which
contains at least one
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functional or structural characteristic of HRAP.
The invention also encompasses polynucleotides which encode HRAP. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence
selected from the group consisting of SEQ 1D N0:17-32, which encodes HRAP.
The invention also encompasses a variant of a polynucleotide sequence encoding
HRAP.
In particular, such a variant polynucleotide sequence will have at least about
70%, more preferably
at least about 85%, and most preferably at least about 95% polynucleotide
sequence identity to the
polynucleotide sequence encoding HRAP. A particular aspect of the invention
encompasses a
variant of a polynucleotide sequence comprising a sequence selected from the
group consisting of
SEQ ID N0:17-32 which has at least about 70%, more preferably at least about
85%, and most
preferably at least about 95% polynucleotide sequence identity to a nucleic
acid sequence selected
from the group consisting of SEQ ID N0:17-32. Any one of the polynucleotide
variants described
above can encode an amino acid sequence which contains at least one functional
or structural
characteristic of HRAP.
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 HRAP, 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 colon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring HRAP, and all such variations
are to be considered
as being specifically disclosed.
Although nucleotide sequences which encode HRAP and its variants are
preferably
capable of hybridizing to the nucleotide sequence of the naturally occurring
HRAP under
appropriately selected conditions of stringency, it may be advantageous to
produce nucleotide
sequences encoding HRAP or its derivatives possessing a substantially
different colon usage, e.g.,
inclusion of non-naturally occurring colons. Colons 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 colons are utilized by the host. Other
reasons for
substantially altering the nucleotide sequence encoding HRAP 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 HRAP
and
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I-IRAP 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 HRAP 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
N0:17-32 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.) For example, stringent salt concentration will ordinarily be
less than about 750 mM
NaCI and 75 mM trisodium citrate, preferably less than about 500 mM NaCI and
50 mM trisodium
citrate, and most preferably less than about 250 mM NaCI and 25 mM trisodium
citrate. Low
stringency hybridization can be obtained in the absence of organic solvent,
e.g., formamide, while
high stringency hybridization can be obtained in the presence of at least
about 35% formamide,
and most preferably at feast about 50% formamide. Stringent temperature
conditions will
ordinarily include temperatures of at least about 30°C, more preferably
of at least about 37°C, and
most preferably of at least about 42°C. Varying additional parameters,
such as hybridization time,
the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the
inclusion or exclusion
of carrier DNA, are well known to those skilled in the art. Various levels of
stringency are
accomplished by combining these various conditions as needed. In a preferred
embodiment,
hybridization will occur at 30°C in 750 mM NaCI, 75 mM trisodium
citrate, and 1% SDS. In a
more preferred embodiment, hybridization will occur at 37°C in 500 mM
NaCI, 50 mM trisodium
citrate, 1% SDS, 35% formamide, and 100 ~cg/ml denatured salmon sperm DNA
(ssDNA). In a
most preferred embodiment, hybridization will occur at 42°C in 250 mM
NaCI, 25 mM trisodium
citrate, 1% SDS, 50 % formamide, and 200 ~g/ml ssDNA. Useful variations on
these conditions
will be readily apparent to those skilled in the art.
The washing steps which follow hybridization can also vary in stringency. Wash
stringency conditions can be defined by salt concentration and by temperature.
As above, wash
stringency can be increased by decreasing salt concentration or by increasing
temperature. For
example, stringent salt concentration for the wash steps will preferably be
less than about 30 mM
NaCI and 3 mM trisodium citrate, and most preferably less than about 15 mM
NaCI and 1.5 mM
trisodium citrate. Stringent temperature conditions for the wash steps will
ordinarily include
temperature of at least about 25°C, more preferably of at least about
42°C, and most preferably of
at least about 68°C. In a preferred embodiment, wash steps will occur
at 25°C in 30 mM NaCI, 3
mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps
will occur at
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42°C in 15 mM NaCI, 1.5 mM trisodium citrate, and 0.1 % SDS. In a most
preferred embodiment,
wash steps will occur at 68°C in 15 mM NaCI, 1.5 mM trisodium citrate,
and 0.1% SDS.
Additional variations on these conditions will be readily apparent to those
skilled in the art.
Methods for DNA sequencing are well known in the art and may be used to
practice any
of the embodiments of the invention. ' The methods may employ such enzymes as
the Klenow
fragment of DNA polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq
polymerise
(Perkin-Elmer), thermostable T7 polymerise (Amersham Phanmacia Biotech,
Piscataway NJ), or
combinations of polymerises and proofreading exonucleases such as those found
in the
ELONGASE amplification system (Life Technologies, Gaithersburg MD).
Preferably, sequence
preparation is automated with machines such as the Hamilton MICROLAB 2200
(Hamilton, Reno
NV), Pettier Thermal Cycler 200 (PTC200; MJ Research, Watertown MA) and the
ABI
CATALYST 800 (Perkin-Elmer). Sequencing is then carried out using either ABI
373 or 377
DNA sequencing systems (Perkin-Elmer) or the MEGABACE 1000 DNA sequencing
system
(Molecular Dynamics, Sunnyvale CA). The resulting sequences are analyzed using
a variety of
algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997)
Short Protocols in
Molecular BioloQV, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A.
(1995) Molecular
Biology and BiotechnoloQV, Wiley VCH, New York NY, pp. 856-853.)
The nucleic acid sequences encoding HRAP may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream
sequences, such as promoters and regulatory elements. For example, one method
which may be
employed, restriction-site PCR, uses universal and nested primers to amplify
unknown sequence
from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. ( 1993) PCR
Methods Applic.
2:318-322.) Another method, inverse PCR, uses primers that extend in divergent
directions to
amplify unknown sequence from a circularized template. The template is derived
from restriction
fragments comprising a known genomic locus and surrounding sequences. (See,
e.g., Triglia, T. et
al. (1988) Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves
PCR
amplification of DNA fragments adjacent to known sequences in human and yeast
artificial
chromosome DNA. (See, e.g., Lagerstrom, M. et al: (1991) PCR Methods Applic.
1:111-119.) In
this method, multiple restriction enzyme digestions and ligations may be used
to insert an
engineered double-stranded sequence into a region 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. 19:3055-306). 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
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junctions. For all PCR-based methods, primers may be designed using
commercially available
software, such as OLIGO 4.06 Primer Analysis software (National Biosciences,
Plymouth MN) or
another appropriate program, to be about 22 to 30 nucleotides in length, to
have a GC content of
about 50% or more, and to anneal to the template at temperatures of about
68°C to 72°C.
When screening for full-length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of
sequence into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to
analyze the size or confirm the nucleotide sequence of sequencing or PCR
products. In particular,
capillary sequencing may employ flowable polymers for electrophoretic
separation, four different
nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled
device camera for
detection of the emitted wavelengths. Output/light intensity may be converted
to electrical signal
using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, 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.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof
which encode HRAP may be cloned in recombinant DNA molecules that direct
expression of
HRAP, 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
The nucleotide sequences of the present invention can be engineered using
methods
generally known in the art in order to alter HRAP-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 colon preference,
produce splice
variants, and so forth.
In another embodiment, sequences encoding HRAP 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) Nucl.
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Acids Res. Symp. Ser. 215-223, and Horn, T. et al. (1980) Nucl. Acids Res.
Symp. Ser. 225-232.)
Alternatively, HRAP 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
HRAP, or any part thereof, may be altered during direct synthesis and/or
combined with sequences
from other proteins, or any part thereof, to produce a variant polypeptide.
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 HRAP, the nucleotide sequences
encoding HRAP
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 HRAP. Such elements may vary in their
strength and
specificity. Specific initiation signals may also be used to achieve more
efficient translation of
sequences encoding HRAP. Such signals include the ATG initiation codon and
adjacent
sequences, e.g. the Kozak sequence. In cases where sequences encoding HRAP and
its initiation
codon and upstream regulatory sequences are inserted into the appropriate
expression vector, no
additional transcripdonal 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 HRAP and appropriate
transcriptional and
transiational 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 Clonin~Laboratorv Manual, Cold Spring Harbor Press, Plainview NY,
ch. 4, 8, and
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16-17; Ausubel, F.M. el al. (1995) Current Protocols in Molecular Bioloev,
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 HRAP. These include, but are not limited to, microorganisms
such as
bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA
expression
vectors; yeast transformed with yeast expression vectors; insect cell systems
infected with viral
expression vectors (e.g., baculovirus); plant cell systems transformed with
viral expression vectors
(e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus,TMV) or with
bacterial expression
vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. 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 HRAP.
For example,
routine cloning, subcloning, and propagation of polynucleotide sequences
encoding HRAP 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 HRAP
into the
vector's multiple cloning site disrupts the IacZ gene, allowing a colorimetric
screening procedure
for identification of transformed bacteria containing recombinant molecules.
In addition, these
vectors may be useful for in vitro transcription, dideoxy sequencing, single
strand rescue with
helper phage, and creation of nested deletions in the cloned sequence. (See,
e.g., Van Heeke, G.
and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When large quantities
of HRAP are
needed, e.g. for the production of antibodies, vectors which direct high level
expression of HRAP
may be used. For example, vectors containing the strong, inducible TS or T7
bacteriophage
promoter may be used.
Yeast expression systems may be used for production of HRAP. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH,
may be used in the yeast Saccharomyces 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;
Grant et al. ( 1987) Methods Enzymol. 153:516-54; and Scorer, C. A. et al. (
1994) Bio/Technology
12:181-184.)
Piant systems may also be used for expression of HRAP. Transcription of
sequences
encoding HRAP 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
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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 Technolo~v (1992) McGi~aw 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 HRAP
may be ligated
into an adenovirus transcription/translation complex consisting of the late
promoter and tripartite
leader sequence. Insertion in a non-essential E 1 or E3 region of the viral
genome may be used to
obtain infective virus which expresses HRAP in host cells. (See, e.g., Logan,
J. and T. Shenk
(1984) Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcription
enhancers, such as the
Rous sarcoma virus (RSV) enhancer, may be used to increase expression in
mammalian host cells.
SV40 or EBV-based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments
of DNA than can be contained in and expressed from a plasmid. HACs of about 6
kb to 10 Mb
are constructed and delivered via conventional delivery methods (liposomes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. (1997) Nat Genet.
15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression of HRAP in cell lines is preferred. For example, sequences encoding
HRAP 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 or apr~ cells, respectively.
(See, e.g., Wigler, M. et
al. ( 1977) Cell I 1:223-232; 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 or pat confer resistance to chlorsulfuron and phosphinotricin
acetyltransferase,
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respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.
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. 85:8047-8051.)
Visible markers,
e.g.,.anthocyanins, green fluorescent proteins (GFP; Clontech),13
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 sequence encoding HRAP is inserted within a marker gene
sequence, transformed
cells containing sequences encoding HRAP can be identified by the absence of
marker gene
function. Alternatively, a marker gene can be placed in tandem with a sequence
encoding HRAP
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 HRAP
and that
express HRAP 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 HRAP 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 HRAP is
preferred, but a
competitive binding assay may be employed. These and other assays are well
known in the art.
(See, e.g., Hampton, R. et al. ( 1990) Serological Methods. a Laboratory
Manual, APS Press, St
Paul MN, Sect. IV; Coligan, J. E. et al. (1997) Current Protocols in
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
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hybridization or PCR probes for detecting sequences related to polynucleotides
encoding HRAP
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled
nucleotide. Alternatively, the sequences encoding HRAP, or any fragments
thereof, may be
cloned into a vector for the production of an mRNA probe. Such vectors are
known in the art, are
commercially available, and may be 'used to synthesize RNA probes in vitro by
addition of an
appropriate RNA polymerise such as T7, T3, or SP6 and labeled nucleotides.
These procedures
may be conducted using a variety of commercially available kits, such as those
provided by
Amersham Phanmacia 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 HRAP may be cultured
under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the
sequence and/or the vector used. As will be understood by those of skill in
the art, expression
vectors containing polynucleotides which encode HRAP may be designed to
contain signal
sequences which direct secretion of HRAP 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"
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, Hel:,a, 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 HRAP 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 HRAP protein
containing a heterologous moiety that can be recognized by a commercially
available antibody
may facilitate the screening of peptide libraries for inhibitors of HRAP
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-
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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
immunoafl~;nity purification of fusion proteins using commercially available
monoclonal and
polyclonal antibodies that specificallyrecognize these epitope tags. A fusion
protein may also be
engineered to contain a proteolytic cleavage site located between the HRAP
encoding sequence
and the heterologous protein sequence, so that HRAP may be cleaved away from
the heterologous
moiety following purification. Methods for fusion protein expression and
purification are
discussed in Ausubel ( 1995, sutira, ch 10). A variety of commercially
available kits may also be
IO used to facilitate expression and purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled HRAP may
be
achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ
extract systems
(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, preferably "S-methionine.
Fragments of HRAP may be produced not only by recombinant production, but also
by
direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton,
sera. pp. 55-60.)
Protein synthesis may be performed by manual techniques or by automation.
Automated synthesis
may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin-
Elmer). Various
fragments of HRAP may be synthesized separately and then combined to produce
the full length
molecule.
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists
between regions of HRAP and human receptor-associated proteins. In addition,
the expression of
HRAP is closely associated with cell proliferative disorders, such as cancer,
and with
inflammation and the immune response. HRAP are expressed in libraries from
cancerous tissues,
hematopoietic tissues, reproductive tissues, cardiovascular tissues, and
gastrointestinal tissues
(Table 3). Therefore, HRAP appears to play a role in cell proliferative,
autoimmune/inflammatory, reproductive, cardiovascular, and gastrointestinal
disorders.
Therefore, in the treatment of disorders associated with increased expression
or activity of HRAP,
it is desirable to decrease the expression or activity of HRAP. In the
treatment of disorders
associated with decreased expression or activity of HRAP, it is desirable to
provide the protein or
to increase the expression of HRAP.
Therefore, in one embodiment, HRAP or a fragment or derivative thereof may be
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administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of HRAP. Examples of such disorders include, but are not limited to,
a cell proliferative
disorder such as actinic keratosis, atherosclerosis, bursitis, cirrhosis,
hepatitis, mixed connective
tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria,
polycythemia vera,
psoriasis, primary thrombocythemia, and cancers including adenocarcinoma,
leukemia,
lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, a
cancer of the
adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall
bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas,
parathyroid, penis,
prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus;
and an
autoimmune/inflammatory disorder such as actinic keratosis, acquired
immunodeficiency
syndrome (AIDS), Addison's disease, adult respiratory distress syndrome,
allergies, ankylosing
spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis,
autoimmune hemolytic
anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis,
cirrhosis, contact dermatitis,
Crohn's disease, atopic denmatitis, dermatomyositis, diabetes mellitus,
emphysema,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's
syndrome, gout, Graves' disease, Hashimoto's thyroiditis, paroxysmal nocturnal
hemoglobinuria,
hepatitis, hypereosinophilia, irritable bowel syndrome, episodic lymphopenia
with
lymphocytotoxins, mixed connective tissue disease (MCTD), multiple sclerosis,
myasthenia
gravis, myocardial or pericardial inflammation, myelofibrosis, osteoarthritis,
osteoporosis,
pancreatitis, polycythemia vera, polymyositis, psoriasis, Reiter's syndrome,
rheumatoid arthritis,
scleroderma, Sjdgren's syndrome, systemic anaphylaxis, systemic lupus
erythematosus, systemic
sclerosis, primary thrombocythemia, thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial,
fungal, parasitic, protozoal, and helminthic infections, and trauma.
In another embodiment, a vector capable of expressing HRAP 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 HRAP including, but not limited to, those described
above.
In a further embodiment, a pharmaceutical composition comprising a
substantially
purified HRAP 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 HRAP
including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of HRAP
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of HRAP including, but not limited to, those listed above.
