Note: Descriptions are shown in the official language in which they were submitted.
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MEMBRANE TRANSPORT PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of membrane
transport
proteins and to the use of these sequences in the diagnosis, treatment, and
prevention of membrane
transport disorders; immune/inflammatory disorders; and cell proliferative
disorders including cancer.
BACKGROUND OF THE INVENTION
Eukaryotic cells are bound by a lipid bilayer membrane and subdivided into
functionally
distinct, membrane bound compartments. The membranes maintain essential
differences between the
cytosol, the extracellular environment, and the contents of intracellular
organelles such as the Golgi or
the endoplasmic reticulum. As lipid membranes are highly impermeable to most
polar molecules,
transport of essential nutrients; metal ions such as K', NH,', P;, SOa'--;
sugars; vitamins; metabolic
waste products; cell signaling molecules; drugs; peptides: and proteins and
other macromolecules
across lipid membranes and between organelles must be mediated by a variety of
transport molecules.
Many transport mechanisms are substrate specific, with each transport protein
carrying particular
members of a molecular class, such as ions, sugars, or amino acids, across
membranes. For example,
amino acids are imported into cells via specific amino acid permeases.
Transport proteins are multi-pass transmembrane proteins, which either
actively transport
molecules across the membrane or passively allow them to cross. Active
transport involves
directional pumping of a solute across the membrane, usually against an
electrochemical gradient.
Active transport is tightly coupled to a source of metabolic energy, such as
ATP hydrolysis or an
electrochemically favorable ion gradient. Passive transport involves the
movement of a solute down
its electrochemical gradient. Transport proteins can be further classified as
either carrier proteins or
channel proteins. Carrier proteins, which can function in active or passive
transport, bind to a specific
solute to be transported and undergo a conformational change which transfers
the bound solute across
the membrane. Channel proteins, which only function in passive transport, form
hydrophilic pores
across the membrane. When the pores open, specific solutes, such as inorganic
ions, pass through the
membrane and down the electrochemical gradient of the solute.
Transport proteins play roles in antibiotic resistance, toxin secretion, ion
balance, synaptic
neurotransmission, kidney function, intestinal absorption, tumor growth, and
other diverse cell
functions (Griffith, J. and C. Sansom (1998) The Transporter Facts Book,
Academic Press, San Diego
CA, pp. 3-29). A variety of human inherited diseases are caused by mutation of
transport proteins.
For example, cystinuria is an inherited disease that results from the
inability to transport cystine, the
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disulfide-linked dimer of cysteine, from the urine into the blood.
Accumulation of cystine in the urine
leads to the formation of cystine stones in the kidneys. Also, many transport
proteins are composed
of subunits that may confer specificity for the tissue in which the transport
mechanism functions, and
are therefore associated with tissue-specific disorders. Examples of transport
proteins include
facilitative transporters, the secondary active symporters and antiporters
driven by ion gradients, and
active ATP binding cassette transporters involved in multiple-drug resistance
and targeting of
antigenic peptides to MHC Class 1 molecules, and the El-E2 cation transport
ATPases.
Carrier proteins which transport a single solute from one side of the membrane
to the other
are called uniporters. In contrast, coupled transporters link the transfer of
one solute with
l0 simultaneous or sequential transfer of a second solute, either in the same
direction (symport) or in the
opposite direction (antiport). For example, intestinal and kidney epithelium
contains a variety of
symporter systems wherein the movement of sodium into the cell down its
electrochemical gradient
co-transports a second solute into the cell. The sodium gradient that provides
the driving force for
solute uptake is maintained by the ubiquitous Na;/K' ATPase. Sodium-coupled
transporters include
the mammalian glucose transporter (SGLT1 ), iodide transporter (NIS), and
multivitamin transporter
(SMVT). These three transporters have twelve putative transmembrane segments,
extracellular
glycosylation sites, and cytoplasmically-oriented N- and C-termini. NIS plays
a crucial role in the
evaluation, diagnosis, and treatment of various thyroid pathologies because it
is the molecular basis
for radioiodide thyroid-imaging techniques and for specific targeting of
radioisotopes to the thyroid
2o gland (Levy, O. et al. ( 1997) Proc. Natl. Acad. Sci. USA 94:5568-5573).
SMVT is expressed in the
intestinal mucosa, kidney, and placenta, and is implicated in the transport of
the water-soluble
vitamins, e.g., biotin and pantothenate (Prasad, P.D. et al. (1998) J. Biol.
Chem. 273:7501-7506).
The largest and most diverse family of transport proteins is the ATP-binding
cassette (ABC)
transporters. As a family, ABC transporters can transport substances that
differ markedly in chemicat
structure and size, ranging from small molecules such as ions, sugars, amino
acids, peptides, and
phospholipids, to lipopeptides, large proteins, and complex hydrophobic drugs.
Each ABC
transporter consists of four modules: two nucleotide-binding domains
(NBDs),,which hydrolyze ATP
to supply the energy required for transport; and two membrane-spanning domains
(MSDs), which
may form membrane channels. The NBDs consist of approximately two hundred
conserved amino
acid residues while the MSDs each contain six putative transmembrane segments.
(See, e.g., Saurin,
W. et al. (1994) Mol. Microbiol. 12:993-1004; Shani, N. et al. (1996) J. Biol.
Chem. 271:8725-8730;
Koster, W. and B. Bohm ( 1992) Mol. Gen. Genet. 232:399-407.) The four ABC
transporter modules
may be encoded by a single gene, as is the case for the cystic fibrosis
transmembrane conductance
regulator (CFTR), or by separate genes. When encoded by separate genes, each
gene product
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contains a single NBD and MSD. These "half molecules" form homo- and
heterodimers, such as
Tapl and Tap2, the endoplasmic reticulum-based major histocompatibility (MHC')
peptide transport
system associated with antigen processing (Androlewicz, M.J. et al. ( 1994)
Proc. Natl. Acad. Sci.
USA 91:12716-12720).
Several genetic diseases are attributed to defects in ABC transporters,
including the following
diseases and their corresponding proteins: cystic fibrosis (CFTR, an ion
channel; Welsh, M.J. and
A.E. Smith (1993) Cell 73:1251-1254); X-linked adrenoleukodystrophy, an inborn
error of
peroxisomal ~i-oxidation of very long chain fatty acids (adrenoleukodystrophy
protein, ALDP);
Zellweger syndrome, an inborn error of peroxisome biogenesis (peroxisomal
membrane protein-70,
PMP70); and hyperinsulinemic hypoglycemia (sulfonylurea receptor, SUR). The
ABC transporters
known as P-glycoproteins, or multidrug resistance (MDR) proteins; are
associated with resistance to a
wide range of hydrophobic drugs (MDR1; Gottesman, M.M. and I. Pastan (1993)
Annu. Rev.
Biochem. 62:385-427) or with phosphatidylcholine transport (MDR2; Ruetz, S.
and P. Gros (1994)
Cell 77:1071-1081 ). MDR is common in cancer cells, and contributes to low
efficacy or failure of
chemotherapy (Taglight, D. and S. Michaelis (1998) Methods Enzymol. 292:131-
163). MDR is
mediated by transporters, e.g., P-glycoproteins or the multidrug resistance-
associated protein MRP,
that normally function in the liver, intestines, and kidney to move toxic
substances from the cytosol
into the bile, intestinal lumen, or urine. In cancerous cells, these
transporters extrude
chemotherapeutic agents into the extracellular space, thereby conferring drug
resistance. Recently, an
ABC transporter-type protein was isolated from a human leukemia cell line.
This transporter, termed
the anthracycline resistance associated protein (GI 1279457, SEQ ID N0:42), is
overexpressed in a
multidrug resistant leukemia cell sub-line, and has sequence homology with
other multidrug-
resistance associated proteins including MRP (Longhurst, T.J. et al. (1996)
Br. J. Cancer 74:1331-
1335).
Transport of fatty acids across the plasma membrane can occur by diffusion, a
high capacity,
low affinity process. However, under normal physiological conditions a
significant fraction of fatty
acid transport appears to occur via a high affinity, low capacity protein-
mediated transport process.
Fatty acid transport protein (FATP), an integral membrane protein with four
transmembrane
segments, is expressed in tissues exhibiting high levels of plasma membrane
fatty acid flux, such as
muscle, heart, and adipose. Expression of FATP is upregulated in 3T3-L1 cells
during adipose
conversion, and expression in COS7 fibroblasts elevates the cells' uptake of
long-chain fatty acids.
Expression studies suggest a role for FATP in lipid metabolism, obesity, and
type II diabetes mellitus
(Hui, T.Y. et al. (1998) J. Biol. Chem. 273:27420-27429).
EI-E2 (or P-type) ATPases constitute a superfamily of cation transporters
present in both
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prokaryotes and eukaryotes that mediate membrane flux of all biologically
relevant cations. These
ATPases are postulated to exist in two different conformational states,
designated EI and E2, during
the course of the ATP hydrolysis reaction, and to conserve the energy from ATP
hydrolysis in the
form of an acyl phosphate, primarily an aspartyl phosphate. Members of this
family are divided into
four major groups; the Ca+Z-transporting ATPases, Na+/K+ -and gastric H+/K+-
transporting ATPases,
plasma membrane H+-transporting ATPases (proton pumps), and the bacterial P-
type ATPases
(BLOCKS: BL00154, P-type cation-transporting ATPase superfamily signature).
The metabolism of amino acids is complex and highly regulated. While cells are
capable of
creating most amino acids de novo, the import of amino acids into cells via
specific amino acid
permease proteins is vital for maintaining the appropriate and complete
availability of all necessary
amino acids. This is particularly important during cell proliferation and
differentiation. In addition to
their role as protein building blocks, amino acids also serve as precursors
for a variety of other
important macromolecules. For example, the hormone thyroxine, the pigment
melanin, and the
neurotransmitters histamine, epinephrine, and serotonin are produced from
various amino acid
IS precursors, including histidine, tyrosine, and tryptophan. A component of
sphingolipid formation,
sphingosine, is derived from serine. Porphyrin rings, which are components of
heme molecules, use
glycine as a nitrogen donor. Significant portions of the ring structures of
purines and pyrimidines,
components of nucleic acids, are formed from the breakdown of numerous amino
acids. Amino acids
are also important in energy metabolism. Unlike fatty acids and glucose, amino
acids cannot be
stored in the cell, so excess amino acids are fed into the citric acid cycle
to produce energy molecules
including fatty acids, ketone bodies, and glucose. Thus, precise control of
amino acid metabolism is
extremely important to both proliferating and non-proliferating cells.
The E16 gene, cloned from human peripheral blood lymphocytes, encodes a 241
amino acid
integral membrane protein with multiple predicted transmembrane domains
(Gaugitsch, H.W. et al.
(1992) J. Biol. Chem. 267:11267-11273). E16 gene expression is closely linked
to cellular activation
and division. In myeloid and lymphoid cells, E16 transcripts are rapidly
induced and rapidly
degraded after stimulation. This pattern of expression resembles the kinetics
seen for proto-
oncogenes and lymphokines in the T cell system. Elevated levels of E16
expression were detected in
colonic, gastric, and breast adenocarcinomas, and in lymphoma, while little or
no E16 expression was
detected in normal (non-cancerous) human tissues such as adult brain, lung,
liver, colon, esophagus,
stomach, or kidney, nor in four-month fetal brain, lung, liver, or kidney
(Wolf, D.A. et al. (1996)
Cancer Res. 56:5012-5022; Gaugitsch et al., supra). E16 was detected in every
cell line tested. Its
presence in rapidly dividing cell lines and its absence in human tissues with
low proliferative potential
suggest that E16 is directly involved in the cell division process, where it
helps provide important
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building blocks for energy metabolism, biochemical synthetic pathways, and
protein synthesis.
Post-translationa) modification of polypeptides occurs in the lumen of the'
Golgi apparatus.
Such modifications include, for example, the addition of sugar molecules by
enzymes such as N-
acetylglucosaminyltransferase, to produce glycoproteins. The sugar-donating
molecules in this
reaction are typically nucleotide sugars, such as uridine diphosphate-
galactose (UDP-Gal). UPD-Gal
and other nucleotide sugars are transported from the cytosol into the Golgi
apparatus by specific
transporter molecules. The availability of these nucleotide sugars can
regulate which glycoproteins
are synthesized, and therefore has a significant impact on cellular function
(Toms, L. et al. (1996) J.