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In a further embodiment, an antagonist of HRAP may be administered to a
subject to treat
or prevent a disorder associated with increased expression or activity of
HRAP. Examples of such
disorders include, but are not limited to, a cell proliferative disorder such
as actinic keratosis,
atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue
disease (MCTD),
myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera,
psoriasis, primary
thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma,
melanoma,
myeloma, sarcoma, teratocarcinoma, and, in particular, a cancer of the adrenal
gland, bladder,
bone, bone marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract, heart, kidney,
liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary
glands, skin, spleen,
testis, thymus, thyroid, and uterus; an autoimmune/inflammatory disorder such
as actinic keratosis,
acquired immunodeficiency syndrome (AIDS), Addison's disease, adult
respiratory distress
syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia,
arteriosclerosis, asthma,
atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis,
bronchitis, bursitis,
cholecystitis, cirrhosis, contact dermatitis, Crohn's disease, atopic
dermatitis, dermatomyositis,
diabetes mellitus, emphysema, erythroblastosis fetalis, erythema nodosum,
atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis,
paroxysmal nocturnal hemoglobinuria, hepatitis, hypereosinophilia, irritable
bowel syndrome,
episodic lymphopenia with lymphocytotoxins, mixed connective tissue disease
(MCTD), multiple
sclerosis, myasthenia gravis, myocardial or pericardial inflammation,
myelofibrosis, osteoarthritis,
osteoporosis, pancreatitis, polycythemia vera, polymyositis, psoriasis,
Reiter's syndrome,
rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis,
systemic lupus
erythematosus, systemic sclerosis, primary thrombocythemia, thrombocytopenic
purpura,
ulcerative colitis, uveitis, Wemer syndrome, complications of cancer,
hemodialysis, and
extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal,
and heiminthic infections,
and trauma; a reproductive disorder such as a disorder of prolactin
production, infertility,
including tubal disease, ovulatory defects, and endometriosis, a disruption of
the estrous cycle, a
disruption of the menstrual cycle, polycystic ovary syndrome, ovarian
hyperstimulation syndrome,
an endometrial or ovarian tumor, a uterine fibroid, an autoimmune disorder, an
ectopic pregnancy,
and teratogenesis, cancer of the breast, fibrocystic breast disease, and
galactorrhea, a disruption of
spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of
the prostate, benign
prostatic hyperplasia, prostatitis, Peyronie's disease, carcinoma of the male
breast, and
gynecomastia; a cardiovascular disorder such as arteriovenous fistula,
atherosclerosis,
hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections,
varicose veins,
thrombophlebitis and phlebothrombosis, and vascular tumors, congestive heart
failure, ischemic
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heart disease, angina pectoris, myocardial infarction, hypertensive heart
disease, degenerative
valvular heart disease, calcific aortic valve stenosis, congenitally bicuspid
aortic valve, mitral
annular calcification, mitral valve prolapse, rheumatic fever and rheumatic
heart disease, infective
endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic
lupus erythematosus,
carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic
heart disease, and
congenital heart disease; and a gastrointestinal disorder such as dysphagia,
peptic esophagitis,
esophageal spasm, esophageal stricture, esophageal carcinoma, dyspepsia,
indigestion, gastritis,
gastric carcinoma, anorexia, nausea, emesis, gastroparesis, antral or pyloric
edema, abdominal
angina, pyrosis, gastroenteritis, intestinal obstruction, an infection of the
intestinal tract, peptic
ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic
carcinoma, biliary tract
disease, hepatitis, hyperbilirubinemia, cirrhosis, passive congestion of the
liver, hepatoma,
infectious colitis, ulcerative colitis, ulcerative proctitis, Crohn's disease,
Whipple's disease,
Mallory-Weiss syndrome, colonic carcinoma, colonic obstruction, irritable
bowel syndrome, short
bowel syndrome, diarrhea, constipation, gastrointestinal hemorrhage, and
acquired
immunodeficiency syndrome (AIDS) enteropathy. In one aspect, an antibody which
specifically
binds HRAP may be used directly as an antagonist or indirectly as a targeting
or delivery
mechanism for bringing a pharmaceutical agent to cells or tissue which express
HRAP.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding HRAP may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of HRAP 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 HRAP may be produced using methods which are generally known
in
the art. In particular, purified HRAP may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind HRAP.
Antibodies to HRAP 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
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inhibit dimer formation) are especially 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 HRAP or with any
fragment or
oiigopeptide 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 lysoiecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH,
and dinitrophenol.
Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and
Corvnebacterium parvum
are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
HRAP have an amino acid sequence consisting of at least about 5 amino acids,
and, more
preferably, 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 sequence of a small, naturally occurring
molecule. Short stretches of
HRAP 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 HRAP may be prepared using any technique which
provides for
the production of antibody molecules by continuous cell lines in culture.
These include, but are
not limited to, the hybridoma technique, the human B-cell hybridoma technique,
and the EBV-
hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497;
Kozbor, D. et al.
(1985) J. Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl.
Acad. Sci.
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., Mornson,
S.L. et al. (1984)
Proc. Natl. Acad. Sci. 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
HRAP-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. 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. 86:
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3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for HRAP 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
HRAP and its specific antibody. A two-site, monoclonal-based immunoassay
utilizing
monoclonal antibodies reactive to two non-interfering HRAP epitopes is
preferred, but a
competitive binding assay may also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay
techniques may be used to assess the affinity of antibodies for HItAP.
Affinity is expressed as an
association constant, K" which is defined as the molar concentration of HRAP-
antibody complex
divided by the molar concentrations of free antigen and free antibody under
equilibrium
conditions. The K, determined for a preparation of polyclonal antibodies,
which are
heterogeneous in their affinities for multiple HRAP epitopes, represents the
average affinity, or
avidity, of the antibodies for HRAP. The K, determined for a preparation of
monoclonal
antibodies, which are monospecific for a particular HRAP epitope, represents a
true measure of
affinity. High-affinity antibody preparations with K, ranging from about 109
to 10'Z L/mole are
preferred for use in immunoassays in which the HItAP-antibody complex must
withstand rigorous
manipulations. Low-affinity antibody preparations with K, ranging from about
106 to 10' L/mole
are preferred for use in immunopurification and similar procedures which
ultimately require
dissociation of HRAP, preferably in active form, from the antibody (Catty, D.
( 1988) Antibodies.
Volume I: A Practical Approach, IItL 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 S-10 mg specific antibody/ml, is preferred for use in procedures
requiring precipitation
of HRAP-antibody complexes. Procedures for evaluating antibody specificity,
titer, and avidity,
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and guidelines for antibody quality and usage in various applications, are
generally available.
(See, e.g., Catty, supra, and Coligan et al. supra.)
In another embodiment of the invention, the polynucleotides encoding HRAP, or
any
fragment or complement thereof, may be used for therapeutic purposes. In one
aspect, the
complement of the polynucleotide encoding HRAP 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 HRAP. Thus, complementary
molecules
or fragments may be used to modulate HRAP 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 contml
regions of sequences
encoding HRAP.
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 HRAP. (See, e.g., Sambrook, supra; Ausubel, 1995,
suvra.)
Genes encoding HRAP can be turned off by transforming a cell or tissue with
expression
vectors which express high levels of a polynucleotide, or fragment thereof,
encoding HRAP. 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 HRAP. 0ligonucleotides derived from
the transcription
initiation site, e.g., between about positions -10 and +10 from the start
site, are preferred.
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 Immunologic 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.
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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 HRAP.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences:
GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides, corresponding to the region of the target gene containing the
cleavage site, may
be evaluated for secondary structural features which may render the
oligonucleotide inoperable.
The suitability of candidate targets may also be evaluated by testing
accessibility to hybridization
with complementary oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared by any method known in the art for the synthesis of nucleic acid
molecules. These
IS include techniques for chemically synthesizing oligonucleotides such as
solid phase
phosphoramidite chemical synthesis. Alternatively, RNA molecules may be
generated by in vitro
and in_, vivo transcription of DNA sequences encoding HRAP. 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.
( 199Z) Nature Biotechnology 15:462-466.)
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Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as dogs, cats, cows,
horses, rabbits,
monkeys, and most preferably, humans.
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 HRAP, antibodies to HRAP, and mimetics, agonists, antagonists, or
inhibitors of I-IRAP. 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,
IS 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 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, slurnes, 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.
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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 identiftcation 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 iipophilic
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 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, tactic,
tartaric, malic, and
succinic acid. Salts tend to be more soluble in aqueous or other protonic
solvents than are the
corresponding free base forms. In other cases, the preferred 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
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appropriate container and labeled for treatment of an indicated condition. For
administration of
HRAP, 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
HRAP or fragments thereof, antibodies of HRAP, and agonists, antagonists or
inhibitors of HRAP,
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 EDT (the dose therapeutically effective in 50% of
the population) or
LDP (the dose lethal to 50% of the population) statistics. The dose ratio of
therapeutic to toxic
effects is the therapeutic index, and it can be expressed as the
ED~/I,Ds° ratio. Pharmaceutical
compositions which exhibit large therapeutic indices are preferred. The data
obtained from cell
culture assays and animal studies are used to formulate a range of dosage for
human use. The
dosage contained in such compositions is preferably within a range of
circulating concentrations
that includes the EDs° with tittle 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. I ~g to 100,000 /cg, up to a
total dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
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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 HRAP may be used for
the
diagnosis of disorders characterized by expression of HRAP, or in assays to
monitor patients being
treated with HRAP or agonists, antagonists, or inhibitors of HRAP. Antibodies
useful for
diagnostic purposes may be prepared in the same manner as described above for
therapeutics.
Diagnostic assays for HRAP include methods which utilize the antibody and a
label to detect
HRAP 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 HRAP, including ELISAs, RIAs, and FACS,
are
known in the art and provide a basis for diagnosing altered or abnormal levels
of HRAP
expression. Normal or standard values for HRAP expression are established by
combining body
fluids or cell extracts taken from normal mammalian subjects, preferably
human, with antibody to
HRAP under conditions suitable for complex formation. The amount of standard
complex
formation may be quantitated by various methods, preferably by photometric
means. Quantities of
HRAP 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 HRAP may
be used
for diagnostic purposes. The polynucleotides which may be used include
oligonucleotide
sequences, complementary RNA and DNA molecules, and PNAs. The polynucleotides
may be
used to detect and quantitate gene expression in biopsied tissues in which
expression of HRAP
may be correlated with disease. The diagnostic assay may be used to determine
absence,
presence, and excess expression of HRAP, and to monitor regulation of HRAP
levels during
therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide sequences, including genomic sequences, encoding HRAP or
closely related
molecules may be used to identify nucleic acid sequences which encode HRAP.
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
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amplification (maximal, high, intermediate, or low), will determine whether
the probe identifies
only naturally occurnng sequences encoding HRAP, allelic variants, or related
sequences.
Probes may also be used for the detection of related sequences, and should
preferably
have at least 50% sequence identity to any of the HRAP encoding sequences. The
hybridization
probes of the subject invention may be DNA or RNA and may be derived from the
sequence of
SEQ ID N0:17-32 or from genomic sequences including promoters, enhancers, and
introns of the
HRAP gene.
Means for producing specific hybridization probes for DNAs encoding HRAP
include the
cloning of polynucleotide sequences encoding HRAP or HRAP 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
polymerises and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as'ZP or'sS, or
by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding HRAP may be used for the diagnosis of
disorders
associated with expression of HRAP. Examples of such disorders include, but
are not limited to, a
cell proliferative disorder such as actinic keratosis, atherosclerosis,
bursitis, cirrhosis, hepatitis,
mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal
hemoglobinuria,
polycythemia vera, psoriasis, primary thrombocythemia, and cancers including
adenocarcinoma,
leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in
particular, a cancer
of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall
bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas,
parathyroid, penis,
prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus;
an
autoimmune/inflammatory disorder such as actinic keratosis, acquired
immunodeficiency
syndrome (AIDS), Addison's disease, adult respiratory distress syndrome,
allergies, ankylosing
spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis,
autoimmune hemolytic
anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis,
cirrhosis, contact dermatitis,
Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus,
emphysema,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's
syndrome, gout, Graves' disease, Hashimoto's thyroiditis, paroxysmal nocturnal
hemoglobinuria,
hepatitis, hypereosinophilia, irritable bowel syndrome, episodic lymphopenia
with
lymphocytotoxins, mixed connective tissue disease (MCTD), multiple sclerosis,
myasthenia
gravis, myocardial or pericardial inflammation, myelofibrosis, osteoarthritis,
osteoporosis,
pancreatitis, polycythemia vera, polymyositis, psoriasis, Reiter's syndrome,
rheumatoid arthritis,
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scleroderma, Sjiigren's syndrome, systemic anaphylaxis, systemic lupus
erythematosus, systemic
sclerosis, primary thrombocythemia, thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial,
fungal, parasitic, protozoal, and helminthic infections, and trauma; a
reproductive disorder such as
a disorder of prolactin production, infertility, including tubal disease,
ovulatory defects, and
endometriosis, a disruption of the estrous cycle, a disruption of the
menstrual cycle, polycystic
ovary syndrome, ovarian hyperstimulation syndrome, an endometrial or ovarian
tumor, a uterine
fibroid, an autoimmune disorder, an ectopic pregnancy, and teratogenesis,
cancer of the breast,
fibrocystic breast disease, and galactorrhea, a disruption of spermatogenesis,
abnormal sperm
physiology, cancer of the testis, cancer of the prostate, benign prostatic
hyperplasia, prostatitis,
Peyronie's disease, carcinoma of the male breast, and gynecomastia; a
cardiovascular disorder
such as arteriovenous fistula, atherosclerosis, hypertension, vasculitis,
Raynaud's disease,
aneurysms, arterial dissections, varicose veins, thrombophlebitis and
phlebothrombosis, and
vascular tumors, congestive heart failure, ischemic heart disease, angina
pectoris, myocardial
infarction, hypertensive heart disease, degenerative valvular heart disease,
calcific aortic valve
stenosis, congenitally bicuspid aortic valve, mitral annular calcification,
mitral valve prolapse,
rheumatic fever and rheumatic heart disease, infective endocarditis,
nonbacterial thrombotic
endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart
disease,
cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, and
congenital heart disease;
and a gastrointestinal disorder such as dysphagia, peptic esophagitis,
esophageal spasm,
esophageal stricture, esophageal carcinoma, dyspepsia, indigestion, gastritis,
gastric carcinoma,
anorexia, nausea, emesis, gastroparesis, antral or pyloric edema, abdominal
angina, pyrosis,
gastroenteritis, intestinal obstruction, an infection of the intestinal tract,
peptic ulcer, cholelithiasis,
cholecystitis, cholestasis, pancreatitis, pancreatic carcinoma, biliary tract
disease, hepatitis,
hyperbilirubinemia, cirrhosis, passive congestion of the liver, hepatoma,
infectious colitis,
ulcerative colitis, ulcerative proctitis, Crohn's disease, Whipple's disease,
Mallory-Weiss
syndrome, colonic carcinoma, colonic obstruction, irritable bowel syndrome,
short bowel
syndrome, diarrhea, constipation, gastrointestinal hemorrhage, and acquired
immunodeficiency
syndrome (AIDS) enteropathy. The polynucleotide sequences encoding HRAP may be
used in
Southern or northern analysis, dot blot, or other membrane-based technologies;
in PCR
technologies; in dipstick, pin, and ELISA assays; and in microarrays utilizing
fluids or tissues
from patients to detect altered lvIRAP expression. Such qualitative or
quantitative methods are
well known in the art.
In a particular aspect, the nucleotide sequences encoding I-IRAP may be useful
in assays
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wo oorosiss ~.~us~n~»~
that detect the presence of associated disorders, particularly those mentioned
above. The
nucleotide sequences encoding I-IRAP may be labeied 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 quantitated
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 HRAP 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
HRAP, 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 I-IRAP, 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
I-iRAP 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 HRAP, or a fragment of a polynucleotide complementary
to the
polynucleotide encoding HRAP, and will be employed under optimized conditions
for
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WO 00/08155 PCT/US99/17777
identification of a specific gene or condition. Oligomers may also be employed
under less
stringent conditions for detection or quantitation of closely related DNA or
RNA sequences.
Methods which may also be used to quantitate the expression of HRAP 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. 229-236.) The
speed of
quantitation of multiple samples may be accelerated by running the assay in an
ELISA format
where the oligomer of interest is presented in various dilutions and a
spectrophotometric or
colorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as targets in a
microamay. 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. 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.
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 HRAP
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., Harnngton,
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: i49-154.)
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) site.
Correlation between the
location of the gene encoding HRAP 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
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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, HRAP, 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 HRAP 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 HRAP, or
fragments thereof, and washed. Bound HRAP is then detected by methods well
known in the art.
Purified HRAP 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 HRAP specifically compete with a
test compound for
binding HRAP. In this manner, antibodies can be used to detect the presence of
any peptide which
shares one or more antigenic determinants with HRAP.
In additional embodiments, the nucleotide sequences which encode HRAP may be
used in
any molecular biology techniques that have yet to be developed, provided the
new techniques rely
on properties of nucleotide sequences that are currently known, including, but
not limited to, such
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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 anyway whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below,
in particular U.S. Ser. No. [Atty. Docket No. PF-0571 P] filed August 7, 1998,
and U.S. Ser. No.
60/098,703, filed September 1, 1998, 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
IS Technologies), a monophasic solution of phenol and guanidine
isothiocyanate. The resulting
lysates were centrifuged over CsCI cushions or extracted with chloroform. RNA
was precipitated
from the lysates with either isopropanol or sodium acetate and ethanol, or by
other routine
methods.
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, Valencia CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
Alternatively,
RNA was isolated directly from tissue lysates using other RNA isolation kits,
e.g., the
POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding
cDNA libraries. Otherwise, cDNA was synthesized and cDNA libraries were
constructed with the
UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life
Technologies),
using the recommended procedures or similar methods known in the art. (See,
e.g., Ausubel,
1997, supra, units 5.1-6.6). Reverse transcription was initiated using oligo
d(T) or random
primers. Synthetic oligonucleotide adapters were ligated to double stranded
cDNA, and the cDNA
was digested with the appropriate restriction enzyme or enzymes. For most
libraries, the cDNA
was size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or
SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or
preparative
agarose gel electrophoresis. cDNAs were ligated into compatible restriction
enzyme sites of the
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polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene),
PSPORTI plasmid
(Life Technologies), or pINCY (Incyte Pharmaceuticals, Palo Alto CA).
Recombinant plasmids
were transformed into competent E. coli cells including XL1-Blue, XL1-BIueMRF,
or SOLR from
Stratagene or DHSa, DH 1 OB, or ElectroMAX DH 1 OB 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 cell lysis. Plasmids were purified using at least one
of the following: a
Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep
pwification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid,
QIAWELL 8
Pius Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the REAL Prep 96
plasmid 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 PCB in
a high-throughput format (Rao, V.B. ( 1994) Anal. Biochem. 216:1-14). Host
cell lysis and
IS 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
The cDNAs were prepared for sequencing using the ABI CATALYST 800 (Perkin-
Elmer)
or the HYDRA microdispenser (Bobbins Scientific) or MICROLAB 2200 (Hamilton)
systems in
combination with the PTC-200 thermal cyclers (MJ Research). The cDNAs were
sequenced using
the ABI PRISM 373 or 377 sequencing systems (Perkin-Elmer) and standard ABI
protocols, base
calling software, and kits. In one alternative, cDNAs were sequenced using the
MEGABACE
1000 DNA sequencing system (Molecular Dynamics). In another alternative, the
cDNAs were
amplified and sequenced using the ABI PRISM BIGDYE Terminator cycle sequencing
ready
reaction kit (Perkin-Elmer). In yet another alternative, cDNAs were sequenced
using solutions
and dyes from Arnersham Pharmacia Biotech. Reading frames for the ESTs were
determined
using standard methods (reviewed in Ausubel, 1997, ssupra, unit 7.7). Some of
the cDNA
sequences were selected for extension using the techniques disclosed in
Example V.
The polynucleotide sequences derived from cDNA, extension, and shotgun
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 software
programs, descriptions,
references, and threshold parameters used. The first column of Table 5 shows
the tools, programs,
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and algorithms used, the second column provides a brief description thereof,
the third column
presents the references which are incorporated by reference herein, 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 probability the
greater the
homology). Sequences were analyzed using. MACDNASIS PRO software (Hitachi
Software
Engineering, S. San Francisco CA) and LASERGENE software (DNASTAR).
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 GenBank primate,
rodent, mammalian,
vertebrate, and eukaryote databases, and BLOCKS 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,
PFAM, and Prosite.
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, s_ upra. 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 database
(Incyte
Pharmaceuticals). This analysis is much faster than multiple membrane-based
hybridizations. In
addition, the sensitivity of the computer search can be modified to determine
whether any
particular match is categorized as exact or similar. The basis of the search
is the product score,
which is defined as:
%~s uence 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
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molecules are usually identified by selecting those which show product scores
between IS and 40,
although lower scores may identify related molecules.
The results of northern analyses are reported a percentage distribution of
libraries in which
the transcript encoding HRAP occurred. Analysis involved the categorization of
cDNA libraries
by organ/tissue and disease. The orgat~/tissue categories included
cardiovascular, dermatologic,
developmental, endocrine, gastrointestinal, hematopoietic/immune,
musculoskeletal, nervous,
reproductive, and urologic. The disease categories included cancer,
inflammation/trauma, fetal,
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 expression are reported in Table 3.
V. Extension of HRAP Encoding Polynucleotides
The full length nucleic acid sequences of SEQ ID N0:17-22 were produced by
extension
of the component fragments described in Table I, Column 5, using
oligonucleotide primers based
on those fragments. One primer was synthesized to initiate extension of an
antisense
polynucleotide, and the other was synthesized to initiate extension of a sense
polynucleotide.
Primers were used to facilitate the extension of the known sequence "outward"
generating
amplicons containing new unknown nucleotide sequence for the region of
interest. The initial
primers were designed from the cDNA 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 (Life Technologies) were used to extend the
sequence. If
more than one extension is necessary or desired, additional sets of primers
are designed to further
extend the known region.
High fidelity amplification was obtained by following the instructions for the
XI,-PCR kit
(Perkin-Elmer Corp.) and thoroughly mixing the enzyme and reaction mix. PCR
was performed
using the PTC-200 thermal cycler (MJ Research), beginning with 40 pmol of each
primer and the
recommended concentrations
of all other
components of
the kit, with
the following
parameters:
Step 1 94 C for 1 min (initial denaturation)
Step 2 65 C for 1 min
Step 3 68 C for 6 min
SAP 4 94 C for 15 sec
Step 5 65 C for 1 min
Step 6 68 C for 7 min
Step 7 Repeat steps 4 through 6 for an additional
15 cycles
Step 8 94 C for 15 sec
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Step 9 65 ° C for 1 min
Step 10 68° C for 7:15 min
Step I I Repeat steps 8 through 10 for an additional 12 cycles
Step 12 72 ° C for 8 min
Step I 3 4 ° C (and holding)
A 5 /cl to 10 ~l aliquot of the 'reaction mixture was analyzed by
electrophoresis on a low
concentration (about 0.6% to 0.8%) agarose mini-gel to determine which
reactions were successful
in extending the sequence. Bands thought to contain the largest products were
excised from the
gel, purified using QIAQUICK purification kit (QIAGEN Inc.), and trimmed of
overhangs using
Klenow enzyme to facilitate religation and cloning.
Ai3er ethanol precipitation, the products were redissolved in 13 ~cl of
ligation buffer, l~l
T4-DNA ligase (IS units) and 1~1 T4 polynucleotide kinase were added, and the
mixture was
incubated at room temperature for 2 to 3 hours, or overnight at 16° C.
Competent E. coli cells (in
40 /cl of appropriate media) were transformed with 3 ul of ligation mixture
and cultured in 80 ~cl
of SOC medium. (See, e.g., Sambrook, supra, Appendix A, p. 2.) After
incubation for one hour at
37°C, the E. coli mixture was plated on Luria Bertani (LB) agar (See,
e.g., Sarnbrook, supra,
Appendix A, p. 1 ) containing carbenicillin (2x carb). The following day,
several colonies were
randomly picked from each plate and cultured in I50 /cl of liquid LB/2x carb
medium placed in an
individual well of an appropriate commercially-available sterile 96-well
microtiter plate. The
following day, 5 ,ul of each overnight culture was transferred into a non-
sterile 96-well plate and,
after dilution 1:10 with water, 5 ~1 from each sample was transferred into a
PCR array.
For PCR amplification, 18 ul of concentrated PCR reaction mix (3.3x)
containing 4 units
of rTth DNA polymerise, a vector primer, and one or both of the gene specific
primers used for
the extension reaction were added to each well. Amplification was performed
using the following
conditions:
Step I 94 C for 60 sec
Step 2 94 C for 20 sec
Step 3 55 C for 30 sec
Step 4 72 C for 90 sec
Step 5 Repeat steps 2 through 4 for an additional
29 cycles
Step 6 72 C for 180 sec
Step 7 4 C (and holding)
Aliquots of the PCR reactions were run on agarose gels together with molecular
weight
markers. The sizes of the PCR products were compared to the original partial
cDNAs, and
appropriate clones were selected, ligated into plasmid, and sequenced.
In like manner, the nucleotide sequences of SEQ ID N0:17-22 is used to obtain
5'
regulatory sequences using the procedure above, oligonucleotides designed for
5' extension, and
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an appropriate genomic library.
The full length nucleic acid sequences of SEQ ID N0:23-32 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 SO% 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)ZSO4, and ~-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 1: 94°C, 3
min; Step 2: 94°C, 15 sec;
Step 3: 60°C, I 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, I 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 ul
PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes,
Eugene OR)
dissolved in 1 XTE and 0.5 pl of undiluted PCR product into each well of an
opaque fluorimeter
plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent. The
plate was
scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the
fluorescence of the
sample and to quantify the concentration of DNA. A S ~cl to 10 ~cl 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
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clones were relegated using T4 ligase (New England Biolabs, Beverly MA) into
pUC 18 vector
(Amersham Pharmacia Biotech), treated with Pfu DNA polymerise (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 polymerise
(Amersham Pharmacia Biotech) and Pfu DNA polymerise (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% dimethysulphoxide (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 sequence of SEQ iD N0:23-32 is used to obtain
5'
regulatory sequences using the procedure above, oligonucleotides designed for
such extension,
and an appropriate genomic library.
VI. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:17-32 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
oiigomer, 250 uCi of
[Y aZp] adenosine triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 supe~ne size exclusion dextrin bead column (Amersham Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-
based hybridization analysis of human genomic DNA digested with one of the
following
endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xbal, 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 increasingly stringent conditions up to 0.1 x saline sodium citrate and
0.5% sodium dodecyl
sulfate. After XOMAT-AR film (Eastman Kodak, Rochester NY) is exposed to the
blots to film
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for several hours, hybridization patterns are compared visually.
VII. 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 complementarily 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; Shalon, 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.
VIII. Complementary Polynncleotides
Sequences complementary~to the HRAP-encoding sequences, or any parts thereof,
are
used to detect, decrease, or inhibit expression of naturally occurring HRAP.
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
HRAP. To inhibit transcription, a complementary oligonucleotide is designed
from the most
unique 5' sequence and used to prevent promoter binding to the coding
sequence. To inhibit
translation, a complementary oligonucleotide is designed to prevent ribosomal
binding to the
HRAP-encoding transcript.
IX. Expression of HRAP
Expression and purification of HRAP is achieved using bacterial or virus-based
expression systems. For expression of HRAP in bacteria, cDNA is subcloned into
an appropriate
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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 HRAP upon induction
with isopropyl beta-
D-thiogalactopyranoside (IPTG). Expression of HRAP in eukaryotic cells is
achieved by infecting
insect or mammalian cell lines with recombinant Autographica californica
nuclear polyhedrosis
virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin
gene of
baculovirus is replaced with cDNA encoding HRAP by either homologous
recombination or
bacterial-mediated transposition involving transfer plasmid intermediates.
Viral infectivity is
maintained and the strong polyhedrin promoter drives high levels of cDNA
transcription.
Recombinant baculovirus is used to infect Spodoptera fru ~~iperda (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
IS 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945.)
In most expression systems, HRAP is synthesized as a fusion protein with,
e.g.,
glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-
His, permitting rapid,
single-step, affinity-based purification of recombinant fusion protein from
crude cell lysates.
GST, a 26-kilodalton enzyme from _Schistosoma ia~onicum, 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 HRAP 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 HRAP obtained by
these methods can be used directly in the following activity assay.
X. Demonstration of HRAP Activity
Receptor activity of HRAP is determined in a ligand-binding assay using
candidate
ligand molecules in the presence of '~I-labeled HRAP. HRAP is labeled with'ZSI
Bolton-Hunter
reagent. (See, e.g., Bolton et al. (1973) Biochem. J. 133:529.) Candidate
ligand molecules
previously arrayed in the wells of a multi-well plate are incubated with the
labeled HRAP, washed,
and any wells with labeled HRAP complex are assayed. Data obtained using
different
concentrations of HRAP are used to calculate values for the number, affinity,
and association of
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HRAP with the ligand molecules.
Alternatively, HRAP activity is determined by measuring the stimulation of DNA
synthesis in Swiss mouse 3T3 cells. Plasmids containing polynucleotides
encoding HRAP are
added to quiescent 3T3 cultured cells using transfection methods well known in
the art and the
transfected cells are then incubated in the presence of [3H]thymidine, a
radioactive DNA
precursor. Varying amounts of HRAP ligand are then added to the cultured
cells. Incorporation
of ['H]thymidine into acid-precipitable DNA is measured over an appropriate
time interval using a
radioisotope counter, and the amount incorporated is directly proportional to
the amount of newly
synthesized DNA. A linear dose-response curve over at least a hundred-fold
HRAP ligand
concentration range is indicative of receptor activity. One unit of activity
per milliliter is defined
as the concentration of HRAP producing a 50% response level, where 100%
represents maximal
incorporation of ['H]thymidine into acid-precipitable DNA (McICay, I. and
Leigh, L, eds. (1993)
Growth Factors: A Practical Approach, Oxford University Press, New York, NY,
page 73).
XI. Functional Assays
HRAP function is assessed by expressing the sequences encoding HRAP 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 (Life Technologies) and
pCR3.1
(Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter.
5-10 ~cg of
recombinant vector are transiently transfected into a human cell line,
preferably of endothelial or
hematopoietic origin, using either liposome formulations or electroporation. 1-
2 ug of an
additional plasmid containing sequences encoding a marker protein are co-
transfected. Expression
of a marker protein provides a means to distinguish transfected cells from
nontransfected cells and
is a reliable predictor of cDNA expression from the recombinant vector. Marker
proteins of
choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a
CD64-GFP fusion
protein. Flow cytometry (FCM), an automated, laser optics-based technique, is
used to identify
transfected cells expressing GFP or CD64-GFP, and to evaluate properties, for
example, their
apoptotic state. 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
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CA 02338385 2001-02-06
wo oorosiss pornrs~n~~~~
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M. G. ( 1994) Flow Cvtometry, Oxford, New York NY.
The influence of HRAP on gene expression can be assessed using highly purified
populations of cells transfected with sequences encoding HRAP 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 HRAP and other genes
of interest can
be analyzed by northern analysis or microarray techniques.
XII. Production of HRAP Specific Antibodies
HRAP 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 HRAP 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, su~,ra, ch. 11.)
Typically, oligopeptides 15 residues in length are synthesized using an ABI
431A
Peptide Synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to ICLH
(Sigma-Aldrich,
St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester
(MBS) to
increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are
immunized with the
oligopeptide-ICI,H complex in complete Freund's adjuvant. Resulting antisera
are tested for
antipeptide activity by, for example, binding the peptide to plastic, blocking
with 1% BSA,
reacting with rabbit antisera, washing, and reacting with radio-iodinated goat
anti-rabbit IgG.
XIII. Purification of Naturally Occurring )(TRAP Using Specific Antibodies
Naturally occurring or recombinant HRAP is substantially purified by
immunoaffinity
chromatography using antibodies specific for HRAP. An immunoaffinity column is
constructed
by covalently coupling anti-HRAP 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 HRAP are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of HRAP (e.g.,
high ionic strength
-53-
CA 02338385 2001-02-06
WO 00/08155 PCTNS99/17777
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/HRAP 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 HR.AP is collected.
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
specific preferred
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.
-54-
CA 02338385 2001-02-06
WO 00/08155 PCT/US99/17777
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czlU G~U ts7U wcn c t~~~ L"~~~., '~
c ~ a .. a ~~~ 4~.~~ ~~~~ s_..'_u
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.=
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.v ~ ~ _ 8
a ._ ~ ~ y~E
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..
wv ~ ~v~~ 0. ~ ~ ~'~~ ~ _ °'
was..
~c ~ ~. ~. a ~.
'u
E
w ~ d
..
a ~a a as u, m a~'""
-66-
CA 02338385 2001-02-06
WO 00/08155 PCT/US99/17777
V
R
r ~ '
a s~'
' ~a
O
vo
II
N
a
- > N eo
.z ~a~a ~
v ~.. oo~~ So
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u~~ ~~ ~~ oa ~ -N =
o°°o ~ '~ (~ ~~ °° E op ° '~ > U
ai ~ 3 v~ o ,~.. c~
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v w ~ ~, N ~ ~~L v rr ~ ~ '~' (~ 3
in1 V at ~ N . ad ~ ~ 00 ''
ivy' Gt;N'~~a ~
V O O. ~ ~ ~ ~ ~~,. . Er
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Z-c~ m~°.t7
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4_~
-67-
CA 02338385 2001-02-06
WO 00/08155 PGT/US99/17777
SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS, INC.
HILLMAN, Jennifer L.
YUE, Henry
LAL, Preeti .
TANG, Y. Tom
GORGONE, Gina A.
GUEGLER, Karl J.
CORLEY, Neil C.
BAUGHN, Mariah R.