Biol. Chem. 271:3897-3901; Guillen, E. et al. (1998) Proc. Natl. Acad. Sci.
USA 95:7888-7892).
The discovery of new membrane transport 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 membrane transport disorders; immune/inflammatory
disorders; and cell
proliferative disorders including cancer.
l5 SUMMARY OF THE INVENTION
The invention features substantially purified polypeptides, membrane transport
proteins,
referred to collectively as "MTRP" and individually as "MTRP-I," "MTRP-2,"
"MTRP-3," "MTRP-
4," "MTRP-S," "MTRP-6," "MTRP-7," "MTRP-8," "MTRP-9," "MTRP-10," "MTRP-11,"
"MTRP-
12," "MTRP-13," "MTRP-14," "MTRP-15," "MTRP-16," and "MTRP-17." In one aspect,
the
invention provides a substantially purified polypeptide comprising an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-17 and fragments thereof. The
invention also includes a
polypeptide comprising an amino acid sequence that differs by one or more
conservative amino acid
substitutions from an amino acid sequence selected from the group consisting
of SEQ ID NO:1-17.
The invention further provides a substantially purified variant having at
least 90% amino acid
identity to at least one of the amino acid sequences selected from the group
consisting of SEQ ID
NO:1-17 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:1-17 and fragments thereof. The invention also includes an isolated
and purified
polynucleotide variant having at least 90% polynucleotide sequence identity to
the poiynucleotide
encoding the polypeptide comprising an amino acid sequence selected from the
group consisting of
SEQ ID NO:I-17 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:1-17 and fragments
thereof. The
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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:1-17 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.
I0 The invention also provides an isolated and purified polynucleotide
comprising a
polynucleotide sequence selected from the group consisting of SEQ ID NO:I 8-34
and fragments
thereof. The invention further provides an isolated and purified
polynucleotide variant having at least
90% polynucleotide sequence identity to the polynucleotide sequence selected
from the group
consisting of SEQ ID N0:18-34 and fragments thereof. The invention also
provides an isolated and
I S purified polynucleotide having a sequence which is complementary to the
polynucleotide comprising
a polynucleotide sequence selected from the group consisting of SEQ ID N0:18-
34 and fragments
thereof.
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
20 consisting of SEQ ID NO:I-17. In another aspect, the expression 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 a polynucleotide of the
invention under conditions suitable for the expression of the polypeptide; and
(b) recovering the
25 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:1-17
and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a
polypeptide selected from
30 the group consisting of SEQ ID NO:1-17 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 MTRP, the method comprising administering
to a subject in need
of such treatment an effective amount of a pharmaceutical composition
comprising a substantially
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purified polypeptide having the amino acid~sequence selected from the group
consisting of SEQ ID
NO:1-17 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 MTRP, 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:1-17 and fragments
thereof.
BRIEF DESCRIPTION OF THE FIGURES AND TABLES
Figures lA, 1B, 1C, and 1D show the amino acid sequence alignment between MTRP-
3
(Incyte Clone ID 1720440; SEQ ID N0:3) and mouse fatty acid transport protein
(GI 2612939; SEQ
ID N0:35), produced using the multisequence alignment program of LASERGENE
software
(DNASTAR, Madison Wl).
Figures 2A, 2B, 2C, and 2D show the amino acid sequence alignment between MTRP-
4
(lncyte Clone ID 2274290; SEQ ID N0:4) and Schistosoma mansoni SMDR1 (GI
425474; SEQ ID
N0:36), produced using the multisequence alignment program of LASERGENE
software
(DNASTAR).
Figures 3A, 3B, 3C, and 3D show the amino acid sequence alignment between MTRP-
5
(Incyte Clone ID 2740029; SEQ ID NO:S) and rat sodium-dependent multivitamin
transporter (GI
3015617; SEQ ID N0:37), produced using the multisequence alignment program of
LASERGENE
software (DNASTAR).
Table 1 shows poiypeptide and nucleotide sequence identification numbers (SEQ
1D NOs),
clone identification numbers (clone IDs), cDNA libraries, and cDNA fragments
used to assemble full-
length sequences encoding MTRP.
Table 2 shows features of each polypeptide sequence, including potential
motifs, homologous
sequences, and methods, algorithms, and searchable databases used for analysis
of MTRP.
Table 3 shows the tissue-specific expression patterns of each nucleic acid
sequence as
determined by northern analysis; diseases, disorders, or conditions associated
with these tissues; and
the vector into which each cDNA was cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which
cDNA clones
encoding MTRP were isolated.
Table 5 shows the tools, programs, and algorithms used to analyze MTRP, along
with
applicable descriptions, references, and threshold parameters.
DESCRIPTION OF THE INVENTION
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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. Al) 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
"MTRP" refers to the amino acid sequences of substantially purified MTRP
obtained from
any species, particularly a mammalian species, including bovine, ovine,
porcine, murine, equine, and
human, and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
MTRP: Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of MTRP either by
directly interacting with
MTRP or by acting on components of the biological pathway in which MTRP
participates.
An "allelic variant" is an alternative form of the gene encoding MTRP. Allelic
variants may
result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in
polypeptides whose structure or function may or may not be altered. A gene may
have none, one, or
many allelic variants of its naturally occurring form. Common mutational
changes which give rise to
allelic variants are generally ascribed to natural deletions, additions, or
substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the
others, one or more times
in a given sequence.
"Altered" nucleic acid sequences encoding MTRP include those sequences with
deletions,
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insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as MTRP or a
polypeptide with at least one functional characteristic of MTRP. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe
of the polynucleotide encoding MTRP, and improper or unexpected hybridization
to allelic variants,
with a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding
MTRP. 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 MTRP. 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 MTRP is
retained. For example,
negatively charged amino acids may include aspartic acid and glutamic acid,
and positively charged
amino acids may include lysine and arginine. Amino acids with uncharged polar
side chains having
similar hydrophilicity values may include: asparagine and glutamine; and
serine and threonine.
Amino acids with uncharged side chains having similar hydrophilicity values
may include: leucine,
1 S isoleucine, and valine; glycine and alanine; and phenylalanine and
tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or synthetic
molecules. Where "amino acid sequence" is recited to refer to an amino acid
sequence of a naturally
occurring protein molecule, "amino acid sequence" and like terms are not meant
to limit the amino
acid sequence to the complete native amino acid sequence associated with the
recited protein
molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well
known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of MTRP. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of MTRP either by
directly interacting with MTRP or by acting on components of the biological
pathway in which MTRP
participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab'),, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind MTRP 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
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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 (KLH). The coupled peptide is
then used to immunize
the animal.
The tenor "antigenic determinant" refers to that region of a molecule (i.e.,
an epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures
on the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition 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"
or "minus" can refer to the
antisense strand, and the designation "positive" or "plus" 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 MTRP, or of any
oligopeptide thereof, to induce a
specific immune response in appropriate animals or cells and to bind with
specific antibodies.
The terms "complementary" and "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 S'." 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 acid strands, and in the design and use of
peptide nucleic acid
(PNA) molecules.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising
a given amino acid sequence" refer broadly to any composition containing the
given polynucleotide or
amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding MTRP or fragments of
MTRP may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
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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 the XL-PCR kit (Perkin-Elmer, Norwalk
CT) in the S' and/or
the 3' direction, and resequenced, or which has been assembled from the
overlapping sequences of
one or more Incyte Clones and, in some cases, one or more public domain ESTs,
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.
"Conservative amino acid substitutions" are those substitutions that, when
made, least
interfere with the properties of the original protein, i.e., the structure and
especially the function of the
protein is conserved and not significantly changed by such substitutions. The
table below shows
amino acids which may be substituted for an original amino acid in a protein
and which are regarded
as conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gin, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
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The term "derivative" refers to the chemical modification of a polypeptide
sequence, or a
polynucleotide sequence. Chemical modifications of a polynucleotide sequence
c'an include, for
example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group.
A derivative
polynucleotide encodes a polypeptide which retains at least one biological or
immunological function
of the natural molecule. A derivative polypeptide is one modified by
glycosylation, pegylation, or any
similar process that retains at least one biological or immunological function
of the polypeptide from
which it was derived.
A "fragment" is a unique portion of MTRP or the polynucleotide encoding MTRP
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example, a
fragment may comprise from S to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10, 15,
20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid residues
in length. Fragments may be preferentially selected from certain regions of a
molecule. For example,
a polypeptide fragment may comprise a certain length of contiguous amino acids
selected from the
first 250 or 500 amino acids (or first 25% or SO% of a polypeptide) as shown
in a certain defined
sequence. Clearly these lengths are exemplary, and any length that is
supported by the specification,
including the Sequence Listing, tables, and figures, may be encompassed by the
present embodiments.
A fragment of SEQ ID NO:I 8-34 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:18-34, for example, as distinct from any
other sequence in the
same genome. A fragment of SEQ ID N0:18-34 is useful, for example, in
hybridization and
amplification technologies and in analogous methods that distinguish SEQ ID
NO:I 8-34 from related
polynucleotide sequences. The precise length of a fragment of SEQ ID N0:18-34
and the region of
SEQ ID N0:18-34 to which the fragment corresponds are routinely determinable
by one of ordinary
skill in the art based on the intended purpose for the fragment.
A fragment of SEQ ID NO:I-l7 is encoded by a fragment of SEQ ID N0:18-34. A
fragment
of SEQ ID NO:I-17 comprises a region of unique amino acid sequence that
specifically identifies
SEQ 1D NO:I-17. For example, a fragment of SEQ ID NO:1-17 is useful as an
immunogenic peptide
for the development of antibodies that specifically recognize SEQ ID NO:1-17.
The precise length of
a fragment of SEQ ID NO:1-17 and the region of SEQ ID NO:1-17 to which the
fragment
corresponds are routinely determinable by one of ordinary skill in the art
based on the intended
purpose for the fragment.
The term "similarity" refers to a degree of complementarity. There may be
partial similarity
or complete similarity. The word "identity" may substitute for the word
"similarity." A partially
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complementary sequence that at least partially inhibits an identical sequence
from hybridizing to a
target nucleic acid is referred to as "substantially similar." The inhibition
of hybridization of the
completely complementary sequence to the target sequence may be examined using
a hybridization
assay (Southern or northern blot, solution hybridization, and the like) under
conditions of reduced
stringency. A substantially similar sequence or hybridization probe will
compete for and inhibit the
binding of a completely similar (identical) sequence to the target sequence
under conditions of
reduced 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" and "% identity," as applied to polynucleotide
sequences, refer
to the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps
in the sequences being compared in order to optimize alignment between two
sequences, and
therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The
"weighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
similarity" between aligned polynucleotide sequence pairs.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
is provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment
Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410),
which is available from
several sources, including the NCBI, Bethesda, MD, and on the Internet at
http://ww<v.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence analysis
programs including "blastn," that is used to align a known polynucleotide
sequence with other
polynucleotide sequences from a variety of databases. Also available is a tool
called "BLAST 2
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Sequences" that is used-for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.go'v/gorf/bl2.html. The
"BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø9 (May-07-1999) set at default parameters. Such default parameters may be,
for example:
Matrix: BLOSUM62
Reward for match: I
Penalty for mismatch: -2
Open Gap: S and Extension Gap: 2 penalties
Gap x drop-off SO
Expect: 1 D
Word Size: I1
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length supported
by the sequences shown herein, in the tables, figures, or Sequence Listing,
may be used to describe a
length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some
alignment methods take into account conservative amino acid substitutions.
Such conservative
substitutions, explained in more detail above, generally preserve the
hydrophobicity and acidity at the
site of substitution, thus preserving the structure (and therefore function)
of the polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
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polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø9
(May-07-1999) with blastp set at default parameters. Such default parameters
may be, for example:
Matrix: BLOSUM62
Open Gap: 11 and Extension Gap: 1 penalties
Gap x drop-off. 50
Expect. 10
Word Size: 3
Filter.' on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ ID number, or may be measured over
a shorter length, for
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at Least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the
elements required for
stable mitotic chromosome segregation and maintenance.