<120> HUMAN RECEPTOR-ASSOCIATED PROTEINS
<130> PF-0571 PCT
<140> To Be Assigned
<141> Herewith
<150> 09/130,884; Unassigned; 60/098,703
<151> 1998-08-07; 1998-08-07; 1998-09-O1
<160> 32
<170> PERL Program
<2I0> 1
<211> 345
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> incyte Clone No: 1877651
<400> 1
Met His Leu Val Gly Gly Ser Cys Glu Val Trp Phe Pro Asp Val
1 5 10 15
Leu Gln Gln Val Pro Leu Pro Cys Leu Trp Ala Pro Ser Met Ala
20 25 30
Asn Ser Ala Met Asp Thr Arg Val Leu Cys Cys Ala Val Ile Cys
35 40 45
Leu Leu Gly Ala Gly Leu Ser Asn Ala Gly Val Met Gln Asn Pro
50 55 60
Arg His Leu Val Arg Arg Arg Gly Gln Glu Ala Arg Leu Arg Cys
65 70 75
Ser Pro Met Lys Gly His Ser His Val Tyr Trp Tyr Arg Gln Leu
80 85 90
Pro Glu Glu Gly Leu Lys Phe Met Val Tyr Leu Gln Lys Glu Asn
95 100 105
Ile Ile Asp Glu Ser Gly Met Pro Lys Glu Arg Phe Ser Ala Glu
110 115 120
Phe Pro Lys Glu Gly Pro Ser Ile Leu Arg Ile Gln Gln Val Val
125 130 135
Arg Gly Asp Ser Ala Ala Tyr Phe Cys Ala Ser Ser Pro His Ser
140 145 150
1~g
CA 02338385 2001-02-06
wo oorosiss pcrius~n~~~7
Lys Gln Ala Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val
155 160 165
Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe
170 175 180
Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu
185 190 195
Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser
200 205 210
Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp
215 220 225
Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr
230 235 240
Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn
245 250 255
Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser
260 265 270
Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln
275 280 285
Ile Val Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr
290 295 300
Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr
305 310 315
Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser
320 325 330
Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp Ser Arg Gly
335 340 345
<210> 2
<211> 487
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2906971
<400> 2
Met Ala Ser Ser Ala Glu Gly Asp Glu Gly Thr Val Val Ala Leu
1 5 10 15
Ala Gly Val Leu Gln Ser Gly Phe Gln Glu Leu Ser Leu Asn Lys
20 25 30
Leu Ala Thr Ser Leu Gly Ala Ser Glu GIn Ala Leu Arg Leu Ile
35 40 45
Ile Ser Ile Phe Leu Gly Tyr Pro Phe Ala Leu Phe Tyr Arg His
50 55 60
Tyr Leu Phe Tyr Lys Glu Thr Tyr Leu Ile His Leu Phe His Thr
65 70 75
Phe Thr Gly Leu Ser Ile Ala Tyr Phe Asn Phe Gly Asn Gln Leu
80 85 90
Tyr His Ser Leu Leu Cys Ile Val Leu Gln Phe Leu Ile Leu Arg
95 100 105
Leu Met Gly Arg Thr Ile Thr Ala Val Leu Thr Thr Phe Cys Phe
I10 115 120
Gln Met Ala Tyr Leu Leu Ala Gly Tyr Tyr Tyr Thr Ala Thr Gly
2/28
CA 02338385 2001-02-06
wo oorosiss poi,~usml~~~~
125 130 135
Asn Tyr Asp Ile Lys Trp Thr Met Pro His Cys Val Leu Thr Leu
140 145 150
Lys Leu Ile Gly Leu Ala Val Asp Tyr Phe Asp Gly Gly Lys Asp
155 160 165
Gln Asn Ser Leu Ser Ser Glu Gln Gln Lys Tyr Ala Ile Arg Gly
170 . 175 180
Val Pro Ser Leu Leu Glu Val Ala Gly Phe Ser Tyr Phe Tyr Gly
185 190 195
Ala Phe Leu Val Gly Pro Gln Phe Ser Met Asn His Tyr Met Lys
200 205 210
Leu Val Gln Gly Glu Leu Ile Asp Ile Pro Gly Lys Ile Pro Asn
215 220 225
Ser Ile Ile Pro Ala Leu Lys Arg Leu Ser Leu Gly Leu Phe Tyr
230 235 240
Leu Val Gly Tyr Thr Leu Leu Ser Pro His Ile Thr Glu Asp Tyr
245 250 255
Leu Leu Thr Glu~Asp Tyr Asp Asn His Pro Phe Trp Phe Arg Cys
260 265 270
Met Tyr Met Leu Ile Trp Gly Lys Phe Val Leu Tyr Lys Tyr Val
275 280 285
Thr Cys Trp Leu Val Thr Glu Gly Val Cys Ile Leu Thr Gly Leu
290 295 300
Gly Phe Asn Gly Phe Glu Glu Lys Gly Lys Ala Lys Trp Asp Ala
305 310 315
Cys Ala Asn Met Lys Val Trp Leu Phe Glu Thr Asn Pro Arg Phe
320 325 330
Thr Gly Thr Ile Ala Ser Phe Asn Ile Aen Thr Asn Ala Trp Val
335 340 345
Ala Arg Tyr Ile Phe Lys Arg Leu Lys Phe Leu Gly Asn Lys Glu
350 355 360
Leu Ser Gln Gly Leu Ser Leu Leu Phe Leu Ala Leu Trp His Gly
365 370 375
Leu His Ser Gly Tyr Leu Val Cys Phe Gln Met Glu Phe Leu Ile
380 385 390
Val Ile Val Glu Arg Gln Ala Ala Arg Leu Ile Gln Glu Ser Pro
395 400 405
Thr Leu Ser Lys Leu Ala Ala Ile Thr Val Leu Gln Pro Phe Tyr
410 415 420
Tyr Leu Val Gln Gln Thr Ile His Trp Leu Phe Met Gly Tyr Ser
425 430 435
Met Thr Ala Phe Cys Leu Phe Thr Trp Asp Lys Trp Leu Lys Val
440 445 450
Tyr Lys Ser Ile Tyr Phe Leu Gly His Ile Phe Phe Leu Ser Leu
455 460 465
Leu Phe Ile Leu Pro Tyr Ile His Lys Ala Met Val Pro Arg Lys
470 475 480
Glu Lys Leu Lys Lys Met Glu
485
<210> 3
<211> 312
<212> PRT
<213> Homo sapiens
3/28
CA 02338385 2001-02-06
wo ooiosiss ~,ius99n7~77
<220>
<221> misc_feature
<223> Incyte Clone No: 2907954
<400> 3
Met Gly Thr Arg Leu Leu Cys Leu Leu
Trp Ala Ala Leu Cys Gly
1 5 - l0 15
Ala Glu Leu Thr Glu Ala Gly Arg Tyr
Val Ala Gln Ser Pro Lys
20 25 30
Ile Ile Glu Lys Arg Gln Ser Phe Trp Asn Pro
Val Ala Cys Ile
35 40 45
Ser Gly His Ala Thr Leu Tyr Gln Gln Leu Gly
Trp Tyr Ile Gln
50 55 60
Gly Pro Lys Leu Leu Ile Gln Asn Asn Val Val
Phe Gln Gly Asp
65 70 75
Aep Ser Gln Leu Pro Lys Asp Ser Ala Arg Leu
Arg phe Glu Lys
80 85 90
Gly Val Asp Ser Thr Leu Lys Pro Ala Leu Glu
Ile Gln Lys Asp
95 100 105
Ser Ala Val Tyr Leu Cys Ala Phe Leu Arg Asn
Ser Ser Asp Asn
110 115 120
Glu Gln Phe Phe Gly Pro Gly Leu Thr Leu Glu
Thr Arg Val Asp
125 130 135
Leu Lys Asn Val Phe Pro Pro Ala Val Glu Pro
Glu Val Phe Ser
140 145 150
Glu Ala Glu Ile Ser His Thr Ala Thr Val Cys
Gln Lys Leu Leu
155 160 165
Ala Thr Gly Phe Tyr Pro Asp Glu Leu Trp Trp
His Val Ser Val
170 175 180
Asn Gly Lys Glu Val His Ser Ser Thr Pro Gln
Gly Val Asp Pro
185 190 195
Leu Lys Glu Gln Pro Ala Leu Ser Arg Cys Leu
Asn Asp Tyr Ser
200 205 210
Ser Arg Leu Arg Val Ser Ala Trp Gln Pro Arg
Thr Phe Asn Asn
215 220 225
His Phe Arg Cys Gln Val Gln Gly Leu Glu Asn
Phe Tyr Ser Asp
230 235 240
Glu Trp Thr Gln Asp Arg Ala Val Thr Ile Val
Lys Pro Gln Ser
245 250 255
Ala Glu Ala Trp Gly Arg Ala Gly Phe Ser Glu
Asp Cys Thr Ser
260 265 270
Tyr Gln Gln Gly Val Leu Ser Ile Leu Glu Ile
Ala Thr Tyr Leu
275 280 285
Leu Gly Lys Ala Thr Leu Tyr Leu Val
Ala Val Ser Ala
Leu Val
290 295 300
Leu Met Ala Met Val Lys Arg Ser Arg
Lys Asp Gly
305 310
<210> 4
<211> 309
<212> PRT
<213> Homo sapiens
<220>
4/28
CA 02338385 2001-02-06
wo oo/u8iss
PCT/US99/17777
<221> misc_feature
<223> Incyte Clone No: 3083742
<400> 4
Met Asn Gly Thr Tyr Asn Thr Ser Ser Asp Leu Trp
Cys Gly Thr
1 5 10 15
Pro Pro Ala Ile Lys Leu Gly Ala Tyr Leu Gly Leu
Phe Tyr Val
20 25 30
Leu Val Leu Gly Leu Leu Leu Leu Ala Leu Trp Phe
Asn Ser Val
35 40 45
Cys Cys Arg Met Gln Gln Trp Thr Arg Ile Tyr Thr
Thr Glu Met
50 55 60
Asn Leu Ala Val Ala Asp Leu Leu Cys Thr Leu Phe
Cys Leu Pro
65 70 75
Val Leu His Ser Leu Arg Asp Asp Thr Pro Leu Gln
Thr Ser Cys
80 85 90
Leu Ser Gln Gly Ile Tyr Leu Arg Tyr Met Ser Ser
Thr Asn Ile
95 100 105
Leu Val Thr Ala Ile Ala Val Tyr Val Ala Val His
Asp Arg Arg
110 115 120
Pro Leu Arg Ala Arg Gly Leu Pro Arg Gln Ala Ala
Arg Ser Ala
125 130 135
Val Cys Ala Val Leu Trp Val Ile Gly Ser Leu Ala
Leu Val Val
140 145 150
Arg Trp Leu Leu Gly Ile Gln Gly Phe Cys Phe Ser
Glu Gly Arg
155 160 165
Thr Arg His Asn Phe Asn Ser Phe Pro Leu Leu Phe
Met Ala Gly
170 175 180
Tyr Leu Pro Leu Ala Val Val Cys Ser Leu Lys Val
Val Phe Val
185 190 195
Thr Ala Leu Ala Gln Arg Pro Asp Val Gly Gln Glu
Pro Thr Ala
200 205 210
Ala Thr Arg Lys Ala Ala Arg Trp Ala Asn Leu Val
Met Val Leu
215 220 225
Phe Val Val Cys Phe Leu Pro Val Gly Leu Thr Arg
Leu His Val
230 235 240
Leu Ala Val Gly Trp Asn Ala Leu Leu Glu Thr Arg
Cys Ala Ile
245 250 255
Arg Ala Leu Tyr Ile Thr Ser Ser Asp Ala Asn Cys
Lys Leu Cys
260 265 270
Leu Asp Ala Ile Cys Tyr Tyr Ala Lys Glu Phe Glu
Tyr Met Gln
275 280 285
Ala Ser Ala Leu Ala Val Ala Ser
Pro Arg Ala Lys Ala His Lys
290 295 300
Gln Asp Ser Leu Cys Val Thr
Leu Ala
305
<210> 5
<211> 367
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
size
CA 02338385 2001-02-06
wo oorosiss po,I,NS99n~77~
<223> Incyte Clone No: 3407686
<400> 5
Met Ile Arg Asn Trp Leu Thr Ile Phe Ile Leu Phe Pro Leu Lys
1 . 5 10 15
Leu Val Glu Lys Cys Glu Ser Ser Val Ser Leu Thr Val Pro Pro
20 . 25 30
Val Val Lys Leu Glu Asn Gly Ser Ser Thr Asn Val Ser Leu Thr
35 40 45
Leu Arg Pro Pro Leu Asn Ala Thr Leu Val Ile Thr Phe Glu Ile
50 55 60
Thr Phe Arg Ser Lys Asn Ile Thr Ile Leu Glu Leu Pro Asp Glu
65 70 75
Val Val Val Pro Pro Gly Val Thr Asn Ser Ser Phe Gln Val Thr
80 85 90
Ser Gln Asn Val Gly Gln Leu Thr Val Tyr Leu His Gly Asn His
95 100 105
Ser Asn Gln Thr Gly Pro Arg Ile Arg Phe Leu Val Ile Arg Ser
110 115 120
Ser Ala Ile Ser Ile Ile Asn Gln Val Ile Gly Trp Ile Tyr Phe
125 130 135
Val Ala Trp Ser Ile Ser Phe Tyr Pro Gln Val Ile Met Asn Trp
140 145 150
Arg Arg Lys Ser Val Ile Gly Leu Ser Phe Asp Phe Val Ala Leu
155 160 165
Asn Leu Thr Gly Phe Val Ala Tyr Ser Val Phe Asn Ile Gly Leu
170 175 180
Leu Trp Val Pro Tyr Ile Lys Glu Gln Phe Leu Leu Lys Tyr Pro
185 190 195
Asn Gly Val Asn Pro Val Asn Ser Asn Asp Val Phe Phe Ser Leu
200 205 210
His Ala Val Val Leu Thr Leu Ile Ile Ile Val Gln Cys Cys Leu
215 220 225
Tyr Glu Arg Gly Gly Gln Arg Val Ser Trp Pro Ala Ile Gly Phe
230 235 240
Leu Val Leu Ala Trp Leu Phe Ala Phe Val Thr Met Ile Val Ala
245 250 255
Ala Val Gly Val Ile Thr Trp Leu Gln Phe Leu Phe Cys Phe Ser
260 265 270
Tyr Ile Lys Leu Ala Val Thr Leu Val Lys Tyr Phe Pro Gln Ala
275 280 285
Tyr Met Asn Phe Tyr Tyr Lys Ser Thr Glu Gly Trp Ser Ile Gly
290 295 300
Asn Val Leu Leu Asp Phe Thr Gly Gly Ser Phe Ser Leu Leu Gln
305 310 315
Met Phe Leu Gln Ser Tyr Asn Asn Asp Gln Trp Thr Leu Ile Phe
320 325 330
Gly Asp Pro Thr Lys Phe Gly Leu Gly Val Phe Ser Ile Val Phe
335 340 345
Asp Val Val Phe Phe Ile Gln His Phe Cys Leu Tyr Arg Lys Arg
350 355 360
Pro Gly Tyr Asp Gln Leu Asn
365
6/28
CA 02338385 2001-02-06
WO 00/08155 PCT/US99/17777
<210> 6
<211> 386
<212> PRT
<213> Homo sapiena
<220>
<221> misc_feature
<223> Incyte Clone No: 3472455
<400> 6
Met Gly Leu Trp Gly Gln Ser Val Pro Thr Ala Ser Ser Ala Arg
1 5 10 15
Ala Gly Arg Tyr Pro Gly Ala Arg Thr Ala Ser Gly Thr Arg Pro
20 25 30
Trp Leu Leu Asp Pro Lys Ile Leu Lys Phe Val Val Phe Iie Val
35 40 45
Ala Val Leu Leu Pro Val Arg Val Asp Ser Ala Thr Ile Pro Arg
50 55 60
Gln Asp Glu Val Pro Gln Gln Thr Val Ala Pro Gln Gln Gln Arg
65 70 75
Arg Ser Leu Lys Glu Glu Glu Cys Pro Ala Gly Ser His Arg Ser
B0 B5 90
Glu Tyr Thr Gly Ala Cys Asn Pro Cys Thr Glu Gly Val Asp Tyr
95 100 105
Thr Ile Ala Ser Asn Asn Leu Pro Ser Cys Leu Leu Cys Thr Val
110 115 120
Cys Lys Ser Gly Gln Thr Asn Lys Ser Ser Cys Thr Thr Thr Arg
125 130 135
Asp Thr Val Cys Gln Cys Glu Lys Gly Ser Phe Gln Asp Lys Asn
140 145 150
Ser Pro Glu Met Cys Arg Thr Cys Arg Thr Gly Cys Pro Arg Gly
155 160 165
Met Val Lys Val Ser Asn Cys Thr Pro Arg Ser Asp Ile Lys Cys
170 175 1B0
Lys Asn Glu Ser Ala Ala Ser Ser Thr Gly Lys Thr Pro Ala Ala
185 190 195
Glu Glu Thr Val Thr Thr Ile Leu Gly Met Leu Ala Ser Pro Tyr
200 205 210
His Tyr Leu Ile Ile Ile Val Val Leu Val Ile Ile Leu Ala Val
215 220 225
Val Val Val Gly Phe Ser Cys Arg Lys Lys Phe Ile Ser Tyr Leu
230 235 240
Lys Gly Ile Cys Ser Gly Gly Gly Gly Gly Pro Glu Arg Val His
245 250 255
Arg Val Leu Phe Arg Arg Arg Ser Cys Pro Ser Arg Val Pro Gly
260 265 270
Ala Glu Asp Asn Ala Arg Asn Glu Thr Leu Ser Asn Arg Tyr Leu
275 280 285
Gln Pro Thr Gln Val Ser Glu Gln Glu Ile Gln Gly Gln Glu Leu
290 295 300
Ala Glu Leu Thr Gly Val Thr Val Glu Ser Pro Glu Glu Pro Gln
305 310 315
Arg Leu Leu Glu Gln Ala Glu Ala Glu Gly Cys Gln Arg Arg Arg
320 325 330
Leu Leu Val Pro Val Asn Asp Ala Asp Ser Ala Asp Ile Ser Thr
335 340 345
Leu Leu Asp Ala Ser Ala Thr Leu Glu Glu Gly His Ala Lys Glu
7/28
CA 02338385 2001-02-06
WO 00/08155 PGTNS99/17777
350 355 360
Thr Ile Gln Asp Gln Leu Val Gly Ser Glu Lys Leu Phe Tyr Glu
365 370 375
Glu Asp Glu Ala Gly Ser Ala Thr Ser Cys Leu
380 385
<210> 7
<211> 346
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 786873
<400> 7
Met Glu Ser Asn Leu Ser Gly Leu Val Pro Ala Ala Gly Leu Val
1 5 10 15
Pro Ala Leu Pro Pro Ala Val Thr Leu Gly Leu Thr Ala Ala Tyr
20 25 30
Thr Thr Leu Tyr Ala Leu Leu Phe Phe Ser Val Tyr Ala Gln Leu
35 40 45
Trp Leu Val Leu Leu Tyr Gly His Lys Arg Leu Ser Tyr Gln Thr
50 55 60
Val Phe Leu Ala Leu Cys Leu Leu Trp Ala Ala Leu Arg Thr Thr
65 70 75
Leu Phe Ser Phe Tyr Phe Arg Asp Thr Pro Arg Ala Asn Arg Leu
80 85 90
Gly Pro Leu Pro Phe Trp Leu Leu Tyr Cys Cys Pro Val Cys Leu
95 100 105
Gln Phe Phe Thr Leu Thr Leu Met Asn Leu Tyr Phe Ala Gln Val
110 115 120
Val Phe Lys Ala Lys Val Lys Arg Arg Pro Glu Met Ser Arg Gly