The term "humanized antibody" refers to antibody molecules in which the amino
acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of identity. Specific
hybridization complexes form under permissive annealing conditions and remain
hybridized after the
"washing" step(s). The washing steps) is particularly important in determining
the stringency of the
hybridization process, with more stringent conditions allowing less non-
specific binding, i.e., binding
between pairs of nucleic acid strands that are not perfectly matched.
Permissive conditions for
annealing of nucleic acid sequences are routinely determinable by one of
ordinary skill in the art and
CA 02349818 2001-05-03
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may be consistent among hybridization experiments, whereas wash conditions may
be varied among
experiments to achieve the desired stringency, and therefore hybridization
specificity. Permissive
annealing conditions occur, for example, at 68°C in the presence of
about 6 x SSC, about 1 % (w/v)
SDS, and about 100 pg/ml denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Generally, such wash temperatures
are selected to be about
S°C to 20°C lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which SO% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating T," and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook et al., 1989,
Molecular Cloning: A Laboratory Manual, 2"° ed., vol. 1-3, Cold Spring
Harbor Press, Plainview NY;
specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present invention
include wash conditions of 68°C in the presence of about 0.2 x SSC and
about 0.1 % SDS, for 1 hour.
Alternatively, temperatures of about 65°C, 60°C, 55°C, or
42°C may be used. SSC concentration
may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1
%. Typically, blocking
reagents are used to block non-specific hybridization. Such blocking reagents
include, for instance,
denatured salmon sperm DNA at about 100-200 pg/ml. Organic solvent, such as
formamide at a
concentration of about 35-50% v/v, may also be used under particular
circumstances, such as for
RNA:DNA hybridizations. Useful variations on these wash conditions will be
readily apparent to
those of ordinary skill in the art. Hybridization, particularly under high
stringency conditions, may be
suggestive of evolutionary similarity between the nucleotides. Such similarity
is strongly indicative
of a similar role for the nucleotides and their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., C°t or Rot
analysis) or formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
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cellular and systemic defense systems.
The term "microarray" refers to an arrangement of distinct polynucleotide"s on
a substrate.
The terms "element" and "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 MTRP. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
biological, functional, or immunological properties of MTRP.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with the second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
IS sequence. Generally, operably linked DNA sequences may be in close
proximity or contiguous and,
where necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop
transcript elongation,
and may be pegylated to extend their lifespan in the cell.
"Probe" refers to nucleic acid sequences encoding MTRP, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents,
and enzymes.
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be
annealed to a target
polynucleotide by complementary base-pairing. The primer may then be extended
along the target
DNA strand by a DNA polymerase enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence, e.g., by the polymerase chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least IS contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
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specification, includingthe tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook et al., 1989, Molecular Clonine: A Laboratory Manual, 2"d
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel et al.,1987, Current Protocols in
Molecular BioloQV,
Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis et al., 1990,
PCR Protocols. A
Guide to Methods and Applications, Academic Press, San Diego CA. PCR primer
pairs can be
derived from a known sequence, for example, by using computer programs
intended for that purpose
such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to
5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer
selection programs have incorporated additional features for expanded
capabilities. For example, the
PrimOU primer selection program (available to the public from the Genome
Center at University of
I S Texas South West Medical Center, Dallas TX) is capable of choosing
specific primers from megabase
sequences and is thus useful for designing primers on a genome-wide scope. The
Primer3 primer
selection program (available to the public from the Whitehead lnstitute/M1T
Center for Genome
Research, Cambridge MA) allows the user to input a "mispriming library," in
which sequences to
avoid as primer binding sites are user-specified. Primer3 is useful, in
particular, for the selection of
oligonucleotides for microarrays. (The source code for the latter two primer
selection programs may
also be obtained from their respective sources and modified to meet the user's
specific needs.) The
PrimeGen program (available to the public from the UK Human Genome Mapping
Project Resource
Centre, Cambridge UK) designs primers based on multiple sequence alignments,
thereby allowing
selection of primers that hybridize to either the most conserved or least
conserved regions of aligned
nucleic acid sequences. Hence, this program is useful for identification of
both unique and conserved
oligonucleotides and polynucleotide fragments. The oligonucleotides and
polynucleotide fragments
identified by any of the above selection methods are useful in hybridization
technologies, for
example, as PCR or sequencing primers, microarray elements, or specific probes
to identify fully or
partially complementary polynucleotides in a sample of nucleic acids. Methods
of oligonucleotide
selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
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such as those described .in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic' acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter
sequence. Such a recombinant nucleic acid may be part of a vector that is
used, for example, to
transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
The term "sample" is used in its broadest sense. A sample suspected of
containing nucleic
acids encoding MTRP, or fragments thereof, or MTRP itself, may comprise a
bodily fluid; an extract
from a cell, chromosome, organelle, or membrane isolated from a cell; a cell;
genomic DNA, RNA, or
cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular
structure of the protein, e.g., the antigenic determinant or epitope,
recognized by the binding
molecule. For example, if an antibody is specific for epitope "A," the
presence of a polypeptide
containing the epitope A, or the presence of free unlabeled A, in a reaction
containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the
antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 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,
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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 a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
95% or at least 98% or
greater sequence identity over a certain defined length. A variant may be
described as, for example,
an "allelic" (as defined above), "splice," "species," or "polymorphic"
variant. A splice variant may
have significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternate splicing of exons during mRNA processing. The
corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
IS reference molecule. Species variants are polynucleotide sequences that vary
from one species to
another. The resulting polypeptides generally will have significant amino acid
identity relative to
each other. A polymorphic variant is a variation in the polynucleotide
sequence of a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least
98% or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human membrane transport
proteins (MTRP),
the polynucleotides encoding MTRP, and the use of these compositions for the
diagnosis, treatment,
or prevention of membrane transport disorders; immune/inflammatory disorders;
and cell proliferative
disorders including cancer.
Table I lists the Incyte clones used to assemble full length nucleotide
sequences encoding
MTRP. Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs) of
the polypeptide
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and nucleotide sequences, respectively. Column 3 shows the clone 1Ds of the
Incyte clones in which
nucleic acids encoding each MTRP were identified, and column 4 shows the cDNA
libraries from
which these clones were isolated. Column 5 shows lncyte clones and their
corresponding cDNA
libraries. Clones for which cDNA libraries are not indicated were derived from
pooled cDNA
libraries. The Incyte clones in column S were used to assemble the consensus
nucleotide sequence of
each MTRP and are useful as fragments in hybridization technologies.
The columns of Table 2 show various properties of each of the polypeptides of
the invention:
column 1 references the SEQ ID NO; column 2 shows the number of amino acid
residues in each
polypeptide; column 3 shows potential phosphorylation sites; column 4 shows
potential glycosylation
sites; column 5 shows the amino acid residues comprising signature sequences
and motifs; column 6
shows homologous sequences as identified by BLAST analysis; and column 7 shows
analytical
methods and in some cases, searchable databases to which the analytical
methods were applied. The
methods of column 7 were used to characterize each polypeptide through
sequence homology and
protein motifs.
As shown in Figures 1 A, 1 B, 1 C, and 1 D, MTRP-3 has chemical and structural
similarity
with mouse fatty acid transport protein (FATP; GI 2612939; SEQ ID N0:35). In
particular, MTRP-3
and FATP share 65% identity. As shown in Figures 2A, 2B, 2C, and 2D, MTRP-4
has chemical and
structural similarity with Schistosoma mansoni ATP-binding cassette family
protein, SMDR-1 (GI
425474; SEQ ID N0:36). In particular, MTRP-4 and SMDR-1 share 38% identity. As
shown in
Figures 3A, 3B, 3C, and 3D, MTRP-5 has chemical and structural similarity with
rat sodium-
dependent multivitamin transporter (SMVT; GI 3015617; SEQ ID N0:37). In
particular, MTRP-5
and SMVT share 82% identity.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or
conditions
associated with nucleotide sequences encoding MTRP. The first column of Table
3 lists the
nucleotide SEQ ID NOs. Column 2 lists tissue categories which express MTRP as
a fraction of total
tissues expressing MTRP. Column 3 lists diseases, disorders, or conditions
associated with those
tissues expressing MTRP as a fraction of total tissues expressing MTRP. Column
4 lists the vectors
used to subclone each cDNA library.
Of particular note are the expression patterns of SEQ ID N0:30 and SEQ ID
N0:31. SEQ ID
N0:30 is expressed in only five libraries, of which at least four (80%) are
associated with cell
proliferation and at least one (20%) with inflammation. Two (40%) ofthe five
libraries are associated
with cardiovascular tissue, and one each (20%) with gastrointestinal, nervous,
and reproductive
tissues. SEQ ID N0:31 is expressed in only four libraries, of which at least
three (75%) are
associated with cell proliferation and at least two (50%) with inflammation or
the immune response.
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WO 00/26245 PCT/US99/26048
Two (50%) of the four libraries are associated with hematopoietic/immune
tissue, and one each (25%)
with cardiovascular and reproductive tissues.
The following fragments of the nucleotide sequences encoding MTRP are useful,
for
example, in hybridization or amplification technologies to identify SEQ ID
N0:18-34 and to
distinguish between SEQ ID N0:18-34 and related polynucieotide sequences. The
useful fragments
include the fragment of SEQ ID N0:18 from about nucleotide 110 to about
nucleotide 154; the
fragment of SEQ ID N0:19 from about nucleotide 759 to about nucleotide 839;
the fragment of SEQ
ID N0:20 from about nucleotide 1531 to about nucleotide 1578; the fragment of
SEQ ID N0:21 from
about nucleotide 538 to about nucleotide 597; the fragment of SEQ 1D N0:22
from about nucleotide
2241 to about nucleotide 2294; the fragment of SEQ ID N0:23 from about
nucleotide 116 to about
nucleotide 145; the fragment of SEQ ID N0:24 from about nucleotide 60 to about
nucleotide 89; the
fragment of SEQ ID N0:25 from about nucleotide 160 to about nucleotide 189;
the fragment of SEQ
ID N0:26 from about nucleotide 763 to about nucleotide 792; the fragment of
SEQ ID N0:27 from
about nucleotide 43 to about nucleotide 72; the fragment of SEQ ID N0:28 from
about nucleotide 361
to about nucleotide 405; the fragment of SEQ ID N0:29 from about nucleotide 35
to about nucleotide
79; the fragment of SEQ ID N0:30 from about nucleotide 206 to about nucleotide
250; the fragment
of SEQ ID N0:31 from about nucleotide 71 to about nucleotide 1 I5; the
fragment of SEQ ID N0:32
from about nucleotide 161 to about nucleotide 205; the fragment of SEQ ID
N0:33 from about
nucleotide 364 to about nucleotide 408; and the fragment of SEQ ID N0:34 from
about nucleotide 18
to about nucleotide 62. The polypeptides encoded by the specified fragments of
SEQ ID NO:20-30
and SEQ ID N0:32-34 are useful, for example, as immunogenic peptides.
The columns of Table 4 show descriptions of the tissues used to construct the
cDNA libraries
from which cDNA clones encoding MTRP were isolated. Column 1 references the
nucleotide SEQ
1D NOs, column 2 shows the cDNA libraries from which these clones were
isolated, and column 3
shows the tissue origins and other descriptive information relevant to the
cDNA libraries in column 2.
The invention also encompasses MTRP variants. A preferred MTRP variant is one
which has
at least about 80%, or alternatively at least about 90%, or even at least
about 95% amino acid
sequence identity to the MTRP amino acid sequence, and which contains at least
one functional or
structural characteristic of MTRP.
The invention also encompasses polynucleotides which encode MTRP. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:18-34, which encodes MTRP.
The invention also encompasses a variant of a polynucleotide sequence encoding
MTRP. In
particular, such a variant polynucleotide sequence will have at least about
75%, or alternatively at
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CA 02349818 2001-05-03
WO 00/26245 PC'T/US99/26048
least about 85%, or even at least about 95% polynucleotide sequence identity
to the polynucleotide
sequence encoding MTRP. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID
N0:18-34 which has at least about 75%, or alternatively at least about 85%, or
even at least about
95% polynucleotide sequence identity to a nucleic acid sequence selected from
the group consisting
of SEQ ID N0:18-34. Any one of the polynucleotide variants described above can
encode an amino
acid sequence which contains at least one functional or structural
characteristic of MTRP.
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 MTRP, some
bearing minimal
similarity to the polynucleotide sequences of any known and naturally
occurring gene, may be
produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
sequence that could be made by selecting combinations based on possible codon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring MTRP, and all such variations
are to be considered as
being specifically disclosed.
Although nucleotide sequences which encode MTRP and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally occurring MTRP under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding MTRP or
its derivatives possessing a substantially different codon usage, e.g.,
inclusion of non-naturally
occurring codons. Codons may be selected to increase the rate at which
expression of the peptide
occurs in a particular prokaryotic or eukaryotic host in accordance with the
frequency with which
particular codons are utilized by the host. Other reasons for substantially
altering the nucleotide
sequence encoding MTRP 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 MTRP
and
MTRP 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 MTRP 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
N0:18-34 and fragments thereof under various conditions of stringency. (See,
e.g., Wahi, G.M. and
S.L. Berger ( 1987) Methods Enzymol. 152:399-407; Kimmel, A.R. ( 1987) Methods
Enzymol.
23
CA 02349818 2001-05-03
WO OOI26245 PCT/US99/Z6048
152:507-511.) Hybridization conditions, including annealing and wash
conditions, are described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerise 1, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise
(Perkin-
Elmer), thermostable T7 polymerise (Amersham Pharmacia Biotech, Piscataway
NJ), or
combinations of polymerises and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
(Perkin-Elmer). Sequencing is then carried out using either the ABI 373 or 377
DNA sequencing
system (Perkin-Elmer), the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics,
Sunnyvale CA), or other systems known in the art. The resulting sequences are
analyzed using a
variety of algorithms which are well known in the art. (See, e.g., Ausubel,
F.M. (1997) Short
Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY, unit 7.7;
Meyers, R.A. ( 1995)
Molecular Biology and Biotechnolo>sy, Wiley VCH, New York NY, pp. 856-853.)
The nucleic acid sequences encoding MTRP 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. i 9:3055-3060).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries
(Clontech, Palo
Alto CA) to walk genomic DNA. This procedure avoids the need to screen
libraries and is useful in
finding intron/exon junctions. For all PCR-based methods, primers may be
designed using
commercially available software, such as OLIGO 4.06 Primer Analysis software
(National
24
CA 02349818 2001-05-03
WO 00/26245 PCT/US99/26048
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 S' 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 S' 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
I S 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 MTRP may be cloned in recombinant DNA molecules that direct
expression of MTRP,
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 MTRP.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter MTRP-encoding sequences for a variety of
purposes including, but
not limited to, modification of the cloning, processing, and/or expression of
the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction
sites, alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
In another embodiment, sequences encoding MTRP may be synthesized, in whole or
in part,
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
al. ( 1980) Nucleic Acids
Symp. Ser. 7:215-223; and Horn, T. et al. ( 1980) Nucleic Acids Symp. Ser.
7:225-232.)
Alternatively, MTRP 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.,
CA 02349818 2001-05-03
WO 00/26245 PCT/US99/26048
Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated synthesis may be
achieved using the
ABI 431 A peptide synthesizer (Perkin-Elmer). Additionally, the amino acid
sequence of MTRP, 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 Proaerties,
WH Freeman, New
York NY.)
In order to express a biologically active MTRP, the nucleotide sequences
encoding MTRP 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
IS encoding MTRP. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
MTRP. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding MTRP and its initiation codon and upstream regulatory
sequences are inserted
into the appropriate expression vector, no additional transcriptional or
translational control signals
may be needed. However, in cases where only coding sequence, or a fragment
thereof, is inserted,
exogenous translational control signals including an in-frame ATG initiation
codon should be
provided by the vector. Exogenous translational elements and initiation codons
may be of various
origins, both natural and synthetic. The efficiency of expression may be
enhanced by the inclusion of
enhancers appropriate for the particular host cell system used. (See, e.g.,
Scharf, D. et al. ( 1994)
Results Probl. Cell Differ. 20:125-162.)
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding MTRP and appropriate transcriptional and
translational
control elements. These methods include in vitro recombinant DNA techniques,
synthetic techniques,
and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. ( 1989)
Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-
17; Ausubel, F.M. et
al. ( 1995) Current Protocols in Molecular Biolo~y, John Wiley & Sons, New
York NY, ch. 9, 13, and
16.}
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding MTRP. These include, but are not limited to, microorganisms such as
bacteria transformed
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CA 02349818 2001-05-03
WO 00/26245 PCT/US99/26048
with recombinant bacteriophage, plasmid, or cosmid DNA. expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vector's (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 MTRP. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding MTRP 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 MTRP into the
vector's multiple
cloning site disrupts the lacZ gene, allowing a colorimetric screening
procedure for identification of
transformed bacteria containing recombinant molecules. 1n 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 MTRP are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of MTRP 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 MTRP. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, 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, su ra; Bitter, G.A. et al. ( 1987} Methods Enzymol. 153:516-544; and
Scorer, C.A, et al. ( 1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of MTRP. Transcription of
sequences
encoding MTRP may be driven viral promoters, e.g., the 355 and 19S promoters
of CaMV used alone
or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
6:307-311 ). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-
1680; Brogue, 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 Technoloev
(1992) McGraw Hill, New York NY, pp. 191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
27
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WO 00/26245 PCT/US99/26048
where an adenovirus is used as an expression vector, sequences encoding MTRP
may be ligated into
an adenovirus transcription/translation complex consisting of the late
promoter an'~ tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses MTRP in host cells. (See, e.g., Logan, J. and
T. Shenk ( 1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalian host
cells. SV40 or EBV-
based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. (1997) Nat. Genet.
15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression of
MTRP in cell lines is preferred. For example, sequences encoding MTRP 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, cel Is may be allowed to grow for about 1 to 2
days in enriched media
before being switched to selective media. The purpose of the selectable marker
is to confer resistance
to a selective agent, and its presence allows growth and recovery of cells
which successfully express
the introduced sequences. Resistant clones of stably transformed cells may be
propagated using tissue
culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines:
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk- and apr~ cells, respectively.
(See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic,
or herbicide resistance can be used as the basis for selection. For example,
dhfr confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al: (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. (1981)
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hisD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan ( 1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech), b glucuronidase and its substrate (3-glucuronide, or
iuciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
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CA 02349818 2001-05-03
WO 00/26245 PCT/US99/26048
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 MTRP is inserted within a marker gene sequence, transformed
cells containing
sequences encoding MTRP can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding MTRP 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 MTRP
and that express
MTRP 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.
lmmunological methods for detecting and measuring the expression of MTRP 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 MTRP is
preferred, but a
competitive binding assay may be employed. These and other assays are well
known in the art. (See,
e.g., Hampton, R. et al. ( 1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN,
Sect. 1V; Coligan, J.E. et al. (1997) Current Protocols in Immunoloay, Greene
Pub. Associates and
Wiley-Interscience, New York NY; and Pound, J.D. (1998) lmmunochemical
Protocols, Humana
Press, Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled
hybridization or PCR probes for detecting sequences related to polynucleotides
encoding MTRP
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide.
Alternatively, the sequences encoding MTRP, 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 availabie,
and may be used to synthesize RNA probes in vitro by addition of an
appropriate RNA polymerase
such as T7, T3, or SP6 and labeled nucleotides. These procedures may be
conducted using a variety
of commercially available kits, such as those provided by Amersham Pharmacia
Biotech, Promega
(Madison WI), and US Biochemical. Suitable reporter molecules or labels which
may be used for
29
CA 02349818 2001-05-03
WO OO/Z6245 PCT/US99/26048
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 MTRP may be cultured
under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
S 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 MTRP may be designed to contain signal
sequences which
direct secretion of MTRP through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of
the polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro" or
"pro" form of the protein may also be used to specify protein targeting,
folding, and/or activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are
available from the
American Type Culture Collection (ATCC, Manassas VA) and may be chosen to
ensure the correct
modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding MTRP 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 MTRP protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of MTRP activity.
Heterologous protein and
peptide moieties may also facilitate purification of fusion proteins using
commercially available
affinity matrices. Such moieties include, but are not limited to, glutathione
S-transferase (GST),
maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide
(CBP), 6-His, FLAG,
c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification of their cognate
fusion proteins on immobilized glutathione, .maltose, phenylarsine oxide,
calmodulin, and metal-
chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable
immunoaffinity
purification of fusion proteins using commercially available monoclonal and
polyclonal antibodies
that specifically recognize these epitope tags. A fusion protein may also be
engineered to contain a
proteolytic cleavage site located between the MTRP encoding sequence and the
heterologous protein
sequence, so that MTRP may be cleaved away from the heterologous moiety
following purification.
Methods for fusion protein expression and purification are discussed in
Ausubel ( 1995, suara, ch. 10).
A variety of commercially available kits may also be used to facilitate
expression and purification of
CA 02349818 2001-05-03
WO 00/26245 PCT/US99/26048
fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled MTR~' may
be achieved in
vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These
systems couple transcription and translation of protein-coding sequences
operably associated with the
T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
precursor, for example, 'SS-methionine.
Fragments of MTRP may be produced not only by recombinant means, but also by
direct
peptide synthesis using solid-phase techniques. (See, e.g., Creighton, supra,
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
MTRP may be synthesized separately and then combined to produce the ful)
length molecule.
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists between
regions of MTRP and membrane transport proteins, including amino acid
transporters, ABC
transporters, nucleotide-sugar transporters, transmembrane carrier proteins,
and ATP-dependent
transporter proteins. In addition, the expression of MTRP is closely
associated with nervous,
reproductive, and gastrointestinal tissues; cancer and other cell
proliferative conditions; and with
inflammation and the immune response. Therefore, MTRP appears to play a role
in membrane
transport disorders; immune/inflammatory disorders; and cell proliferative
disorders including cancer.
In the treatment of disorders associated with increased MTRP expression or
activity, it is desirable to
decrease the expression or activity of MTRP. In the treatment of disorders
associated with decreased
MTRP expression or activity, it is desirable to increase the expression or
activity of MTRP.
Therefore, in one embodiment, MTRP 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 MTRP. Examples of such disorders include, but are not limited to,
a membrane transport
disorder such as cystinuria, dibasicaminoaciduria, hypercystinuria, lysinuria,
hartnup disease,
tryptophan malabsorption, methionine malabsorption, histidinuria,
iminoglycinuria,
dicarboxylicaminoaciduria, cystinosis, renal glycosuria, glucose-galactose
malabsorption, familial
hypercholesterolemia, hypouricemia, familial hypophophatemic rickets,
congenital chloridorrhea,
cystic fibrosis, familial goiter, distal renal tubular acidosis, Menkes'
disease, lethal diarrhea,
nephrogenic diabetes insipidus, juvenile pernicious anemia, folate
malabsorption,
adrenoleukodystrophy, hereditary myoglobinuria, Zellweger syndrome,
hyperinsulinemic
hypoglycemia, akinesia, amyotrophic lateral sclerosis, ataxia telangiectasia,
cystic fibrosis, Becker's
muscular dystrophy, Bell's palsy, Charcot-Marie Tooth disease, diabetes
mellitus, diabetes insipidus,
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diabetic neuropathy, Duchenne muscular dystrophy, hyperkalemic periodic
paralysis, normokalemic
periodic paralysis, Parkinson's disease, malignant hyperthermia, multidrug
resistance, myasthenia
gravis, myotonic dystrophy, catatonia, tardive dyskinesia, dystonias,
peripheral neuropathy, cerebral
neoplasms, and prostate cancer; a cardiac disorder associated with transport
such as angina,
bradyarrythmia, tachyarrythmia, hypertension, Long QT syndrome, myocarditis,
cardiomyopathy,
nemaline myopathy, centronuclear myopathy, lipid myopathy, mitochondrial
myopathy, thyrotoxic
myopathy, ethanol myopathy, dermatomyositis, inclusion body myositis,
infectious myositis, and
polymyositis; a neurological disorder associated with transport such as
Alzheimer's disease, amnesia,
bipolar disorder, dementia, depression, epilepsy, Tourette's disorder,
paranoid psychoses, and
schizophrenia; and an other disorder associated with transport such as
neurofibromatosis, postherpetic
neuralgia, trigeminal neuropathy, sarcoidosis, sickle cell anemia, Wilson's
disease, cataracts,
infertility, pulmonary artery stenosis, sensorineural autosomal deafness,
hyperglycemia,
hypoglycemia, Grave's disease, goiter, Cushing's disease, Addison's disease,
glucose-galactose
malabsorption syndrome, and hypercholesterolemia; an immune/inflammatory
disorder such as
acquired immunodeficiency syndrome (AIDS), Addison's disease, adult
respiratory distress
syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma,
atherosclerosis,
autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune
polyenodocrinopathy-
candidiasis-ectodermal dystrophy (APECED), bronchitis, chotecystitis, contact
dermatitis, Crohn's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema,
episodic lymphopenia
with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic
gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis,
hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's
syndrome, rheumatoid arthritis, scferoderma, Sjogren's syndrome, systemic
anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation; a viral, bacterial,
fungal, parasitic, protozoal, or helminthic infection; and trauma; a cell
proliferative disorder such as
actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis,
hepatitis, mixed connective tissue
disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria,
polycythemia vera, psoriasis,
primary thrombocythemia; and a cancer 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.