125 130 135
Leu Leu Ala Val Arg Gly Ala Phe Val Gly Ala Ser Leu Leu Phe
140 145 150
Leu Leu Val Asn Val Leu Cys Ala Val Leu Ser His Arg Arg Arg
155 160 165
Ala Gln Pro Trp Ala Leu Leu Leu Val Arg Val Leu Val Ser Asp
170 175 180
Ser Leu Phe Val Ile Cys Ala Leu Ser Leu Ala Ala Cys Leu Cys
185 190 195
Leu Va1 Ala Arg Arg Ala Pro Ser Thr Ser Ile Tyr Leu Glu Ala
200 205 210
Lys Ala Asp Leu Val Asn Asp Leu Gly Asn Lys Gly Tyr Leu Val
215 220 225
Phe Gly Leu Ile Leu Phe Val Trp Glu Leu Leu Pro Thr Thr Leu
230 235 240
Leu Val Gly Phe Phe Arg Val His Arg Pro Pro Gln Asp Leu Ser
245 250 255
Thr Ser His Ile Leu Asn Gly Gln Val Phe Ala Ser Arg Ser Tyr
260 265 270
Phe Phe Asp Arg Ala'Gly His Cys Glu Asp Glu Gly Cys Ser Trp
275 280 285
CA 02338385 2001-02-06
WO 00/08155 PCT/US99/17777
Glu His Ser Arg Gly Glu Ser Thr Ser Met Ser Gly Ser Leu Gly
290 295 300
Ser Gly Ser Trp Tyr Gly Ala Ile Gly Arg Glu Pro Gly Trp Tyr
305 310 315
Gly Gly Ser Gln Thr Lys Thr Thr Pro Leu Leu Phe Ser Gln Val
320 325 330
Pro,Gly Pro Gly Gly His His His Ser Leu Tyr Ser Thr Pro Gln
335 340 345
Thr
<210> 8
<211> 241
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1220371
<400> 8
Met Gly Thr Ala Ser Arg Ser Asn Ile Ala Arg His Leu Gln Thr
1 5 10 15
Asn Leu Ile Leu Phe Cys Val Gly Ala Val Gly Ala Cys Thr Leu
20 25 30
Ser Val Thr Gln Pro Trp Tyr Leu Glu Val Asp Tyr Thr His Glu
35 40 45
Ala Val Thr Ile Lys Cys Thr Phe Ser Ala Thr Gly Cys Pro Ser
50 55 60
Glu Gln Pro Thr Cys Leu Trp Phe Arg Tyr Gly Ala His Gln Pro
65 70 75
Glu Asn Leu Cys Leu Asp Gly Cys Lys Ser Glu Ala Asp Lys Phe
80 85 90
Thr Val Arg Glu Ala Leu Lys Glu Asn Gln Val Ser Leu Thr Val
95 100 105
Asn Arg Val Thr Ser Asn Asp Ser Ala Ile Tyr Ile Cys Gly Ile
110 115 120
Ala Phe Pro Ser Val Pro Glu Ala Arg Ala Lys Gln Thr Gly Gly
125 130 135
Gly Thr Thr Leu Val Val Arg Glu Ile Lys Leu Leu Ser Lys Glu
140 145 150
Leu Arg Ser Phe Leu Thr Ala Leu Vai Ser Leu Leu Ser Val Tyr
155 160 165
Val Thr Gly Val Cys Val Ala Phe Ile Leu Leu Ser Lys Ser Lys
170 175 180
Ser Asn Pro Leu Arg Asn Lys Glu Ile Lys Glu Asp Ser Gln Lys
185 190 195
Lys Lys Ser Ala Arg Arg Ile Phe Gln Glu Ile Ala Gln Glu Leu
200 205 210
Tyr His Lys Arg His Val Glu Thr Asn Gln Gln Ser Glu Lys Asp
215 220 225
Asn Asn Thr Tyr Glu Asn Arg Arg Val Leu Ser Asn Tyr Glu Arg
230 235 240
Pro
9/28
CA 02338385 2001-02-06
WO 00/08155 PGT/US99/17777
<210> 9
<211> 450
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1258785
<400> 9
Met Gly Glu Thr Met Ser Lys Arg Leu Lys Leu His Leu Gly Gly
1 5 10 15
Glu Ala Glu Met Glu Glu Arg Ala Phe Val Asn Pro Phe Pro Asp
20 25 30
Tyr Glu Ala Ala Ala Gly Ala Leu Leu Ala Ser Gly Ala Ala Glu
35 40 45
Glu Thr Gly Cys Val Arg Pro Pro Ala Thr Thr Asp Glu Pro Gly
50 55 60
Leu Pro Phe His Gln Asp Gly Lys Ile Ile His Asn Phe Ile Arg
65 70 75
Arg Ile Gln Thr Lys Ile Lys Asp Leu Leu Gln Gln Met Glu Glu
80 85 90
Gly Leu Lys Thr Ala Asp Pro His Asp Cys Ser Ala Tyr Thr Gly
95 100 105
Trp Thr Gly Ile Ala Leu Leu Tyr Leu Gln Leu Tyr Arg Val Thr
110 115 120
Cys Asp Gln Thr Tyr Leu Leu Arg Ser Leu Asp Tyr Val Lys Arg
125 , 130 135
Thr Leu Arg Asn Leu Asn Gly Arg Arg Val Thr Phe Leu Cys Gly
140 145 150
Asp Ala Gly Pro Leu Ala Val Gly Ala Val Ile Tyr His Lys Leu
155 160 165
Arg Ser Asp Cys Glu Ser Gln Glu Cys Val Thr Lys Leu Leu Gln
170 175 180
Leu Gln Arg Ser Val Val Cys Gln Glu Ser Asp Leu Pro Asp Glu
185 190 195
Leu Leu Tyr Gly Arg Ala Gly Tyr Leu Tyr Ala Leu Leu Tyr Leu
200 205 210
Asn Thr Glu Ile Gly Pro Gly Thr Val Cys Glu Ser Ala Ile Lys
215 220 225
Glu Val Val Asn Ala Ile Ile Glu Ser Gly Lys Thr Leu Ser Arg
230 235 240
Glu Glu Arg Lys Thr Glu Arg Cys Pro Leu Leu Tyr Gln Trp His
245 250 255
Arg Lys Gln Tyr Val Gly Ala Ala His Gly Met Ala Gly Ile Tyr
260 265 270
Tyr Met Leu Met Gln Pro Ala Ala Lys Val Asp Gln Glu Thr Leu
275 280 285
Thr Glu Met Val Lys Pro Ser Ile Asp Tyr Val Arg His Lys Lys
290 295 300
Phe Arg Ser Gly Asn Tyr Pro Ser Ser Leu Ser Asn Glu Thr Asp
305 310 315
Arg Leu Val His Trp Cys His Gly Ala Pro Gly Val Ile His Met
320 325 330
Leu Met Gln Ala Tyr Lys Val Phe Lys Glu Glu Lys Tyr Leu Lys
1 U/28
CA 02338385 2001-02-06
WO 00/08155 PCT/US99/17777
335 340 345
Glu Ala Met Glu Cys Ser Asp Val Ile Trp Gln Arg Gly Leu Leu
350 355 360
Arg Lys Gly Tyr Gly Ile Cys His Gly Thr Ala Gly Asn Gly Tyr
365 370 375
Ser Phe Leu Ser Leu Tyr Arg Leu Thr Gln Asp Lys Lys Tyr Leu
380 ~ 385 390
Tyr Arg Ala Cys Lys Phe Ala Glu Trp Cys Leu Asp Tyr Gly Ala
395 400 405
His Gly Cys Arg Ile Pro Asp Arg Pro Tyr Ser Leu Phe Glu Gly
410 415 420
Met Ala Gly Ala Ile His Phe Leu Ser Asp Val Leu Gly Pro Glu
425 430 435
Thr Ser Arg Phe Pro Ala Phe Glu Leu Asp Ser Ser Lys Arg Asp
440 445 450
<210> 10
<211> 269
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1361202
<400> 10
Met Glu Thr Leu Leu Gly Leu Leu ile Leu Trp Leu Gln Leu Gln
1 5 10 15
Trp Val Ser Ser Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu
20 25 30
Ser Val Pro Glu Gly Glu Asn Leu Val Leu Asn Cys Ser Phe Thr
35 40 45
Asp Ser Ala Ile Tyr Asn Leu Gln Trp Phe Arg Gln Asp Pro Gly
50 55 60
Lys Gly Leu Thr Ser Leu Leu Leu Ile Gln Ser Ser Gln Arg Glu
65 70 75
Gln Thr Ser Gly Arg Leu Asn Ala Ser Leu Asp Lys Ser Ser Gly
80 85 90
Arg Ser Thr Leu Tyr Ile Ala Ala Ser Gln Pro Gly Asp Ser Ala
95 100 105
Thr Tyr Leu Cys Ala Val Arg Asp Asn Asp Met Arg Phe Gly Ala
110 115 120
Gly Thr Arg Leu Thr Val Lys Pro Asn Ile Gln Asn Pro Asp Pro
125 130 135
Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val
140 145 150
Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser
155 160 165
Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met
170 175 180
Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn
185 190 195
Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile
200 205 210
11/28
CA 02338385 2001-02-06
WO 00/08155 PCT/US99/17777
Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val
215 220 225
Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe
230 235 240
Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val
245 250 255
Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser
260 265
<210> 11
<211> 190
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2132846
<400> 11
Met Gly Lys Ser Asn Ser Lys Leu Thr Pro Glu Val Val Glu Glu
1 5 10 15
Leu Thr Arg Lys Thr Tyr Phe Thr Glu Lys Glu VaI Gln Gln Trp
20 25 30
Tyr Lys Gly Phe Ile Lys Asp Cys Pro Ser Gly Gln Leu Asp Ala
35 40 45
Ala Gly Phe Gln Lys Ile Tyr Lys Gln Phe Phe Pro Phe Gly Asp
50 55 60
Pro Thr Lys Phe Ala Thr Phe Val Phe Asn Val Phe Asp Glu Asn
65 70 75
Lys Asp Gly Arg Ile Glu Phe Ser Glu Phe Ile Gln Ala Leu Ser
80 85 90
Val Thr Ser Arg Gly Thr Leu Asp Glu Lys Leu Arg Trp Ala Phe
95 100 105
Lys Leu Tyr Asp Leu Asp Asn Asp Gly Tyr Ile Thr Arg Asn Glu
110 115 120
Met Leu Asp Ile Val Asp Ala Ile Tyr Gln Met Val Gly Asn Thr
125 130 135
Val Glu Leu Pro Glu Glu Glu Asn Thr Pro Glu Lys Arg Val Asp
140 145 150
Arg Ile Phe Ala Met Met Asp Lys Asn Ala Asp Gly Lys Leu Thr
155 160 165
Leu Gln Glu Phe Gln Glu Gly Ser Lys Ala Asp Pro Ser Ile Val
170 175 180
Gln Ala Leu Ser Leu Tyr Asp Gly Leu Val
185 190
<210> 12
<211> 450
<212> PRT
<213> Homo sapiens
12/28
CA 02338385 2001-02-06
WO 00/08155 PGT/US99/17777
<220>
<221> misc_feature
<223> Incyte Clone No: 2539294
<400> 12
Met Ser Asp Met Glu Asp Asp Phe Met Cys Asp Asp Glu Glu Asp
1 . 5 ~ 10 15
Tyr Asp Leu Glu Tyr Ser Glu Asp Ser Asn Ser Glu Pro Asn Val
20 25 30
Asp Leu Glu Asn Gln Tyr Tyr Asn Ser Lys Ala Leu Lys Glu Asp
35 40 45
Asp Pro Lys Ala Ala Leu Ser Ser Phe Gln Lys Val Leu Glu Leu
50 55 60
Glu Gly Glu Lys Gly Glu Txp Gly Phe Lys Ala Leu Lys Gln Met
65 70 75
Ile Lys Ile Asn Phe Lys Leu Thr Asn Phe Pro Glu Met Met Asn
80 85 90
Arg Tyr Lys Gln Leu Leu Thr Tyr Ile Arg Ser Ala Val Thr Arg
95 100 105
Asn Tyr Ser Glu Lys Ser Ile Asn Ser Ile Leu Asp Tyr Ile Ser
110 115 120
Thr Ser Lys Gln Asn Ser Asp Phe Leu Cys Gln Met Asp Leu Leu
125 130 135
Gln Glu Phe Tyr Glu Thr Thr Leu Glu Ala Leu Lys Asp Ala Lys
140 145 150
Asn Asp Arg Leu Trp Phe Lys Thr Asn Thr Lys Leu Gly Lys Leu
155 160 165
Tyr Leu Glu Arg Glu Glu Tyr Gly Lys Leu Gln Lys Ile Leu Arg
170 175 180
Gln Leu His Gln Ser Cys Gln Thr Asp Asp Gly Glu Asp Asp Leu
i85 190 195
Lys Lys Gly Thr Gln Leu Leu Glu Ile Tyr Ala Leu'Glu Ile Gln
200 205 210
Met Tyr Thr Ala Gln Lys Asn Asn Lys Lys Leu Lys Ala Leu Tyr
215 220 225
Glu Gln Ser Leu His Ile Lys Ser Ala Ile Pro His Pro Leu Ile
230 235 240
Met Gly Val Ile Arg Glu Cys Gly Gly Lys Met His Leu Arg Glu
245 250 255
Gly Glu Phe Glu Lys Ala His Thr Asp Phe Phe Glu Ala Phe Lys
260 265 270
Asn Tyr Asp Glu Ser Gly Ser Pro Arg Arg Thr Thr Cys Leu Lys
275 280 285
Tyr Leu Val Leu Ala Asn Met Leu Met Lys Sex Gly Ile Asn Pro
290 295 300
Phe Asp Ser Gln Glu Ala Lys Pro Tyr Lys Asn Asp Pro Glu Ile
305 310 315
Leu Ala Met Thr Asn Leu Val Ser Ala Tyr Gln Asn Asn Asp Ile
320 325 330
Thr Glu Phe Glu Lys Ile Leu Lys Thr Asn His Ser Asn Ile Met
335 340 345
Asp Asp Pro Phe Ile Arg Glu His Ile Glu Glu Leu Leu Arg Asn
350 355 360
Ile Arg Thr Gln Val Leu Ile Lys Leu Ile Lys Pro Tyr Thr Arg
365 370 375
Ile His Ile Pro Phe Ile Ser Lys Glu Leu Asn Ile Asp Val Ala
380 385 390
13/28
CA 02338385 2001-02-06
wo oorosiss rcrius~n~~~~
Asp Val Glu Ser Leu Leu Val Gln Cys Ile Leu Asp Asn Thr Ile
395 400 405
His Gly Arg Ile Asp Gln Val Asn Gln Leu Leu Glu Leu Asp His
410 415 420
Gln Lys Arg Gly Gly Ala Arg Tyr Thr Ala Leu Asp Lys Trp Thr
425 430 435
Asn Gln Leu Asn Ser Leu Asn Gin Ala Val Val Ser Lys Leu Ala
440 445 450
<210> 13
<211> 240
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2589371
<400> 13
Met Leu Leu Gln Ser Gln Thr Met Gly Val Ser His Ser Phe Thr
1 5 10 15
Pro Lys Gly Ile Thr Ile Pro Gln Arg Glu Lys Pro Gly His Met
20 25 30
Tyr Gln Asn Glu Asp Tyr Leu Gln Asn Gly Leu Pro Thr Glu Thr
35 40 45
Thr Val Leu Gly Thr Val Gln Ile Leu Cys Cys Leu Leu Ile Ser
50 55 60
Ser Leu Gly Ala Ile Leu Val Phe Ala Pro Tyr Pro Ser His Phe
65 70 75
Asn Pro Ala Ile Ser Thr Thr Leu Met Ser Gly Tyr Pro Phe Leu
80 ~ 85 90
Gly Ala Leu Cys Phe Gly Ile Thr Gly Ser Leu Ser Ile Ile Ser
95 100 105
Gly Lys Gln Ser Thr Lys Pro Phe Asp Leu Ser Ser Leu Thr Ser
110 115 120
Asn Ala Val Ser Ser Val Thr Ala Gly Ala Gly Leu Phe Leu Leu
125 130 135
Ala Asp Ser Met Val Ala Leu Arg Thr Ala Ser Gln His Cys Gly
140 145 150
Ser Glu Met Asp Tyr Leu Ser Ser Leu Pro Tyr Ser Glu Tyr Tyr
155 160 165
Tyr Pro Ile Tyr Glu Ile Lys Asp Cys Leu Leu Thr Ser Val Ser
170 175 180
Leu Thr Gly Val Leu Val Val Met Leu Ile Phe Thr Val Leu Glu
185 190 195
Leu Leu Leu Ala Ala Tyr Ser Ser Val Phe Trp Trp Lys Gln Leu
200 205 210
Tyr Ser Asn Asn Pro Gly Ser Ser Phe Ser Ser Thr Gln Ser Gln
215 220 225
Asp His Ile Gln Gln Val Lys Lys Ser Ser Ser Arg Ser Trp Ile
230 235 240
14/28
CA 02338385 2001-02-06
WO 00/08155 PCT/US99/17777
<210> 14
<211> 316
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2656082
<400> 14
Met Gly Cys Arg Leu Leu Cys Cys Ala Val Leu Cys Leu Leu Gly
1 5 10 15
Ala Val Pro Met Glu Thr Gly Val Thr Gln Thr Pro Arg His Leu
20 25 30
Val Met Gly Met Thr Asn Lys Lys Ser Leu Lys Cys Glu Gln His
35 40 45
Leu Gly His Asn Ala Met Tyr Trp Tyr Lys Gln Ser Ala Lys Lys
50 55 60
Pro Leu Glu Leu Met Phe Val Tyr Ser Leu Glu Glu Arg Val Glu
65 70 75
Asn Asn Ser Val Pro Ser Arg Phe Ser Pro Glu Cys Pro Asn Ser
80 85 90
Ser His Leu Phe Leu His Leu His Thr Leu Gln Pro Glu Asp Ser
95 100 105
Ala Leu Tyr Leu Cys Ala Ser Ser Gln Val His Pro Gly Leu Ala
110 115 120
Gly Gly Leu Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr
125 130 135
Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val
140 145 150
Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr
155 160 165
Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu
170 175 180
Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr
185 190 195
Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg
200 205 210
Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln
215 220 225
Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu
230 235 240
Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr
245 250 255
Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe
260 265 270
Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu
275 280 285
Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val
290 295 300
Ser Ala Leu ~Val Leu Met Ala Met Val Lys Arg Lys Asp Ser Arg
305 310 315
Gly
15/28
CA 02338385 2001-02-06
WO 00/08155 PCT/US99/17777
<210> 15
<211> 172
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature .