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In another embodiment, a vector capable of expressing MTRP 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 MTRP including, but not limited to, those described
above.
In a further embodiment, a pharmaceutical composition comprising a
substantially purified
MTRP 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 MTRP
including, but not
limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of MTRP
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of MTRP including, but not limited to, those listed above.
In a further embodiment, an antagonist of MTRP may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of MTRP.
Examples of such
disorders include, but are not limited to, those membrane transport disorders;
immune/inflammatory
disorders; and cell proiiferative disorders including cancer described above.
In one aspect, an
antibody which specifically binds MTRP may be used directly as an antagonist
or indirectly as a
targeting or delivery mechanism for bringing a pharmaceutical agent to cells
or tissues which express
MTRP.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding MTRP may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of MTRP 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 MTRP may be produced using methods which are generally known
in the
art. In particular, purified MTRP may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind MTRP.
Antibodies to MTRP may
also be generated using methods that are well known in the art. Such
antibodies may include, but are
not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies,
Fab fragments, and
fragments produced by a Fab expression library. Neutralizing antibodies (i.e.,
those which inhibit
dimer formation) are generally preferred for therapeutic use.
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For the production of antibodies, various hosts including goats, rabbits,
rats, mice, humans,
and others may be immunized by injection with MTRP or with any fragment or
oligopeptide thereof
which has immunogenic properties. Depending on the host species, various
adjuvants may be used to
increase immunological response. Such adjuvants include, but are not limited
to, Freund's, mineral
gels such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants
used in humans,
BCG (bacilli Calmette-Guerin) and Cor_vnebacterium ~arvum are especially
preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
MTRP have an amino acid sequence consisting of at least about 5 amino acids,
and generally will
consist of at least about 10 amino acids. It is also preferable that these
oligopeptides, peptides, or
fragments are identical to a portion of the amino acid sequence of the natural
protein and contain the
entire amino acid sequence of a small, naturally occurring molecule. Short
stretches of MTRP 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 MTRP may be prepared using any technique which
provides for the
production of antibody molecules by continuous cell lines in culture. These
include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma technique, and
the EBV-hybridoma
technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. ( 1983) Proc. Natl. Acad. Sci.
USA 80:2026-2030; and
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and Takeda,
S. et al. ( 1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the art, to produce
MTRP-specific single
chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be
generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g.,
Burton, D.R. ( 1991 ) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte
population or by screening immunoglobulin libraries or panels of highly
specific binding reagents as
disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl.
Acad. Sci. USA
86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for MTRP may also be
generated.
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WO 00/26245 PCT/US99/26048
For example, such fragments include, but are not limited to, F(ab'), fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab')2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.)
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
MTRP and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies
reactive to two non-interfering MTRP epitopes is generally used, 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 MTRP. Affinity
is expressed as an
I S association constant, K" which is defined as the molar concentration of
MTRP-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 MTRP epitopes, represents the average affinity, or
avidity, of the antibodies for
MTRP. The K8 determined for a preparation of monoclonal antibodies, which are
monospecific for a
particular MTRP epitope, represents a true measure of affinity. High-affinity
antibody preparations
with Ka ranging from about 10''to 10''- L/mole are preferred for use in
immunoassays in which the
MTRP-antibody complex must withstand rigorous manipulations. Low-affinity
antibody preparations
with Ke ranging from about 106 to 10' L/mole are preferred for use in
immunopurification and similar
procedures which ultimately require dissociation of MTRP, preferably in active
form, from the
antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL
Press, Washington, DC;
Liddell, J.E. and Cryer, A. (1991} A Practical Guide to Monoclonal Antibodies,
John Wiley & Sons,
New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
determine the quality and suitability of such preparations for certain
downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml, preferably
5-10 mg specific antibody/ml, is generally employed in procedures requiring
precipitation of MTRP-
antibody complexes. Procedures for evaluating antibody specificity, titer, and
avidity, and guidelines
for antibody quality and usage in various applications, are generally
available. (See, e.g., Catty,
supra, and Coligan et al. supra.)
CA 02349818 2001-05-03
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In another embodiment of the invention, the polynucleotides encoding MTRP, or
any
fragment or complement thereof, may be used for therapeutic purposes. In one
aspect, the
complement of the polynucleotide encoding MTRP 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 MTRP. Thus, complementary
molecules or
fragments may be used to modulate MTRP activity, or to achieve regulation of
gene function. Such
technology is now well known in the art, and sense or antisense
oligonucleotides or larger fragments
can be designed from various locations along the coding or control regions of
sequences encoding
MTRP.
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
MTRP. (See, e.g., Sambrook, su ra; Ausubel, 1995, supra.)
Genes encoding MTRP can be turned off by transforming a cell or tissue with
expression
vectors which express high levels of a polynucleotide, or fragment thereof,
encoding MTRP. 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 MTRP. Oligonucleotides derived from
the transcription
initiation site, e.g., between about positions -10 and +10 from the start
site, may be employed.
Similarly, inhibition can be achieved using triple helix base-pairing
methodology. Triple helix pairing
is useful because it causes inhibition of the ability of the double helix to
open sufficiently for the
binding of polymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances
using triplex DNA have been described in the literature. (See, e.g., Gee, J.E.
et al. (1994) in Huber,
B.E. and B.I. Carr, Molecular and Immunolo i; ~ c Approaches, Futura
Publishing, Mt. Kisco NY, pp.
163-177.) A complementary sequence or antisense molecule may also be designed
to block
translation of mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
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WO 00/26245 PCT/US99/26048
molecule to complementary target RNA, followed by endor~ucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding MTRP.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared
by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA
sequences encoding MTRP. 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,
cel Is, or tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3' ends
of the molecule, or the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages
within the backbone of the molecule. This concept is inherent in the
production of PNAs and can be
extended in all of these molecules by the inclusion of nontraditional bases
such as inosine, queosine,
and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified
forms of adenine, cytidine,
guanine, thymine, and uridine which are not as easily recognized by endogenous
endonucleases.
Many methods for introducing vectors into cells or tissues are available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
Biotechnol. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
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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 MTRP,
antibodies to MTRP, and mimetics, agonists, antagonists, or inhibitors of
MTRP. 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, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
In addition to the active ingredients, these pharmaceutical compositions may
contain suitable
pharmaceutically-acceptable carriers comprising excipients and auxiliaries
which facilitate processing
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 Remin tg on's
Pharmaceutical Sciences (Maack Publishing, Easton PA).
Pharmaceutical compositions for oral administration can be formulated using
pharmaceutically acceptable carriers well known in the art in dosages suitable
for oral administration.
Such carriers enable the pharmaceutical compositions to be formulated as
tablets, pills, dragees,
capsules, liquids, gels, syrups, slurries, suspensions, and the like, for
ingestion by the patient.
Pharmaceutical preparations for oral use can be obtained through combining
active
compounds with solid excipient and processing the resultant mixture of
granules (optionally, after
grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be
added, if desired. Suitable
excipients include carbohydrate or protein fillers, such as sugars, including
lactose, sucrose, mannitol,
and sorbitol; starch from corn, wheat, rice, potato, or other plants;
cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums,
including arabic and
tragacanth; and proteins, such as gelatin and collagen. if desired,
disintegrating or solubilizing agents
may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and
alginic acid or a salt thereof,
such as sodium alginate.
Dragee cores may be used in conjunction with suitable coatings, such as
concentrated sugar
solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone,
carbopo) gel, polyethylene
glycol, and/or titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee coatings for
product identification or to
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characterize the quantity of active compound, i.e., dosage. .
Pharmaceutical preparations which can be used orally include push-fit capsules
made of
gelatin, as well as soft, sealed capsules made of gelatin and a coating, such
as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers or
binders, such as lactose or
starches, lubricants, such as talc or magnesium stearate, and, optionally,
stabilizers. In soft capsules,
the active compounds may be dissolved or suspended in suitable liquids, such
as fatty oils, liquid, or
liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration may be
formulated in
aqueous solutions, preferably in physiologically compatible buffers such as
Hanks' solution, Ringer's
solution, or physiologically buffered saline. Aqueous injection suspensions
may contain substances
which increase the viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or
dextran. Additionally, suspensions of the active compounds may be prepared as
appropriate oily
injection suspensions. Suitable lipophilic solvents or vehicles include fatty
oils, such as sesame oil, or
synthetic fatty acid esters, such as ethyl oieate, triglycerides, or
liposomes. Non-lipid polycationic
IS amino polymers may also be used for delivery. Optionally, the suspension
may also contain suitable
stabilizers or agents to increase the solubility of the compounds and allow
for the preparation of
highly concentrated solutions.
For topical or nasal administration, penetrants appropriate to the particular
barrier to be
permeated are used in the formulation. Such penetrants are generally known in
the art.
The pharmaceutical compositions of the present invention may be manufactured
in a manner
that is known in the art, e.g., by means of conventional mixing, dissolving,
granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping, or
lyophilizing processes.
The pharmaceutical composition may be provided as a salt and can be formed
with many
acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic,
tartaric, malic, and succinic
acids. Salts tend to be more soluble in aqueous or other protonic solvents
than are the corresponding
free base forms. In other cases, the preparation may be a lyophilized powder
which may contain any
or all ofthe following: I mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to
7% mannitol, at a
pH range of 4.5 to 5.5, that is combined with buffer prior to use.
After pharmaceutical compositions have been prepared, they can be placed in an
appropriate
container and labeled for treatment of an indicated condition. For
administration of MTRP, 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.
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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 MTRP
or fragments thereof, antibodies of MTRP, and agonists, antagonists or
inhibitors of MTRP, 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 LDS° (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDS°/EDs°
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
little or no toxicity. The dosage varies within this range depending upon the
dosage form employed,
the sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and
response to therapy. Long-acting pharmaceutical compositions may be
administered every 3 to 4
days, every week, or biweekly depending on the half life and clearance rate of
the particular
formulation.
Normal dosage amounts may vary from about 0.1 ~g to 100,000 ~cg, up to a total
dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind MTRP may be used for
the
diagnosis of disorders characterized by expression of MTRP, or in assays to
monitor patients being
CA 02349818 2001-05-03
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treated with MTRP or agonists, antagonists, or inhibitors of MTRP. Antibodies
useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics: Diagnostic assays
for MTRP include methods which utilize the antibody and a label to detect MTRP
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 MTRP, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
MTRP expression. Normal
or standard values for MTRP expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibody to MTRP under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of MTRP
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 MTRP may
be used
for diagnostic purposes. The polynucleotides which may be used include
oligonucleotide sequences,
complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used
to detect
and quantify gene expression in biopsied tissues in which expression of MTRP
may be correlated
with disease. The diagnostic assay may be used to determine absence, presence,
and excess
expression of MTRP, and to monitor regulation of MTRP levels during
therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding MTRP or closely related
molecules may be used
to identify nucleic acid sequences which encode MTRP. The specificity of the
probe, whether it is
made from a highly specific region, e.g., the 5' regulatory region, or from a
Less specific region, e.g., a
conserved motif, and the stringency of the hybridization or amplification will
determine whether the
probe identifies only naturally occurring sequences encoding MTRP, allelic
variants, or related
sequences.
Probes may also be used for the detection of related sequences, and may have
at least 50%
sequence identity to any of the MTRP encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:18-34 or from
genomic sequences including promoters, enhancers, and introns of the MTRP
gene.
Means for producing specific hybridization probes for DNAs encoding MTRP
include the
cloning of polynucleotide sequences encoding MTRP or MTRP derivatives into
vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and may
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be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes
maybe labeled by a
variety of reporter groups, for example, by radionuclides such as''-P 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 MTRP may be used for the diagnosis of
disorders
associated with expression of MTRP. Examples of such disorders include, but
are not limited to, a
membrane transport disorder such as cystinuria, dibasicaminoaciduria,
hypercystinuria, lysinuria,
hartnup disease, tryptophan malabsorption, methionine malabsorption,
histidinuria, iminoglycinuria,
dicarboxylicaminoaciduria, cystinosis, renal glycosuria, glucose-galactose
malabsorption, familial
hypercholesterolemia, hypouricemia, familial hypophophatemic rickets,
congenital chloridorrhea,
cystic fibrosis, familial goiter, distal renal tubular acidosis, Menkes'
disease, lethal diarrhea,
nephrogenic diabetes insipidus, juvenile pernicious anemia, folate
malabsorption,
adrenoleukodystrophy, hereditary myoglobinuria, Zellweger syndrome,
hyperinsulinemic
hypoglycemia, akinesia, amyotrophic lateral sclerosis, ataxia telangiectasia,
cystic fibrosis, Becker's
IS muscular dystrophy, Bell's palsy, Charcot-Marie Tooth disease, diabetes
mellitus, diabetes insipidus,
diabetic neuropathy, Duchenne muscular dystrophy, hyperkalemic periodic
paralysis, normokalemic
periodic paralysis, Parkinson's disease, malignant hyperthermia, multidrug
resistance, myasthenia
gravis, myotonic dystrophy, catatonia, tardive dyskinesia, dystonias,
peripheral neuropathy, cerebral
neoplasms, and prostate cancer; a cardiac disorder associated with transport
such as angina,
bradyarrythmia, tachyarrythmia, hypertension, Long QT syndrome, myocarditis,
cardiomyopathy,
nemaline myopathy, centronuclear myopathy, lipid myopathy, mitochondria)
myopathy, thyrotoxic
myopathy, ethanol myopathy, dermatomyositis, inclusion body myositis,
infectious myositis, and
polymyositis; a neurological disorder associated with transport such as
Alzheimer's disease, amnesia,
bipolar disorder, dementia, depression, epilepsy, Tourette's disorder,
paranoid psychoses, and
schizophrenia; and an other disorder associated with transport such as
neurofibromatosis, postherpetic
neuralgia, trigeminal neuropathy, sarcoidosis, sickle cell anemia, Wilson's
disease, cataracts,
infertility, pulmonary artery stenosis, sensorineural autosomal deafness,
hyperglycemia,
hypoglycemia, Grave's disease, goiter, Cushing's disease, Addison's disease,
glucose-galactose
malabsorption syndrome, and hypercholesterolemia; an immune/inflammatory
disorder such as
acquired immunodeficiency syndrome (AIDS), Addison's disease, adult
respiratory distress
syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma,
atherosclerosis,
autoimmune hemolytic anemia, autoimrriune thyroiditis, autoimmune
polyenodocrinopathy-
candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact
dermatitis, Crohn's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema,
episodic lymphopenia
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with lymphocytotoxins; erythroblastosis fetalis, erythema nodosum, atrophic
gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto s
thyroiditis,
hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's
syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation; a viral, bacterial,
fungal, parasitic, protozoal, or helminthic infection; and trauma; a cell
proliferative disorder such as
actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis,
hepatitis, mixed connective tissue
disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria,
polycythemia vera, psoriasis,
primary thrombocythemia; and a cancer including adenocarcinoma, leukemia,
lymphoma, melanoma,
myeloma, sarcoma, teratocarcinoma; and, in particular, a cancer of the adrenal
gland, bladder, bone,
bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal
tract, heart, kidney, liver,
lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands,
skin, spleen, testis,
thymus, thyroid, and uterus. The polynucleotide sequences encoding MTRP may be
used in Southern
or northern analysis, dot blot, or other membrane-based technologies; in PCR
technologies; in
dipstick, pin, and muttiformat ELISA-like assays; and in microarrays utilizing
fluids or tissues from
patients to detect altered MTRP expression. Such qualitative or quantitative
methods are well known
in the art.
In a particular aspect, the nucleotide sequences encoding MTRP may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding MTRP may be labeled by standard methods and added to a
fluid or tissue sample
from a patient under conditions suitable for the formation of hybridization
complexes. After a
suitable incubation period, the sample is washed and the signal is quantified
and compared with a
standard value. If the amount of signal in the patient sample is significantly
altered in comparison to a
control sample then the presence of altered levels of nucleotide sequences
encoding MTRP 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
MTRP, 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 MTRP, under conditions suitable for
hybridization or
amplification. Standard hybridization may be quantified by comparing the
values obtained from
43
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WO 00/26245 PCT/US99/26048
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.
p 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
I S or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding MTRP
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 MTRP, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
20 MTRP, and will be employed under optimized conditions for identification of
a specific gene or
condition. Oligomers may also be employed under less stringent conditions for
detection or
quantification of closely related DNA or RNA sequences.
Methods which may also be used to quantify the expression of MTRP include
radiolabeling
or biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
25 standard curves. (See, e.g., Melby, P.C. et al. ( 1993) J. Immunol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by running the assay in a high-throughput format where the
oligomer of interest is
presented in various dilutions and a spectrophotometric or colorimetric
response gives rapid
quantitation.
30 In further embodiments, oligonucleotides or longer fragments derived from
any of the
polynucleotide sequences described herein may be used as targets in a
microarray. The microarray
can be used to monitor the expression level of large numbers of genes
simultaneously and to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, and to develop and
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monitor the activities of therapeutic agents.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Bfennan, T.M. et at. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalon, D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-
2155; and Heller, M.J. et al. ( 1997) U.S. Patent No. 5,605,662.)
In another embodiment of the invention, nucleic acid sequences encoding MTRP
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 (PACs), bacterial artificial chromosomes (BACs), bacterial P1
constructions, or single
chromosome cDNA libraries. (See, e.g., Harrington, J.J. et al. (1997) Nat.
Genet. 15:345-355; Price,
C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J. (1991) Trends Genet. 7:149-
154.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
chromosome
t5 mapping techniques and genetic map data. (See, e.g., Heinz-Ulrich, et al.
(1995) in Meyers, supra,
pp. 965-968.) Examples of genetic map data can be found in various scientific
journals or at the
Online Mendelian Inheritance in Man (OMIM) World Wide Web site. Correlation
between the
location of the gene encoding MTRP on a physical chromosomal map and a
specific disorder, or a
predisposition to a specific disorder, may help define the region of DNA
associated with that disorder.
The nucleotide sequences of the invention may be used to detect differences in
gene sequences among
normal, carrier, and affected individuals.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
may reveal associated markers even if the number or arm of a particular human
chromosome is not
known. New sequences can be assigned to chromosomal arms by physical mapping.
This provides
valuable information to investigators searching for disease genes using
positional cloning or other
gene discovery techniques. Once the disease or syndrome has been crudely
localized by genetic
linkage to a particular genomic region, e.g., ataxia-telangiectasia to 11 q22-
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, MTRP, its catalytic or immunogenic
fragments, or
CA 02349818 2001-05-03
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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 MTRP 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.) Ln this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with MTRP, or
fragments thereof,
and washed. Bound MTRP is then detected by methods well known in the art.
Purified MTRP 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 MTRP specifically compete with a test compound
for binding MTRP.
In this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with MTRP.
In additional embodiments, the nucleotide sequences which encode MTRP may be
used in
any molecular biology techniques that have yet to be developed, provided the
new techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following preferred specific
embodiments are, therefore, to be construed as merely illustrative, and not
limitative ofthe remainder
of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below, in
particular U.S. Ser. No. [Attorney Docket No. PF-0633 P, filed November 4,
1998], U.S. Ser. No.
[Attorney Docket No. PF-0645 P, filed November 24, 1998], U.S. Ser. No.
[Attorney Docket No. PF-
0657 P, filed December 22, 1998], and U.S. Ser. No. 60/121,896, 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
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tissues were homogenized and lysed in guanidinium isothiocyanate, while others
were homogenized
and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL
(Life Technologies), a
monophasic solution of phenol and guanidine isothiocyanate. The resulting
lysates were centrifuged
over CsCI cushions or extracted with chloroform. RNA was precipitated from the
lysates with either
isopropanoi or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A+) RNA was isolated
using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonueleotide 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 51000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), or
pINCY (Incyte
Pharmaceuticals, Palo Alto CA). Recombinant plasmids were transformed into
competent E. coli
cells including XLI-Blue, XLI-BIueMRF, or SOLR from Stratagene or DHSa, DHIOB,
or
ElectroMAX DHIOB from Life Technologies.
II. Isolation of cDNA Clones
Plasmids were recovered from host cells by in vivo excision using the UNIZAP
vector system
(Stratagene) or by cell lysis. Plasmids were purified using at least one of
the following: a Magic or
WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep
purification kit (Edge
Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid,
QIAWELL 8
Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid
purification kit from QIAGEN.
Following precipitation, plasmids were resuspended in 0.1 ml of distilled
water and stored, with or
without lyophiiization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
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high-throughput format(Rao, V.B. (1994) Anal. Biochem. 216:1-14): Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
proces"sed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically
using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence
scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
cDNA sequencing reactions were processed using standard methods or high-
throughput
instrumentation such as the ABI CATALYST 800 (Perkin-Elmer) thermal cycler or
the PTC-200
thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser
(Robbins Scientific) or
the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Pharmacia Biotech or supplied in ABl
sequencing kits such as
the ABI PR1SM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-
Elmer).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carried out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics}; the
IS ABI PRISM 373 or 377 sequencing system (Perkin-Elmer) in conjunction with
standard ABI
protocols and base calling software; or other sequence analysis systems known
in the art. Reading
frames within the cDNA sequences were identified using standard methods
(reviewed in Ausubel,
1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension
using the techniques
disclosed in Example V.
The polynucleotide sequences derived from cDNA sequencing were assembled and
analyzed
using a combination of software programs which utilize algorithms well known
to those skilted in the
art. Table S summarizes the tools, programs, and algorithms used and provides
applicable
descriptions, references, and threshold parameters. The first column of Table
5 shows the tools,
programs, and algorithms used, the second column provides brief descriptions
thereof, the third
column presents appropriate references, all of which are incorporated by
reference herein in their
entirety, and the fourth column presents, where applicable, the scores,
probability values, and other
parameters used to evaluate the strength of a match between two sequences (the
higher the score, the
greater the homology between two sequences). Sequences were analyzed using
MACDNASIS PRO
software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE
software
(DNASTAR). Polynucleotide and polypeptide sequence alignments were generated
using the default
parameters specified by the clustal algorithm as incorporated into the
MEGALIGN multisequence
alignment program (DNASTAR), which also calculates the percent identity
between aligned
sequences.