<223> Incyte Clone No: 2762182
<400> 15
Met Ala Thr Ile Thr Glu Lys Glu Val Gln Gln Trp Tyr Lys Gly
1 5 10 15
Phe Ile Lys Asp Cys pro Ser Gly Gln Leu Asp Ala Ala Gly Phe
20 25 30
Gln Lys Ile Tyr Lys Gln Phe Phe Pro Phe Gly Asp Pro Thr Lys
35 40 45
Phe Ala Thr Phe Val Phe Asn Val Phe Asp Glu Asn Lys Asp Gly
50 55 60
Arg Ile Glu Phe Ser Glu Phe Ile Gln Ala Leu Ser Val Thr Ser
65 70 75
Arg Gly Thr Leu Asp Glu Lys Leu Arg Trp Ala Phe Lys Leu Tyr
80 85 90
Asp Leu Asp Asn Asp Gly Tyr Ile Thr Arg Asn Glu Met Leu Asp
95 100 105
Ile Val Asp Ala Ile Tyr Gln Met Val Gly Asn Thr Val Glu Leu
110 115 120
Pro Glu Glu Glu Asn Thr Pro Glu Lys Arg Val Asp Arg Ile Phe
125 130 135
Ala Met Met Asp Lys Asn Ala Asp Gly Lys Leu Thr Leu Gln Glu
140 145 150
Phe Gln Glu Gly Ser Lys Ala Asp Pro Ser Ile Val Gln Ala Leu
155 160 165
Ser Leu Tyr Asp Gly Leu Val
170
<210> 16
<211> 364
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 3140659
<400> 16
Met Ser Val Met Val Val Arg Lys Lys Val Thr Arg Lys Trp Glu
1 5 10 15
Lys Leu Pro Gly Arg Asn Thr Phe Cys Cys Asp Gly Arg Val Met
20 25 30
Met Ala Arg Gln Lys Gly Ile Phe Tyr Leu Thr Leu Phe Leu Ile
35 40 45
Leu Gly Thr Cys Thr Leu Phe Phe Ala Phe Glu Cys Arg Tyr Leu
50 55 60
Ala Val Gln Leu Ser Pro Ala Ile Pro Val Phe Ala Ala Met Leu
16/28
CA 02338385 2001-02-06
WO 00/08155 PGTNS99/17777
65 70 75
Phe Leu Phe Ser Met Ala Thr Leu Leu Arg Thr Ser Phe Ser Asp
80 85 90
Pro Gly Val Ile Pro Arg Ala Leu Pro Asp Glu Ala Ala Phe Ile
95 100 105
Glu Met Glu Ile Glu Ala Thr Asn Gly Ala Val Pro Gln Gly Gln
110 ~ 115 120
Arg Pro Pro Pro Arg Ile Lys Asn Phe Gln Ile Asn Asn Gln Ile
125 130 135
Val Lys Leu Lys Tyr Cys Tyr Thr Cys Lys Ile Phe Arg Pro Pro
140 145 150
Arg Ala Ser His Cys Ser Ile Cys Asp Asn Cys Val Glu Arg Phe
155 160 165
Asp His His Cys Pro Trp Val Gly Asn Cys Val Gly Lys Arg Asn
170 175 180
Tyr Arg Tyr Phe Tyr Leu Phe Ile Leu Ser Leu Ser Leu Leu Thr
185 190 195
Ile Tyr Val Phe Ala Phe Asn Ile Val Tyr Val Ala Leu Lys Ser
200 205 210
Leu Lys Ile Gly Phe Leu Glu Thr Leu Lys Glu Thr Pro Gly Thr
215 220 225
Val Leu Glu Val Leu Ile Cys Phe Phe Thr Leu Trp Ser Val Val
230 235 240
Gly Leu Thr Gly Phe His Thr Phe Leu Val Ala Leu Asn Gln Thr
245 250 255
Thr Asn Glu Asp Ile Lys Gly Ser Trp Thr Gly Lys Asn Arg Val
260 265 270
Gln Asn Pro Tyr Ser His Gly Asn Ile Val Lys Asn Cys Cys Glu
275 280 285
Val Leu Cys Gly Pro Leu Pro Pro Ser Val Leu Asp Arg Arg Gly
290 295 300
Ile Leu Pro Leu Glu Glu Ser Gly Ser Arg Pro Pro Ser Thr Gln
305 310 315
Glu Thr Ser Ser Ser Leu Leu Pro Gln Ser Pro Ala Pro Thr Glu
320 325 330
His Leu Asn Ser Asn Glu Met Pro Glu Asp Ser Ser Thr Pro Glu
335 340 345
Glu Met Pro Pro Pro Glu Pro Pro Glu Pro Pro Gln Glu Ala Ala
350 355 360
Glu Ala Glu Lys
<210> 17
<211> 1270
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1877651
<400> 17
gacctcacag gaagatgcat cttgtaggag gcagctgtga ggtctggttc cccgacgtgc 60
tgcagcaagt gcctttgccc tgcctgtggg ctccctccat ggccaactct gctatggaca 120
ccagagtact ctgctgtgcg gtcatctgtc ttctgggggc aggtctctca aatgccggcg 180
17/28
CA 02338385 2001-02-06
WO 00/08155 PCT/US99/17777
tcatgcagaa cccaagacac ctggtcagga ggaggggaca ggaggcaaga ctgagatgca 240
gcccaatgaa aggacacagt catgtttact ggtatcggca gctcccagag gaaggtctga 300
aattcatggt ttatctccag aaagaaaata tcatagatga gtcaggaatg ccaaaggaac 360
gattttctgc tgaatttccc aaagagggcc ccagcatcct gaggatccag caggtagtgc 420
gaggagattc ggcagcttat ttctgtgcca gctcaccaca ttcgaagcag gctgagcagt 480
tcttcgggcc agggacacgg ctcaccgtgc tagaggacct gaaaaacgtg ttcccacccg 540
aggtcgctgt gtttgagcca tcagaagcag.agatctccca cacccaaaag gccacactgg 600
tgtgcctggc cacaggcttc taccccgacc acgtggagct gagctggtgg gtgaatggga 660
aggaggtgca cagtggggtc agcacagacc cgcagcccct caaggagcag cccgccctca 720
atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc tggcagaacc 780
cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat gacgagtgga 840
cccaggatag ggccaaacct gtcacccaga tcgtcagcgc cgaggcctgg ggtagagcag 900
actgtggctt cacctccgag tcttaccagc aaggggtcct gtctgccacc atcctctatg 960
agatcttgct agggaaggcc accttgtatg ccgtgctggt cagtgccctc gtgctgatgg 1020
ccatggtcaa gagaaaggat tccagaggct agctccaaaa ccatcccagg tcattcttca 1080
tcctcaccca ggattctcct gtacctgctc ccaatctgtg ttcctaaaag tgattctcac 1140
tctgcttctc atctcctact tacatgaata cttctctctt ttttctgttt ccctgaagat 1200
tgagctccca acccccaagt acgaaatagg ctaaaccaat aaaaaattgt gtgttgggcc 1260
tggttgcatt
1270
<210> 18
<211> 2234
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2906971
<400> 18
aggttttgcc cgcattcggg gcgcgcggac tggggggtcc ctgtggggct cccggagtta 60
agatggcgtc ctcagcggag ggggacgagg ggactgtggt ggcgctggcg ggggttctgc 120
agtcgggttt ccaggagctg agccttaaca agttggegac gtccctgggc gcgtcagaac 180
aggcgctgcg gctgatcatc tccatcttcc tgggttaccc ctttgctttg ttttatcggc 240
attacctttt ctacaaggag acctacctca tccacctctt ccataccttt acaggcctct 300
caattgctta ttttaacttt ggaaaccagc tctaccactc cctgctgtgt attgtgcttc 360
agttcctcat ccttcgacta atgggccgca ccatcactgc cgtcctcact accttttgct 420
tccagatggc ctaccttctg gctggatact attacactgc caccggcaac tacgatatca 480
agtggacaat gccacattgt gttctgactt tgaagctgat tggtttggct gttgactact 540
ttgacggagg gaaagatcag aattccttgt cctctgagca acagaaatat gccatacgtg 600
gtgttccttc cctgctggaa gttgctggtt tctcctactt ctatggggcc ttcttggtag 660
ggccccagtt ctcaatgaat cactacatga agctggtgca gggagagctg attgacatac 720
caggaaagat accaaacagc atcattcctg ctctcaagcg cctgagtctg ggccttttct 780
acctagtggg ctacacactg ctcagccccc acatcacaga agactatctc ctcactgaag 840
actatgacaa ccaccccttc tggttccgct gcatgtacat gctgatctgg ggcaagtttg 900
tgctgtacaa atatgtcacc tgttggctgg tcacagaagg agtatgcatt ttgacgggcc 960
tgggcttcaa tggctttgaa gaaaagggca aggcaaagtg ggatgcctgt gccaacatga 1020
aggtgtggct ctttgaaaca aacccccgct tcactggcac cattgcctca ttcaacatca 1080
acaccaacgc ctgggtggcc cgctacatct tcaaacgact caagttcctt ggaaataaag 1140
aactctctca gggtctctcg ttgctattcc tggccctctg gcacggcctg cactcaggat 1200
acctggtctg cttccagatg gaattcctca ttgttattgt ggaaagacag gctgccaggc 1260
tcattcaaga gagccccacc ctgagcaagc tggccgccat tactgtcctc cagcccttct 1320
actatttggt gcaacagacc atccactggc tcttcatggg ttactccatg actgccttct 1380
gcctcttcac gtgggacaaa tggcttaagg tgtataaatc catctatttc cttggccaca 1440
tcttcttcct gagcctacta ttcatattgc cttatattca caaagcaatg gtgccaagga 1500
aagagaagtt aaagaagatg gaataatcca tttccctggt ggcctgtgcg ggactggtgc 1560
1$/28
CA 02338385 2001-02-06
WO 00/08155 PCT/US99/17777
agaaactact cgtctccctt ttcacagcac tcctttgccc cagagcagag aatggaaaag 1620
ccagggaggt ggaagatcga tgcttccagc tgtgcctctg ctgccagcca agtcttcatt 1680
tggggccaaa ggggaaactt ttttttggag aaggcgtctt gctttgtcac ccacgctgga 1740
atgcagtggc gggatctcag ctcaccgcaa cctccacctc ctgggttcaa gtgattttcc 1800
tgcctcagcc tcccaagtag ctgggaatac aggcacgcca ccatgcccag ctaatttttg 1860
tattttcagt agaaacggga tttcaccacg ttggccaggc tggtctcgaa ctcctgaccg 1920
caagtgatcc acccgcctcc gcctcccaaa gtgctgggat tacaggcgtg agccaccgtg 1980
cccggcccaa aggggaaact cttgtgggag gagcagaggg gctcacatct cccctctgat 2040
tcccccatgc acattgcctt atctctcccc atctagccag gaatctattg tgtttttctt 2100
ctgccaattt actatgattg tgtatgtgcc gctaccacca ccccccccat gggggggtgg 2160
agaggggtgc aaggccctgc ctgctccact ttttctacct tggaactgta ttagatatgg 2220
tcacttctgt gtgt 2234
<210> 19
<211> 1552
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2907954
<400> 19
tgaccctgat tgggcaaagc tcccatcctt ccctgaccct gccatgggca ccaggctcct 60
ctgctgggcg gccctctgtc tcctgggagc agaactcaca gaagctggag ttgcccagtc 120
tcccagatat aagattatag agaaaaggca gagtgtggct ttttggtgca atcctatatc 180
tggccatgct accctttact ggtaccagca gatcctggga cagggcccaa agcttctgat 240
tcagtttcag aataacggtg tagtggatga ttcacagttg cctaaggatc gattttctgc 300
agagaggctc aaaggagtag actccactct caagatccaa cctgcaaagc ttgaggactc 360
ggccgtgtat ctctgtgcca gcagcttctt agaccgaaac aatgagcagt tcttcgggcc 420
agggacacgg ctcaccgtgc tagaggacct gaaaaacgtg ttcccacccg aggtcgctgt 480
gtttgagcca tcagaagcag agatctccca cacccaaaag gccacactgg tgtgcctggc 540
cacaggcttc taccccgacc acgtggagct gagctggtgg gtgaatggga aggaggtgca 600
cagtggggtc agcacagacc cgcagcccct caaggagcag cccgccctca atgactccag 660
atactgcctg agcagccgcc tgagggtctc ggccaccttc tggcagaacc cccgcaacca 720
cttccgctgt caagtccagt tctacgggct ctcggagaat gacgagtgga cccaggatag 780
ggccaaacct gtcacccaga tcgtcagcgc cgaggcctgg ggtagagcag actgtggctt 840
cacctccgag tcttaccagc aaggggtcct gtctgccacc atcctctatg agatcttgct 900
agggaaggcc accttgtatg ccgtgctggt cagtgccctc gtgctgatgg ccatggtcaa 960
gagaaaggat tccagaggct agctccaaaa ccatcccagg tcattcttca tcctcaccca 1020
ggattctcct gtacctgctc ccaatctgtg ttcctaaaag tgattctcac tctgcttctc 1080
atctcctact tacatgaata cttctctctt ttttctgttt ccctgaagat tgagctccca 1140
acccccaagt acgaaatagg ctaaaccaat aaaaaatggt gtgttgggcc tggttgcatt 1200
tcaggagtgt ctgtggagtt ctgctcatca ctgacttatc ttctgattta gggaaagcag 1260
cattcccttg gacatctgaa gtgacagccc tctttctctc cacccaatgc tgctttctcc 1320
tgttcatcct gatggaagtc ctcaaacacc atttccatac ccaggcattc tgggtcccca 1380
ctggagggtt agtctgaagg gcaatggctg ggctttggaa aaccagcaag atgaggacag 1440
agaggaaggc acacagcaaa ccataagccc ttacccagtg caggacagag gatgcgggca 1500
gacctatggg ttacaatgtc tggtcatttc ccaattccag attaaatatg to 1552
<210> 20
<211> 1369
<212> DNA
<213> Homo sapiens
19/28
CA 02338385 2001-02-06
WO 00/0$155 PGT/US99/17777
<220>
<221> misc_feature
<223> Incyte Clone No: 3083742
<400>. 20
gggtcacaca ctccacccgg gaggccaaag ctgcctgcag gaccatgaat ggcacctaca 60
acacctgtgg ctccagcgac ctcacctggc ccccagcgat caagctgggc ttctacgcct 120
acttgggcgt cctgctggtg ctaggcctgc tgctcaacag cctggcgctc tgggtgttct 180
gctgccgcat gcagcagtgg acggagaccc gcatctacat gaccaacctg gcggtggccg 240
acctctgcct gctgtgcacc ttgcccttcg tgctgcactc cctgcgagac acctcagaca 300
cgccgctgtg ccagctctcc cagggcatct acctgaccaa caggtacatg agcatcagcc 360
tggtcacggc catcgccgtg gaccgctatg tggccgtgcg gcacccgctg cgtgcccgcg 420
ggctgcggtc ccccaggcag gctgcggccg tgtgcgcggt cctctgggtg ctggtcatcg 480
gctccctggt ggctcgctgg ctcctgggga ttcaggaggg cggcttctgc ttcaggagca 540
cccggcacaa tttcaactcc atggcgttcc cgctgctggg attctacctg cccctggccg 600
tggtggtctt ctgctccctg aaggtggtga ctgccctggc ccagaggcca cccaccgacg 660
tggggcaggc agaggccacc cgcaaggctg cccgcatggt ctgggccaac ctcctggtgt 720
tcgtggtctg cttcctgccc ctgcacgtgg ggctga.cagt gcgcctcgca gtgggctgga 780
acgcctgtgc cctcctggag acgatccgtc gcgccctgta cataaccagc aagctctcag 840
atgccaactg ctgcctggac gccatctgct actactacat ggccaaggag ttccaggagg 900
cgtctgcact ggccgtggct ccccgtgcta aggcccacaa aagccaggac tctctgtgcg 960
tgaccctcgc ctaagaggcg tgctgtgggc gctgtgggcc aggtctcggg ggctccggga 1020
ggtgctgcct gccaggggaa gctggaacca gtagcaagga gcccgggatc agccctgaac 1080
tcactgtgta ttctcttgga gccttgggtg ggcagggacg gcccaggtac ctgctctctt 1140
gggaagagag agggacaggg acaagggcaa gaggactgag gccagagcaa ggccaatgtc 1200
agagaccccc gggatggggc ctcacacttg ccacccccag aaccagctca cctggccaga 1260
gtgggttcct gctggccagg gtgcagcctt gatgacacct gccgctgccc ctcggggctg 1320
gaataaaact ccccacccag agtcaaaaaa aaaaaaaaga aaaaaaaaa 1369
<210> 21
<211> 2096
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 3407686
<400> 21
cggacccgcc tctcccaaag tctagccggg caagggaacg cggtgcattc ctgaccggca 60
cctggcgagg ctcatgcgtc ccgtgagggc ggttcctcga gcctgggggc gctcagattg 120
ctttggagac gctgagagag cctttgcgag agcgccggtt gacgtgcgga gtgcggggct 180
ccgggggact gagcagcacg agaccccatc ctcccctccg ggttttcaca ctgggcgaag 240
ggaggactcc tgagctctgc ctcttccagt aacattgagg attactgtgt tttgtgagag 300
ctcgctaggc gccctaagca acagagttct gagaaatcga gaaacatgat aaggaattgg 360
ctgactattt ttatcctttt tcccctgaag ctcgtagaga aatgtgagtc aagcgtcagc 420
ctcactgttc ctcctgtcgt aaagctggag aacggcagct cgaccaacgt cagcctcacc 480
ctgcggccac cattaaatgc aaccctggtg atcacttttg aaatcacatt tcgttccaaa 540
aatattacta tccttgagct ccccgatgaa gttgtggtgc ctcctggagt gacaaactcc 600