The polynucleotide sequences were validated by removing vector, linker, and
polyA
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sequences and by masking ambiguous bases, using algorithms and programs based
on BLAST,
dynamic programing, and dinucleotide nearest neighbor analysis. The sequences
were then queried
against a selection of public databases such as the GenBank primate, rodent,
mammalian, vertebrate,
and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM to acquire
annotation using programs based on BLAST, FASTA, and BLIMPS. The sequences
were assembled
into full length polynucleotide sequences using programs based on Phred,
Phrap, and Consed, and
were screened for open reading frames using programs based on GeneMark, BLAST,
and FASTA.
The full length polynucleotide sequences were translated to derive the
corresponding full length
amino acid sequences, and these full length sequences were subsequently
analyzed by querying
against databases such as the GenBank databases (described above), SwissProt,
BLOCKS, PRINTS,
DOMO, PRODOM, Prosite, and Hidden Markov Model (HMM~based protein family
databases such
as PFAM. HMM is a probabilistic approach which analyzes consensus primary
structures of gene
families. (See, e.g., Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.)
The programs described above for the assembly and analysis of full length
polynucleotide and
amino acid sequences were also used to identify polynucleotide sequence
fragments from SEQ ID
N0:18-34. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
amplification technologies were described in The Invention section above.
IV. Northern Analysis
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
from a particular cell type or tissue have been bound. (See, e.g., Sambrook,
s. upra, ch. 7; Ausubel,
1995, suara, 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 (lncyte
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 ofthe search is the product score, which is
defined as:
sequence identity x % maximum BLAST score
100
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. For example, with a product score of 40, the
match will be exact within
a 1% to 2% error, and, with a product score of 70, the match will be exact.
Similar molecules are
usually identified by selecting those which show product scores between i 5
and 40, although lower
scores may identify related molecules.
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The results of northern analyses are reported as a percentage distribution of
libraries in which
the transcript encoding MTRP occurred. Analysis involved the categorization
oi=cDNA libraries by
organ/tissue and disease. The organ/tissue categories included cardiovascular,
dermatologic,
developmental, endocrine, gastrointestinal, hematopoietic/immune,
musculoskeletal, nervous,
reproductive, and urologic. The disease/condition categories included cancer,
inflammation, trauma,
cell proliferation, neurological, and pooled. For each category, the number of
libraries expressing the
sequence of interest was counted and divided by the total number of libraries
across all categories.
Percentage values of tissue-specific and disease- or condition-specific
expression are reported in
Table 3
V. Extension of MTRP Encoding Polynucleotides
The full length nucleic acid sequences of SEQ ID NO: I 8-34 were produced by
extension of
an appropriate fragment of the full length molecule using oligonucleotide
primers designed from this
fragment. One primer was synthesized to initiate 5' extension of the known
fragment, and the other
primer, to initiate 3' extension of the known fragment. The initial primers
were designed using
OLIGO 4.06 software (National Biosciences}, or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68°C to about 72°C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg=', (NH,,)ZS04,
and ~3-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerase (Stratagene), with the following
parameters for primer
pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C,
2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5
min; Step 7: storage at 4°C. In the
alternative, the parameters for primer pair T7 and SK+ were as follows: Step
1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 pl
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1 X TE
and 0.5 pl of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to hind to the reagent. The plate was scanned in a
Fluoroskan II
CA 02349818 2001-05-03
WO 00/26245 PCT/US99/26048
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ~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 relegation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones
were relegated using T4 ligase (New England Biolabs, Beverly MA) into pUC I 8
vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in
restriction site
overhangs, and transfected into competent E. coli cells. Transformed cells
were selected on
antibiotic-containing media, individual colonies were picked and cultured
overnight at 37°C in 384-
well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the
following
parameters: Step 1: 94°C, 3 min; Step 2: 94°C, IS sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step
7: storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted with
20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer
sequencing
primers and the DYENAMIC DIRECT kit (Arnersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
In like manner, the nucleotide sequences of SEQ ID NO:I 8-34 are used to
obtain S'
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 N0:18-34 are employed to screen
cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide
fragments. Oligonucleotides are designed using state-of the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 ~Ci of
[y-'ZP] adenosine triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
51
CA 02349818 2001-05-03
WO 00/26245 PCT/US99/26048
SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typic"al membrane-based
hybridization analysis of human genomic DNA digested with one of the following
endonucleases:
Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
VII. Microarrays
A chemical coupling procedure and an ink jet device can be used to synthesize
array
elements -)? -on the surface of a substrate. (See, e.g., Baldeschweiler,
supra.) An array analogous to a dot
or slot blot may also be used to arrange and link elements to the surface of a
substrate using thermal,
UV, chemical, or mechanical bonding procedures. A typical array may be
produced by hand or using
available methods and machines and contain any appropriate number of elements.
After
hybridization, nonhybridized probes are removed and a scanner used to
determine the levels and
patterns of fluorescence. The degree of complementarity and the relative
abundance of each probe
which hybridizes to an element on the microarray may be assessed through
analysis of the scanned
images.
Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may
comprise
the elements of the microarray. Fragments suitable for hybridization can be
selected using software
well known in the art such as LASERGENE software (DNASTAR). Full-length cDNAs,
ESTs, or
fragments thereof corresponding to one of the nucleotide sequences of the
present invention, or
selected at random from a cDNA library relevant to the present invention, are
arranged on an
appropriate substrate, e.g., a glass slide. The cDNA is fixed to the slide
using, e.g., UV cross-linking
followed by thermal and chemical treatments and subsequent drying. {See, e.g.,
Schena, M. et al.
(1995) Science 270:467-470; 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 Polynucleotides
Sequences complementary to the MTRP-encoding sequences, or any parts thereof,
are used
to detect, decrease, or inhibit expression of naturally occurring MTRP.
Although use of
oligonucleotides comprising from about 15 to 30 base pairs is described,
essentially the same
52
CA 02349818 2001-05-03
WO 00/26245 PCT/US99/Z6048
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 MTRP. 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 MTRP-encoding
transcript.
IX. Expression of MTRP
Expression and purification of MTRP is achieved using bacterial or virus-based
expression
systems. For expression of MTRP in bacteria, cDNA is subcloned into an
appropriate vector
containing an antibiotic resistance gene and an inducible promoter that
directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the
trp-lac (tac) hybrid
promoter and the TS or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21(DE3).
Antibiotic resistant bacteria express MTRP upon induction with isopropyl beta-
D-
thiogalactopyranoside (IPTG). Expression of MTRP in eukaryotic cells is
achieved by infecting
insect or mammalian cell lines with recombinant Autographica californica
nuclear polyhedrosis virus
(AcMNPV), commonly known as bacuiovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding MTRP 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 Snodoptera frugi~erda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus. (See Engelhard, E.K.
et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al.
(1996) Hum. Gene Ther.
7:1937-1945.)
In most expression systems, MTRP 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 ja onp icum, enables the purification of
fusion proteins on
immobilized glutathione under conditions that maintain protein activity and
antigenicity (Amersham
Pharmacia Biotech). Following purifECation, the GST moiety can be
proteolytically cleaved from
MTRP at specii~ically 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 MTRP obtained by these methods can be used
directly in the
53
CA 02349818 2001-05-03
WO 00/26245 PCT/US99/26048
following activity assay,
X. Demonstration of MTRP Activity
ATPase activity associated with MTRP can be measured by hydrolysis of
radiolabeled ATP-
[y-''-P], separation of the hydrolysis products by chromatographic methods,
and quantitation of the
recovered''-P using a scintillation counter. The reaction mixture contains ATP-
[y-3'P] and varying
amounts of MTRP in a suitable buffer incubated at 37°C for a suitable
period of time. The reaction is
terminated by acid precipitation with trichloroacetic acid and then
neutralized with base, and an
aliquot of the reaction mixture is subjected to membrane or filter paper-based
chromatography to
separate the reaction products. The amount of'ZP liberated is counted in a
scintillation counter. The
amount of radioactivity recovered is proportional to the ATPase activity of
MTRP in the assay.
MTRP transport activity is assayed by measuring uptake of labeled substrates
into Xenopus
laevis oocytes. Oocytes at stages V and VI are injected with MTRP mRNA (10 ng
per oocyte) and
incubated for 3 days at 18°C in OR2 medium (82.SmM NaCI, 2.5 mM KCI,
1mM CaCI,, 1mM
MgCI,, 1 mM Na~HP04, 5 mM Hepes, 3.8 mM NaOH , SOp.g/ml gentamycin, pH 7.8) to
allow
expression of MTRP protein. Oocytes are then transferred to standard uptake
medium ( 100mM NaCI,
2 mM KCI, 1mM CaCl2, 1mM MgClz, 10 mM Hepes/Tris, pH 7.5). Uptake of various
substrates
(e.g., amino acids, sugars, drugs, and neurotransmitters) is initiated by
adding a'H-labeled substrate
to the oocytes. After 30 minutes of incubation, uptake is terminated by
washing the oocytes three
times in Na+-free medium. Incorporation of 'H is measured, and compared with
controls. MTRP
transport activity is proportional to the level of internalized'H-labeled
substrate.
XI. Functional Assays
MTRP function is assessed by expressing the sequences encoding MTRP 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 ,ug of recombinant vector are
transiently transfected into
a human cell line, for example, an endothelial or hematopoietic cell line,
using either liposome
formulations or electroporation. 1-2 ug of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate
the apoptotic state of the cells and other cellular properties. FCM detects
and quantifies the uptake of
54
CA 02349818 2001-05-03
WO 00/26245 PCTNS99/26048
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
piopidium iodide;
changes in cell size and granularity as measured by forward light scatter and
90 degree side light
scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as
measured by reactivity with
specific antibodies; and alterations in plasma membrane composition as
measured by the binding of
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M.G. ( 1994) Flow C ometrv, Oxford, New York NY.
The influence of MTRP on gene expression can be assessed using highly purified
populations of cells transfected with sequences encoding MTRP 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
I S art. Expression of mRNA encoding MTRP and other genes of interest can be
analyzed by northern
analysis or microarray techniques.
XII. Production of MTRP Specific Antibodies
MTRP 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 MTRP 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 of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to KLH
(Sigma-Aldrich, St.
Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
to increase
immunogenicity. (See, e.g., Ausubel, 1995, su ra.) Rabbits are immunized with
the oligopeptide-
KLH complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide and anti-
MTRP activity by, for example, binding the peptide or MTRP to a substrate,
blocking with 1% BSA,
reacting with rabbit antisera, washing, and reacting with radio-iodinated goat
anti-rabbit IgG.
XIII. Purification of Naturally Occurring MTRP Using Specific Antibodies
Naturally occurring or recombinant MTRP is substantially purified by
immunoaffinity
CA 02349818 2001-05-03
PCT/US99/26048
WO 00/26245
chromatography using antibodies specific for MTRP. An immunoaffinity column is
constructed by
covalently coupling anti-MTRP 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 MTRP are passed over the~immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of MTRP (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/MTRP 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 MTRP is collected.
XIV. Identification of Molecules Which Interact with MTRP
MTRP, or biologically active fragments thereof, are labeled with '251 Bolton-
Hunter reagent.
(See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.)
Candidate molecules
previously arrayed in the wells of a multi-well plate are incubated with the
labeled MTRP, washed,
and any wells with labeled MTRP complex are assayed. Data obtained using
different concentrations
of MTRP are used to calculate values for the number, affinity, and association
of MTRP with the
candidate molecules.
Various modifications and variations of the described methods and systems of
the invention
will be apparent to those skilled in the art without departing from the scope
and spirit of the invention.
Although the invention has been described in connection with certain
embodiments, it should be
understood that the invention as claimed should not be unduly limited to such
specific embodiments.
Indeed, various modifications of the described modes for carrying out the
invention which are
obvious to those skilled in molecular biology or related fields are intended
to be within the scope of
the following claims.
56
CA 02349818 2001-05-03
WO 00/26245 PCTNS99/26048
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SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS, INC.
HILLMAN, Jennifer L.
YUE, Henry
TANG, Y. Tom
LAL, Preeti
CORLEY, Neil C.
GUEGLER, Karl J.
BAUGHN, Mariah R.
AZIMZAI, Yalda
LU, Dyung Aina M.
<120> MEMBRANE TRANSPORT PROTEINS
<130> PF-0633 PCT
<140> To Be Assigned
<141> Herewith
<150> 09/186,778; unassigned; 09/200,277; unassigned; 09/221,405;
unassigned; 60/121,896
<151> 1998-11-04; 1998-11-04; 1998-11-24; 1998-11-24; 1998-12-22;
1998-12-22; 1999-02-26
<160> 42
<170> PERL Program
<210> 1
<211> 384
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 961344CD1
<400> 1
Met Leu Thr Gly Asp Lys Leu Glu Thr Ala Thr Cys Ile Ala Lys
1 5 10 15
Ser Ser His Leu Val Ser Arg Thr Gln Asp Ile His Ile Phe Arg
20 25 30
Gln Val Thr Ser Arg Gly Glu Ala His Leu Glu Leu Asn Ala Phe
35 40 45
Arg Arg Lys His Asp Cys Ala Leu Val Ile Ser Gly Asp Ser Leu
50 55 60
Glu Val Cys Leu Lys Tyr Tyr Glu His Glu Phe Val Glu Leu Ala
65 70 75
Cys Gln Cys Pro Ala Val Val Cys Cys Arg Cys Ser Pro Thr Gln
80 85 90
Lys Ala Arg Ile Val Thr Leu Leu Gln Gln His Thr Gly Arg Arg
95 100 105
1
CA 02349818 2001-05-03
WO 00/26245 PCT/US99/26048
Thr Cys Ala Ile Gly Asp Gly Gly Asn Asp Val Ser Met Ile Gln
110 115 120
Ala Ala Asp Cys Gly Ile Gly Ile Glu Gly Lys Glu Gly Lys Gln
125 130 135
Ala Ser Leu Ala Ala Asp Phe Ser Ile Thr Gln Phe Arg His Ile
140 145 150
Gly Arg Leu Leu Met Val His Gly Arg Asn Ser Tyr Lys Arg Ser
155 160 165
Ala Ala Leu Gly Gln Phe Val Met His Arg Gly Leu IIe Ile Ser
170 175 180
Thr Met Gln Ala Val Phe Ser Ser Val Phe Tyr Phe Ala Ser Val
185 190 195
Pro Leu Tyr Gln Gly Phe Leu Met Val Gly Tyr Ala Thr Ile Tyr
200 205 210
Thr Met Phe Pro Val Phe Ser Leu Val Leu Asp Gln Asp Val Lys
215 220 225
Pro Glu Met Ala Met Leu Tyr Pro Glu Leu Tyr Lys Asp Leu Thr
230 235 240
Lys Gly Arg Ser Leu Ser Phe Lys Thr Phe Leu Ile Trp Val Leu
245 250 255
Ile Ser Ile Tyr Gln Gly Gly Ile Leu Met Tyr Gly Ala Leu Val
260 265 270
Leu Phe Glu Ser Glu Phe Val His Val Val Ala Ile Ser Phe Thr
275 280 285
Ala Leu Ile Leu Thr Glu Leu Leu Met Val Ala Leu Thr Val Arg
290 295 300
Thr Trp His Trp Leu Met Val Val Ala Glu Phe Leu Ser Leu Gly
305 310 315
Cys Tyr Val Ser Ser Leu Ala Phe Leu Asn Glu Tyr Phe Gly Ile
320 325 330
Gly Arg Val Ser Phe Gly Ala Phe Leu Asp Val Ala Phe Ile Thr
335 340 345
Thr Val Thr Phe Leu Trp Lys Val Ser Ala Ile Thr Val Val Ser
350 355 360
Cys Leu Pro Leu Tyr Val Leu Lys Tyr Leu Arg Arg Lys Leu Ser
365 370 375
Pro Pro Ser Tyr Cys Lys Leu Ala Ser
380
<210> 2
<211> 846
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3128782CD1
<400> 2
Met Pro Lys Ala Pro Lys Gln Gln Pro Pro Glu Pro Glu Trp Ile
1 5 10 15
Gly Asp Gly Glu Ser Thr Ser Pro Ser Asp Lys Val Val Lys Lys
20 25 30
Gly Lys Lys Asp Lys Lys Ile Lys Lys Thr Phe Phe Glu Glu Leu
2
CA 02349818 2001-05-03
WO 00/26245 PCT/US99/26048
-35 40 . 45
Ala Val Glu Asp Lys Gln Ala Gly Glu Glu Glu Lys Val Leu Ly$"
50 55 60
Glu Lys Glu Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Lys
65 70 75
Lys Lys Arg Asp Thr Arg Lys Gly Arg Arg Lys Lys Asp Val Asp
80 85 90
Asp Asp Gly Glu Glu Lys Glu Leu Met Glu Arg Leu Lys Lys Leu
95 100 105
Ser Val Pro Thr Ser Asp G,lu Glu Asp Glu Val Pro Ala Pro Lys
110 115 120
Pro Arg Gly Gly Lys Lys Thr Lys Gly Gly Asn Val Phe Ala Ala
125 130 I35
Leu Ile Gln Asp Gln Ser Glu Glu Glu Glu Glu Glu Glu Lys His
140 145 150
Pro Pro Lys Pro Ala Lys Pro Glu Lys Asn Arg Ile Asn Lys Ala
155 160 165
Val Ser Glu Glu Gln Gln Pro Ala Leu Lys Gly Lys Lys Gly Lys
170 175 180
Glu Glu Lys Ser Lys Gly Lys Ala Lys Pro Gln Asn Lys Phe Ala
185 190 195
Ala Leu Asp Asn Glu Glu Glu Asp Lys Glu Glu Glu Ile Ile Lys
200 205 210
Glu Lys Glu Pro Pro Lys Gln Gly Lys Glu Lys Ala Lys Lys Ala
215 220 225
Glu Gln Gly Ser Glu Glu Glu Gly Glu Gly Glu Glu Glu Glu Glu
230 235 240
Glu Gly Gly Glu Ser Lys Ala Asp Asp Pro Tyr Ala His Leu Ser
245 250 255
Lys Lys Glu Lys Lys Lys Leu Lys Lys Gln Met Glu Tyr Glu Arg
260 265 270
Gln Val Ala Ser Leu Lys Ala Ala Asn Ala Ala Glu Asn Asp Phe
275 280 285
Ser Val Ser Gln Ala Glu Met Ser Ser Arg Gln Ala Met Leu Glu
290 295 300
Asn Ala Sex Asp Ile Lys Leu Glu Lys Phe Ser Ile Ser Ala His
305 310 315
Gly Lys Glu Leu Phe Val Asn Ala Asp Leu Tyr Ile Val Ala Gly
320 325 330
Arg Arg Tyr Gly Leu Val Gly Pro Asn Gly Lys Gly Lys Thr Thr
335 340 345
Leu Leu Lys His Ile Ala Asn Arg Ala Leu Ser Ile Pro Pro Asn
350 355 360
Ile Asp Val Leu Leu Cys Glu Gln Glu Val Val Ala Asp Glu Thr
365 370 375
Pro Ala Val Gln Ala Val Leu Arg Ala Asp Thr Lys Arg Leu Lys
380 385 390
Leu Leu Glu Glu Glu Arg Arg Leu Gln Gly Gln Leu Glu Gln Gly
395 400 405
Asp Asp Thr Ala Ala Glu Arg Leu Glu Lys Val Tyr Glu Glu Leu
410 415 420
Arg Ala Thr Gly Ala Ala Ala Ala Glu Ala Lys Ala Arg Arg Ile
425 430 435
Leu Ala Gly Leu Gly Phe Asp Pro Glu Met Gln Asn Arg Pro Thr
440 445 450
3
CA 02349818 2001-05-03
WO 00/26245 PCTNS99/26048
Gln Lys Phe Ser Gly Gly Trp Arg Met Arg Val Ser Leu Ala Arg
455 460 46y
Ala Leu Phe Met Glu Pro Thr Leu Leu Met Leu Asp Glu Pro Thr
470 475 480
Asn His Leu Asp Leu Asn Ala Val Ile Trp Leu Asn Asn Tyr Leu
485 490 495
Gln Gly Trp Arg Lys Thr Leu Leu Ile Val Ser His Asp Gln Gly
500 505 510
Phe Leu Asp Asp Val Cys Thr Asp Ile Ile His Leu Asp Ala Gln
515 520 525
Arg Leu His Tyr Tyr Arg Gly Asn Tyr Met Thr Phe Lys Lys Met
530 535 540
Tyr Gln Gln Lys Gln Lys Glu Leu Leu Lys Gln Tyr Glu Lys Gln
545 550 555
Glu Lys Lys Leu Lys Glu Leu Lys Ala Gly Gly Lys Ser Thr Lys
560 565 570
Gln Ala Glu Lys Gln Thr Lys Glu Ala Leu Thr Arg Lys Gln Gln
5?5 580 585
Lys Cys Arg Arg Lys Asn Gln Asp Glu Glu Ser Gln Glu Ala Pro
590 595 600
Glu Leu Leu Lys Arg Pro Lys Glu Tyr Thr Val Arg Phe Thr Phe
605 610 615
Pro Asp Pro Pro Pro Leu Ser Pro Pro Val Leu Gly Leu His Gly
620 625 630
Val Thr Phe Gly Tyr Gln Gly Gln Lys Pro Leu Phe Lys Asn Leu
635 640 645
Asp Phe Gly Ile Asp Met Asp Ser Arg Ile Cys Ile Val Gly Pro
650 655 660
Asn Gly Val Gly Lys Ser Thr Leu Leu Leu Leu Leu Thr Gly Lys
665 670 675
Leu Thr Pro Thr His Gly Glu Met Arg Lys Asn His Arg Leu Lys
680 685 690
Ile Gly Phe Phe Asn Gln Gln Tyr Ala Glu Gln Leu Arg Met Glu
695 700 705
Glu Thr Pro Thr Glu Tyr Leu Gln Arg Gly Phe Asn Leu Pro Tyr
710 715 720
Gln Asp Ala Arg Lys Cys Leu Gly Arg Phe Gly Leu Glu Ser His
725 730 735
Ala His Thr Ile Gln Ile Cys Lys Leu Ser Gly Gly Gln Lys Ala
740 745 750
Arg Val Val Phe Ala Glu Leu Ala Cys Arg Glu Pro Asp Val Leu
755 760 765
Ile Leu Asp Glu Pro Thr Asn Asn Leu Asp Ile Glu Ser Ile Asp
770 775 780
Ala Leu Gly Glu Ala Ile Asn Glu Tyr Lys Gly Ala Val Ile Val
785 790 795
Val Ser His Asp Ala Arg Leu Ile Thr Glu Thr Asn Cys Gln Leu
800 805 810
Trp Val Val Glu Glu Gln Ser Val Ser Gln Ile Asp Gly Asp Phe
815 820 825
Glu Asp Tyr Lys Arg Glu Val Leu Glu Ala Leu Gly Glu Val Met
830 835 840
Val Ser Arg Pro Arg Glu
845
CA 02349818 2001-05-03
WO 00/Z6245 PCT/US99/26048
<210> 3 '
<211> 511
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1720440CD1
<400> 3
Met Glu Asn Arg Asn Glu Phe Val Gly Leu Trp Leu Gly Met Ala
1 5 10 15
Lys Leu Gly Val Glu Ala Ala Leu Ile Asn Thr Asn Leu Arg Arg
20 25 30
Asp Ala Leu Leu His Cys Leu Thr Thr Ser Arg Ala Arg Ala Leu
35 40 45
Val Phe Gly Ser Glu Met Ala Ser Ala Ile Cys Glu Va1 His Ala
50 55 60
Ser Leu Asp Pro Ser Leu Ser Leu Phe Cys Ser Gly Ser Trp Glu
65 70 75
Pro Gly Ala Val Pro Pro Ser Thr Glu His Leu Asp Pro Leu Leu
80 85 90
Lys Asp Ala Pro Lys His Leu Pro Ser Cys Pro Asp Lys Gly Phe
95 100 105
Thr Asp Lys Leu Phe Tyr Ile Tyr Thr Ser Gly Thr Thr Gly Leu
110 115 120
Pro Lys Ala Ala Ile Val Val His Ser Arg Tyr Tyr Arg Met Ala
125 130 135
Ala Leu Val Tyr Tyr Gly Phe