tcttttcaag tgacatctca aaatgttgga caacttactg tttatctaca tggaaatcac 660
tccaatcaga ccggcccgag gatacgcttt cttgtgatcc gcagcagcgc cattagcatc 720
ataaaccagg tgattggctg gatctacttt gtggcctggt ccatctcctt ctaccctcag 780
gtgatcatga attggaggcg gaaaagtgtc attggtctga gcttcgactt cgtggctctg 840
aacctgacgg gcttcgtggc ctacagtgta ttcaacatcg gcctcctctg ggtgccctac 900
atcaaggagc agtttctcct caaatacccc aacggagtga accccgtgaa cagcaacgac 960
gtcttcttca gcctgcacgc ggttgtcctc acgctgatca tcatcgtgca gtgctgcctg 1020
20/2$
CA 02338385 2001-02-06
WO 00/08155 PCT/US99/17777
tatgagcgcg gtggccagcg cgtgtcctgg cctgccatcg gcttcctggt gctcgcgtgg 1080
ctcttcgcat ttgtcaccat gatcgtggct gcagtgggag tgatcacgtg gctgcagttt 1140
ctcttctgct tctcctacat caagctcgca gtcacgctgg tcaagtattt tccacaggcc 1200
tacatgaact tttactacaa aagcactgag ggctggagca ttggcaacgt gctcctggac 1260
ttcaccgggg gcagcttcag cctcctgcag atgttcctcc agtcctacaa caacgaccag 1320
tggacgctga tcttcggaga cccaaccaag tttggactcg gggtcttctc catcgtcttc 1380
gacgtcgtct tcttcatcca gcacttctgt ttgtacagaa agagaccggg gtatgaccag 1440
ctgaactagc acccagggac ccagtgtacc cagcctctgg cctcgtgccc tgctggggaa 1500
ggcctcaccc agcgaaggcc ggagaagcgg ttgggccctg gcacacaggg ctggctcagt 1560
gtgcggacag aggagaccac tctgctcctg gggccagagg ccattcaata gcctgccttc 1620
gtccgggccc ctcctgggcc tccceggcca ggcacgtggc accgtcgcct tgacaccgcc 1680
atctcttttc tttaaggctt caggcagcgc gcacaggctc tggcagccgt ctcaggcagg 1740
actgggcagc aagcttgcag ccgaagcgtt gccccaaact accagcgttt ctgcaagcag 1800
cttgaagggc tgaccttgca gccgggtgag ccaagggcac tttgctgcca ccgctgcatt 1860
cccagagatc aagcagcccg gtgccgtggc cagtgaactc agaggtgctg gtgggacggg 1920
ctaggacttt ggggttaggc catggggcgt ctttctctgg aggccacttt cctgacgtac 1980
tctctgtaca taattcagcg tccgtgactg cagtaacgag cagccctagc cgaggatttc 2040
tgagccatga ggggcccacg gaattggttc taaattgatc atgcccagcc attaga 2096
<210> 22
<211> 1431
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 3472455
<400> 22
gaacctttgc acgcgcacaa actacgggga cgatttctga ttgcaatcag gcgcattcga 60
tccaccctcc tcccttctca tgggactttg gggacaaagc gtcccgaccg cctcgagcgc 120
tcgagcaggg cgctatccag gagccaggac agcgtcggga accagaccat ggctcctgga 180
ccccaagatc cttaagttcg tcgtcttcat cgtcgcggtt ctgctgccgg tccgggttga 240
ctctgccacc atcccccggc aggacgaagt tccccagcag acagtggccc cacagcaaca 300
gaggcgcagc ctcaaggagg aggagtgtcc agcaggatct catagatcag aatatactgg 360
agcctgtaac ccgtgcacag agggtgtgga ttacaccatt gcttccaaca atttgccttc 420
ttgcctgcta tgtacagttt gtaaatcagg tcaaacaaat aaaagttcct gtaccacgac 480
cagagacacc gtgtgtcagt gtgaaaaagg aagcttccag gataaaaact cccctgagat 540
gtgccggacg tgtagaacag ggtgtcccag agggatggtc aaggtcagta attgtacgcc 600
ccggagtgac atcaagtgca aaaatgaatc agctgccagt tccactggga aaaccccagc 660
agcggaggag acagtgacca ccatcctggg gatgcttgcc tctccctatc actaccttat 720
catcatagtg gttttagtca tcattttagc tgtggttgtg gttggctttt catgtcggaa 780
gaaattcatt tcttacctca aaggcatctg ctcaggtggt ggaggaggtc ccgaacgtgt 840
gcacagagtc cttttccggc ggcgttcatg tccttcacga gttcctgggg cggaggacaa 900
tgcccgcaac gagaccctga gtaacagata cttgcagccc acccaggtct ctgagcagga 960
aatccaaggt caggagctgg cagagctaac aggtgtgact gtagagtcgc cagaggagcc 1020
acagcgtctg ctggaacagg cagaagctga agggtgtcag aggaggaggc tgctggttcc 1080
agtgaatgac gctgactccg ctgacatcag caccttgctg gatgcctcgg caacactgga 1140
agaaggacat gcaaaggaaa caattcagga ccaactggtg ggctccgaaa agctctttta 1200
tgaagaagat gaggcaggct ctgctacgtc ctgcctgtga aagaatctct tcaggaaacc 1260
agagcttccc tcatttacct tttctcctac aaagggaagc agcctggaag aaacagtcca 1320
gtacttgacc catgccccaa caaactctac tatccaatat ggggcagctt accaatggtc 1380
ctagaacttt gttaacgcac ttggagtaat ttttatgaaa tactgcgtgt g 1431
<210> 23
21/28
CA 02338385 2001-02-06
wo oorosiss
<211> 1788
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 786873
<400> 23
cctacgacga ctatagggaa tttggccctc gagcacggac attccggcac gatggcgagc 60
ctccccctcc tccccaggac gacatgaacg accgaggcca gggagtcctc tccttgggcc 120
tctgcatccc cccatccttg gctctggggt aggcccaggg aggagacacc cccaacccct 180
atccggtctg tcctggagaa aagagactgc ccttccatgc ccctgagtga ggggcctggg 240
gcccaggctg cctgtgttcc ccaagggcaa gggtctctct gttgaggagg aggggcctgt 300
cagccacaac ttctttcctc ctgagcgccc catctccctc tctgcaccct gcaattccca 360
cccctccgta tttatttccc tggtcccgcc gacagtccct ccttgtctgt ctccgggatt 420
caggcctccc tccctgacat ggagagtaac ctgtctggcc tggtgcctgc tgccgggctg 480
gtgcctgcgc tgccacctgc tgtgaccctg gggctgacag ctgcctacac caccctgtat 540
gccctgctct tcttctccgt ctatgcccag ctctggctgg tgcttctgta tgggcacaag 600
cgtctcagct atcagacggt gttcctggcc ctctgtctgc tctgggccgc cttgcgtacc 660
accctcttct ccttctactt ccgagatact ccccgcgcca accgcctggg gcccttgccc 720
ttctggcttc tctactgctg ccccgtctgc ctgcagttct tcaccttgac gcttatgaac 780
ctctactttg cccaggtggt gttcaaggcc aaggtgaagc gtcggccgga gatgagccga 840
ggcttgctcg ctgtccgagg ggcctttgtg ggggcctcgc tgctctttct gctggtgaac 900
gtgctgtgtg ctgtgctctc ccatcggcgc cgggcacagc cctgggccct gctgcttgtc 960
cgcgtcctgg tgagcgactc cctgttcgtc atctgcgcgc tgtctcttgc tgcctgcctc 1020
tgcctcgtcg ccaggcgggc gccctccact agcatctacc tggaggccaa ggcggacctg 1080
gtgaatgacc tggggaacaa aggctacctg gtatttggcc tcatcctctt cgtgtgggag 1140
ctactgccca ccaccctgct ggtgggcttc ttccgggtgc accggccccc acaggacctg 1200
agcaccagcc acatcctcaa tgggcaggtc tttgcctctc ggtcctactt ctttgaccgg 1260
gctgggcact gtgaagatga gggctgctcc tgggagcaca gccggggtga gagcaccagt 1320
atgtcgggca gtctaggctc tgggagctgg tatggtgcca tcgggcgtga gccgggctgg 1380
tatgggggca gccagacgaa gaccactcct ctgctcttct cccaggtgcc aggaccaggc 1440
ggccaccacc acagtctcta ctccacccca cagacgtgat ccccctccct cccccacaga 1500
atacccaggc cccagtcccc ctcaccctag gcccctgtgc caagtttgtc tgccgcttct 1560
tgcccaggat cctgggggtc gtggctaccc cctcctctgg ccggctcctt gctgctcctg 1620
tcatagtgag cttgtgccgt ccccctagga tggggggcat ggccctggct gccagatgcc 1680
cacagcaccc tggcatgacc tgccacctct gcttccacac cggagccagc tacctctcct 1740
gtgcctgcca ctcaataaac agtgtctgcg ccccacaaaa aaaaaaaa 1788
<210> 24
<211> 1044
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1220371
<400> 24
gaaagccaac tttcctttca aatacacacc ccaacccgcc ccggcataca cagaaatggg 60
gactgcgagc agaagcaaca tcgctcgcca tctgcaaacc aatctcattc tattttgtgt 120
cggtgctgtg ggcgcctgta ctctctctgt cacacaaccg tggtacctag aagtggacta 180
cactcatgag gccgtcacca taaagtgtac cttctccgca accggatgcc cttctgagca 240
accaacatgc ctgtggtttc gctacggtgc tcaccagcct gagaacctgt gcttggacgg 300
gtgcaaaagt gaggcagaca agttcacagt gagggaggcc ctcaaagaaa accaagtttc 360
22/28
CA 02338385 2001-02-06
wo ooiosiss ~,NS~m,~',~
cctcactgta aacagagtga cttcaaatga cagtgcaatt tacatctgtg gaatagcatt 420
ccccagtgtg ccggaagcga gagctaaaca gacaggagga gggaccacac tggtggtaag 480
agaaattaag ctgctcagca aggaactgcg gagcttcctg acagctcttg tatcactgct 540
ctctgtctat gtgaccggtg tgtgcgtggc cttcatactc ctctccaaat caaaatccaa 600
ccctctaaga aacaaagaaa taaaagaaga ctcacaaaag aagaagagtg ctcggcgtat 660
ttttcaggaa attgctcaag aactatacca taagagacat gtggaaacaa atcagcaatc 720
tgagaaagat aacaacactt atgaaaacag aagagtactt tccaactatg aaaggccata 780
gaaacgtttt aattttcaat gaagtcactg aaaatccaac tccaggagct atggcagtgt 840
taatgaacat atatcatcag gtcttaaaaa aaaataaagg taaactgaaa agacaactgg 900
ctacaaagaa ggatgccaga atgtaaggaa actataacta atagtcatta ccaaaatact 960
aaaacccaac aaaatgcaac tgaaaaatac cttccaaatt tgccaagaaa aaaaattcta 1020
ttttaaactt gtaaaaaaaa aaaa 1044
<210> 25
<211> 1605
<212> DNA
<213> Homo sapiens
<220>
<221> mi.sc_feature
<223> Incyte Clone No: 1258785
<400> 25
gtccccgccc gcgcgccgta ccgcggcgga gatgggcgag accatgtcca agaggctgaa 60
gctccacctg ggaggggagg cagaaatgga ggaacgggcg ttcgtcaacc ccttcccgga 120
ctacgaggcc gccgccgggg cgctgctcgc ctccggagcg gccgaagaga caggctgtgt 180
tcgtcccccg gcgaccacgg atgagcccgg cctccctttt catcaggacg ggaagatcat 240
tcataatttc ataagacgga tccagaccaa aattaaagat cttctgcagc aaatggaaga 300
agggctgaag acggctgatc cccatgactg ctctgcttat actggctgga caggcatagc 360
ccttttgtac ctgcagttgt accgggtcac atgtgaccaa acctacctgc tccgatccct 420
ggattacgta aaaagaacac ttcggaatct gaatggccgc agggtcacct tcctctgtgg 480
ggatgctggc cccctggctg ttggagctgt gatttatcac aaactcagaa gtgactgtga 540
gtcccaggaa tgtgtcacaa aacttttgca gctccagaga tcggttgtct gccaagaatc 600
agaccttcct gatgagctgc tttatggacg ggcaggttat ctgtatgcct tactgtacct 660
gaacacagag ataggtccag gcaccgtgtg tgagtcagct attaaagagg tagtcaatgc 720
tattattgaa tcgggtaaga ctttgtcaag ggaagaaaga aaaacggagc gctgcccgct 780
gttgtaccag tggcaccgga agcagtacgt tggagcagcc catggcatgg ctggaattta 840
ctatatgtta atgcagccgg cagcaaaagt ggaccaagaa accttgacag aaatggtgaa 900
acccagtatt gattatgtgc gccacaaaaa attccgatct gggaattacc catcatcatt 960
aagcaatgaa acagaccggc tggtgcactg gtgccacggc gccccggggg tcatccacat 1020
gctcatgcag gcgtacaagg tctttaagga ggagaagtac ttgaaagagg ccatggagtg 1080
tagcgatgtg atttggcagc gaggtttgct gcggaagggc tacgggatat gccatgggac 1140
tgctggcaac ggctattcct tcctgtccct ttaccgtctc acgcaggata agaagtacct 1200
ctaccgagct tgcaagtttg cagagtggtg tctagattac ggagcacacg ggtgccgcat 1260
tcctgacaga ccctattcgc tctttgaagg catggctggc gctattcact ttctctctga 1320
tgtcctggga ccagagacat cacggtttcc agcatttgaa cttgactctt cgaagaggga 1380
ttaaaaggtg caaaaagaca actaaaatac ccatttggac caaaagccgc cagattgctt 1440
agtgcctgac acagaaacaa ctgggaatcc tgaaagagaa gcagacaccg tcacaggccc 1500
ctctggttag actagcatga gtgaccgaag ccatccatca acattttcta acagcaccct 1560
catcaatata aaatatgact tcttcacata cagaaaaaaa aaaaa 1605
<210> 26
<211> 1464
<212> DNA
<213> Homo sapiens
23/28
CA 02338385 2001-02-06
WO 00/08155
<220>
<221> misc_feature
<223> Incyte Clone No: 1361202
PCTNS99/17777
<400> 26
gagatagcaa agcagagtgt ttatcttgtg agccattctc catatttcag atataagatt 60
tcagttctca gtgagtctaa gtgacagaag gaatggagac cctcttgggc ctgcttatcc 120
tttggctgca gctgcaatgg gtgagcagca aacaggaggt gacacagatt cctgcagctc 180
tgagtgtccc agaaggagaa aacttggttc tcaactgcag tttcactgat agcgctattt 240
acaacctcca gtggtttagg caggaccctg ggaaaggtct cacatctctg ttgcttattc 300
agtcaagtca gagagagcaa acaagtggaa gacttaatgc ctcgctggat aaatcatcag 360
gacgtagtac tttatacatt gcagcttctc agcctggtga ctcagccacc tacctctgtg 420
ctgtgaggga caatgacatg cgctttggag cagggaccag actgacagta aaacccaata 480
tccagaaccc tgaccctgcc gtgtaccagc tgagagactc taaatccagt gacaagtctg 540
tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 600
tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 660
ctgtggcctg gagcaacaaa tctgactttg catgtgcaaa cgccttcaac aacagcatta 720
ttccagaaga caccttcttc cccagcccag aaagttcctg tgatgtcaag ctggtcgaga 780
aaagctttga aacagatacg aacctaaact ttcaaaacct gtcagtgatt gggttccgaa 840
tcctcctcct gaaagtggcc gggtttaatc tgctcatgac gctgcggctg tggtccagct 900
gagatctgca agattgtaag acagcctgtg ctccctcgct ccttcctctg cattgcccct 960
cttctccctc tccaaacaga gggaactctc ccacccccaa ggaggtgaaa gctgctacca 1020
cctctgtgcc cccccggcaa tgccaccaac tggatcctac ccgaatttat gattaagatt 1080
gctgaagagc tgccaaacac tgctgccacc ccctctgttc ccttattgct gcttgtcact 1140
gcctgacatt cacggcagag gcaaggctgc tgcagcctcc cctggctgtg cacattccct 1200
cctgctcccc agagactgcc tccgccatcc cacagatgat ggatcttcag tgggttctct 1260
tgggctctag gtcctgcaga atgttgtgag gggtttattt ttttttaata gtgttcataa 1320
agaaatacat agtattcttc ttctcaagac gtggggggaa attatctcat tatcgaggcc 1380
ctgctatgct gtgtatctgg gcgtgttgta tgtcctgctg ccgatgcctt cattaaaatg 1440
attgggagag cagaaaaaaa aaaa
1464
<210> 27
<211> 960
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2132846
<400> 27
acggccagtg caagctaaaa ttaaccctca ctaaagggaa taagcttgcg ccgccagagg 60
atggggaaat ccaacagcaa gttgacgccc gaagttgtgg aggagctgac caggaagacc 120
tactttaccg agaaggaggt ccagcagtgg tacaaaggct tcatcaagga ctgccccagt 180
gggcagctgg atgcggcagg cttccagaag atctacaagc aattcttccc gttcggagac 240
cccaccaagt ttgccacatt tgttttcaac gtctttgatg aaaacaagga cgggcgaatt 300
gagttctccg agttcatcca ggcgctgtcg gtgacctcac ggggaaccct ggatgagaag 360
ctacggtggg ccttcaagct ctacgacttg gacaatgatg gctacatcac caggaatgag 420
atgctggaca ttgtggatgc catttaccag atggtgggga ataccgtgga gctcccagag 480
gaggagaaca ctcctgagaa gagggtggac cggatctttg ccatgatgga taagaatgcc 540
gacgggaagc tgaccctgca ggagttccag gaggggtcca aggcagaccc gtccattgtg 600
caggcgctgt ccctctacga cgggctggta tagtcccagg ctggagctgg atgcctggga 660
accactcacc tccttctgtg ccatgaggcc acctcagccc tgacaccaac cccgtgcgtc 720
cacccagcct tcttccgcat ccacacacag ccggctgccc ttgacccggg aggccccggc 780
tctcctctcc cctgtcctgc acccatcccc cgcctgaagc caccggctcc aattgccagc 840
aacctctgct tgtccggaaa acgacaacac gaaatggaaa aggctacagc cctctgcata 900
z4ns
CA 02338385 2001-02-06
wo oo~osiss Pcrius99n~7~~
aaccaaggac ttggctgcct cgcaggcagc gtccgttcct cccgctctct tgcgcgtgtg 960
<210> 28
<211> 1990
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2539294
<400> 28
tctcctcccc ctcccggcca agatgtctga catggaggat gatttcatgt gcgatgatga 60
ggaggactac gacctggaat actctgaaga tagtaactcc gagccaaatg tggatttgga 120
aaatcagtac tataattcca aagcattaaa agaagatgac ccaaaagcgg cattaagcag 180
tttccaaaag gttttggaac ttgaaggtga aaaaggagaa tggggattta aagcactgaa 240
acaaatgatt aagattaact tcaagttgac aaactttcca gaaatgatga atagatataa 300
gcagctattg acctatattc ggagtgcagt cacaagaaat tattctgaaa aatccattaa 360
ttctattctt gattatatct ctacttctaa acagaattct gattttttat gtcagatgga 420
tttactgcag gaattctatg aaacaacact ggaagctttg aaagatgcta agaatgatag 480
actgtggttt aagacaaaca caaagcttgg aaaattatat ttagaacgag aggaatatgg 540
aaagcttcaa aaaattttac gccagttaca tcagtcgtgc cagactgatg atggagaaga 600
tgatctgaaa aaaggtacac agttattaga aatatatgct ttggaaattc aaatgtacac 660
agcacagaaa aataacaaaa aacttaaagc actctatgaa cagtcacttc acatcaagtc 720
tgccatccct catccactga ttatgggagt tatcagagaa tgtggtggta aaatgcactt 780
gagggaaggt gaatttgaaa aggcacacac tgattttttt gaagccttca agaattatga 840
tgaatctgga agtccaagac gaaccacttg cttaaaatat ttggtcttag caaatatgct 900
tatgaaatcg ggaataaatc catttgactc acaggaggcc aagccgtaca aaaatgatcc 960
agaaatttta gcaatgacga atttagtaag tgcctatcag aataatgaca tcactgaatt 1020
tgaaaagatt ctaaaaacaa atcacagcaa catcatggat gatcctttca taagagaaca 1080
cattgaagag cttttgcgaa acatcagaac acaagtgctt ataaaattaa ttaagcctta 1140
cacaagaata catattcctt ttatttctaa ggagttaaac atagatgtag ctgatgtgga 1200
gagcttgctg gtgcagtgca tattggataa cactattcat ggccgaattg atcaagtcaa 1260
ccaactcctt gaactggatc atcagaagag gggtggtgca cgatatactg cactagataa 1320
atggaccaac caactaaatt ctctcaacca ggctgtagtc agtaaactgg cttaacagag 1380
aacaagcttt tacagacgtc cttaaggcaa cagtgcagag atgtaatcct taaaagaact 1440
gggaatggca aaactactgt cggttgatgt gtcctgaaaa ttattggagt tatggcagaa 1500
gtgctttttt gatcaactgg tttgtgtttt gctgctgcat ttatcccaag aaaaacagct 1560
ttaatctcca gaagaaaacc saaataccat gggatttatg ctgtattgac atcttgccct 1620
aaacgtacaa catcatagta atttgtcatg ggcaacatga ccagagagaa gatttttgtc 1680
atgattttaa atacactgac acgctactgt tggttaaatt taaacatgtt ttacctgcag 1740
aaattctctc acaaataacc tgcaataact tgaaatgcat acccttttga acacttcctt 1800
ttctcatgta taaattaaaa tgtttgctgc attttgcaaa atgtcaattc tctaaaaatg 1860
tgtccgtata tttctgtacc tgcagtgtag taaaggttta gacgaaaccc cataattata 1920
gtggcatact gtcacttagg tttcaagcag caaaataaac agtgcagctc agaaattgta 1980
aaaaaaaaaa 1990
<210> 29
<211> 1038
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2589371
25/28
CA 02338385 2001-02-06
WO 00/08155 PGT/U899/17777
<400> 29
cataaatact gctctaagaa agggacagga agtctcagag gctggagagc agagcaccaa 60
gatcgttctg gcaggaacag ccagtgggag gttccagctg agcgctcccc agaggtgagc 120
tgatccccag ccacagcaca caggaccagg ctgcgagaac agcatcatca gcatcatgct 1B0
attacaatcc caaaccatgg gggtttctca cagctttaca ccaaagggca tcactatccc 240
tcaaagagag aaacctggac acatgtacca aaacgaagat tacctgcaga acgggctgcc 300
aacagaaacc accgttcttg ggactgtcca gatcctgtgt tgcctgttga tttcaagtct 360
gggggccatc ttggtttttg ctccctaccc ctcccacttc aatccagcaa tttccaccac 420
tttgatgtct gggtacccat ttttaggagc tctgtgtttt ggcattactg gatccctctc 480
aattatctct ggaaaacaat caactaagcc ctttgacctg agcagcttga cctcaaatgc 540
agtgagttct gttactgcag gagcaggcct cttcctcctt gctgacagca tggtagccct 600
gaggactgcc tctcaacatt gtggctcaga aatggattat ctatcctcat tgccttattc 660
ggagtactat tatccaatat atgaaatcaa agattgtctc ctgaccagtg tcagtttaac 720
aggtgtccta gtggtgatgc tcatcttcac tgtgctggag ctcttattag ctgcatacag 780
ttctgtcttt tggtggaaac agctctactc caacaaccct gggagttcat tttcctcgac 840
ccagtcacaa gatcatatcc aacaggtcaa aaagagttct tcacggtctt ggatataagt 900
aactcttggc ctcagaggaa ggaaaagcaa ctcaacactc atggtcaagt gtgattagac 960
tttcctgaaa tctctgccat tttagatact gtgaaacaaa aaaaaaaaaa aaaagctttt 1020
gttttgtaaa aaaaaaaa 1038
<210> 30
<211> 1260
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2656082
<400> 30
gaaaaaccac caaccaaggc caaggagacc agagcccagc acctcaccca gaggacccca 60
gtcagaggcc ccatctcaga cccgaggcta gcatgggctg caggctgctc tgctgtgcgg 120
ttctctgtct cctgggagcg gtccccatgg aaacgggagt tacgcagaca ccaagacacc 180
tggtcatggg aatgacaaat aagaagtctt tgaaatgtga acaacatctg ggtcataacg 240
ctatgtattg gtacaagcaa agtgctaaga agccactgga gctcatgttt gtctacagtc 300
ttgaagaacg ggttgaaaac aacagtgtgc caagtcgctt ctcacctgaa tgccccaaca 360
gctctcactt attccttcac ctacacaccc tgcagccaga agactcggcc ctgtatctct 420
gcgccagcag ccaagtacat ccgggactag cgggaggatt aaatgagcag ttcttcgggc 480
cagggacacg gctcaccgtg ctagaggacc tgaaaaacgt gttcccaccc gaggtcgctg 540
tgtttgagcc atcagaagca gagatctccc acacccaaaa ggccacactg gtgtgcctgg 600
ccacaggctt ctaccccgac cacgtggagc tgagctggtg ggtgaatggg aaggaggtgc 660
acagtggggt cagcacagac ccgcagcccc tcaaggagca gcccgccctc aatgactcca 720
gatactgcct gagcagccgc ctgagggtct cggccacctt ctggcagaac ccccgcaacc 780
acttccgctg tcaagtccag ttctacgggc tctcggagaa tgacgagtgg acccaggata 840
gggccaaacc cgtcacccag atcgtcagcg ccgaggcctg gggtagagca gactgtggct 900
tcacctccga gtcttaccag caaggggtcc tgtctgccac catcctctat gagatcttgc 960
tagggaaggc caccttgtat gccgtgctgg tcagtgccct cgtgctgatg gccatggtca 1020
agagaaagga ttccagaggc tagctccaaa accatcccag gtcattcttc atcctcaccc 1080
aggattctcc tgtacctgct cccaatctgt gttcctaaaa gtgattctca ctctgcttct 1140
catctcctac ttacatgaat acttctctct tttttctgtt tccctgaaga ttgagctccc 1200
aacccccaag tacgaaatag gctaaaccaa taaaaaattg tgtgttggga aaaaaaaaaa 1260
<210> 31
<211> 1551
<212> DNA
26/28
CA 02338385 2001-02-06
WU 00~08I55
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2762182
PGTNS99/17777
<400> 31 ,
agagcccggg ctgaccacag agaggggctg cagggacagg ctgacaggga gggtggtcca 60
cccttctgct gagagctgcc ccttttcagc ctagctcccc ccaccccccc acataggtgg 120
ggcagccgag tgcctgggga agccagggcc ttccctgaca tcccgtcccc agggattgag 180
agatggcaac gattaccgag aaggaggtcc agcagtggta caaaggcttc atcaaggact 240
gccccagtgg gcagctggat gcggcaggct tccagaagat ctacaagcaa ttcttcccgt 300
tcggagaccc caccaagttt gccacatttg ttttcaacgt ctttgatgaa aacaaggacg 360
ggcgaattga gttctccgag ttcatccagg cgctgtcggt gacctcacgg ggaaccctgg 420
atgagaagct acggtgggcc ttcaagctct acgacttgga caatgatggc tacatcacca 480
ggaatgagat gctggacatt gtggatgcca tttaccagat ggtggggaat accgtggagc 540
tcccagagga ggagaacact cctgagaaga gggtggaccg gatctttgcc atgatggata 600
agaatgccga cgggaagctg accctgcagg agttccagga ggggtccaag gcagacccgt 660
ccattgtgca ggcgctgtcc ctctacgacg ggctggtata gtcccaggct ggagctggat 720
gcctgggaac cactcacctc cttctgtgcc atgaggccac ctcagccctg acaccaaccc 780
cgtgcgtcca cccagccttc ttccgcatcc acacacagcc ggctgccctt gacccgggag 840
gccccggctc tcctctcccc tgtcctgcac ccatcccccg cctgaagcca ccggctccaa 900
ttgccagcaa cctctgcttg tccggaaaac gacaacacga aatggaaaag gctacagccc 960
tctgcataaa ccaaggactt ggctgcctcg caggcagcct ccgttcctcc cgctctcttg 1020
cgcgtgtgct tttgtttttt attttgaaca gacgttttaa aagaaaaaaa aacaactacc 1080
ttctgtccta gaagacacag actgacagat ggggtgaagg cctggggacc tcagagaact 1140
ctgccttgcc ctcgtccctc gtccttcggc agccggagag gctgtgggtg ggccgagggt 1200
gtctaggggt tctgcctggt caacgttatt tgtcgtccca tcttttggca gcaaaaccac 1260
ctgcgtggct aggatgatta attatgagga tgatgatttt ttttgtgata acagtattgt 1320
gctttttgtg gggaaagtga ggtttttttt tatatacata tataattgat atctttaatt 1380
tattggttgt taactgttgc tgctgcctgg. tgtgtcctca gctcccaggg ctgcgggccc 1440
accgtttaca tgtgcacgcc ctgacccacc tgcccacgcc gacttgggag gatggtggcc 1500
tgcagcggcc aagaagccaa aaaaaatttt ttttttttca aaaaaaaaaa a 1551
<210> 32
<211> 2889
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No:3140659
<400> 32
gccacagctg gtttagggcc ccgaccactg gggccccttg tcaggaggag acagcctccc 60
ggcccgggga ggacaagtcg ctgccacctt tggctgccga cgtgattccc tgggacggtc 120
cgtttcctgc cgtcagctgc cggccgagtt gggtctccgt ggttcaggcc ggctccccct 180
tcctggtctc ccttctcccg ctgggccggt ttatcgggag gagattgtct tccagggcta 240
gcaattggac ttttgatgat gtttgaccca gcggcaggaa tagcaggcaa cgtgatttca 300
aagctgggct cagcctctgt ttcttctctc gtgtaatcgc aaaacccatt ttggagcagg 360
aattccaatc atgtctgtga tggtggtgag aaagaaggtg acacggaaat gggagaaact 420
cccaggcagg aacacctttt gctgtgatgg ccgcgtcatg atggcccggc aaaagggcat 480
tttctacctg acccttttcc tcatcctggg gacatgtaca ctcttcttcg cctttgagtg 540
ccgctacctg gctgttcagc tgtctcctgc catccctgta tttgctgcca tgctcttcct 600
tttctccatg gctacactgt tgaggaccag cttcagtgac cctggagtga ttcctcgggc 660
gctaccagat gaagcagctt tcatagaaat ggagatagaa gctaccaatg gtgcggtgcc 720
27/28
CA 02338385 2001-02-06
wo oo~osiss
PCT/US99/17777
ccagggccag cgaccaccgc ctcgtatcaa gastttccag ataaacaacc agattgtgaa 780
actgaaatac tgttacacat gcaagatctt ccggcctccc cgggcctccc attgcagcat 840
ctgtgacaac tgtgtggagc gcttcgacca tcactgcccc tgggtgggga attgtgttgg 900
aaagaggaac taccgctact tctacetctt catectttct ctctccctcc tcacaatcta 960
tgtcttcgcc ttcaacatcg tctatgtggc cctcaaatct ttgaaaattg gcttcttgga 1020
gacattgaaa gaaactcctg gaactgttct agaagtcctc atttgcttct ttacactctg 1080
gtccgtcgtg ggactgactg gatttcatac tttcctcgtg gctctcaacc agacaaccaa 1140
tgaagacatc aaaggatcat ggacagggaa gaatcgcgtc cagaatccct acagccatgg 1200
caatattgtg aagaactgct gtgaagtgct gtgtggcccc ttgcccccca gtgtgctgga 1260
tcgaaggggt attttgccac tggaggaaag tggaagtcga cctcccagta ctcaagagac 1320
cagtagcagc ctcttgccac agagcccagc ccccacagaa cacctgaact caaatgagat 1380
gccggaggac agcagcactc ccgaagagat gccacctcca gagcccccag agccaccaca 1440
ggaggcagct gaagctgaga agtagcctat ctatggaaga gacttttgtt tgtgtttaat 1500
tagggctatg agagatttca ggtgagaagt taaacctgag acagagagca agtaagctgt 1560 .
cccttttaac tgtttttctt tggtctttag tcacccagtt gcacactggc attttcttgc 1620
tgcaagcttt tttaaatttc tgaactcaag gcagtggcag aagatgtcag tcacctctga 1680
taactggaaa aatgggtctc ttgggccctg gcactggttc tccatggcct cagccacagg 1740
gtccccttgg accccctctc ttccctccag atcccagccc tcctgcttgg ggtcactggt 1800
ctcattctgg ggctaaaagt ttttgagact ggctcaaatc ctcccaagct gctgcacgtg 1860
ctgagtccag aggcagtcac agagacctct ggccagggga tcctaactgg gttcttgggg 1920
tcttcaggac tgaagaggag ggagagtggg gtcagaagat tctcctggcc accaagtgcc 1980
agcattgccc acaaatcctt ttaggaatgg gacaggtacc ttccacttgt tgtatttatt 2040
agtgtagctt ctcctttgtc tcccatccac tctgacacct aagccccact cttttcccat 2100
tagatatatg taagtagttg tagtagagat aataattgac atttctcgta gactacccag 2160
aaactttttt aatacctgtg ccattctcaa taagaattta tgagatgcca gcggcatagc 2220
ccttcacact ctctgtctca tctctcctcc tttctcatta gcccctttta atttgttttt 2280
ccttttgact cctgctccca ttaggagcag gaatggcagt aataaaagtc tgcactttgg 2340
tcatttcttt tcctcagagg aagcctgagt gctcacttaa acactatccc ctcagactcc 2400
ctgtgtgagg cctgcagagg ccctgaatgc acaaatggga aaccaaggca cagagaggct 2460
ctcctctcct ctcctctccc cegatgtacc ctcaaaaaaa aaaaaaatgc taaccagttc 2520
ttccattaag cctcggctga gtgagggaaa gcccagcact gctgccctct cgggtaactc 2580
accctaaggc ctcggcccac ctctggctat ggtaaccaca ctgggggctt cctccaagcc 2640
ccgctcttcc agcacttcca ccggcagagt cccagagcca cttcaccctg ggggtgggct 2700
gtggccccca gtcagctctg ctcaggacct gctctatttc agggaagaag atttatgtat 2760
tatatgtggc tatatttcct agagcacctg tgttttcctc tttctaagcc agggtcctgt 2820
ctggatgact tatgcggtgg gggagtgtaa accggaactt ttcatetatt tgaaggcgat 2880
taaactgtg
2889
28128
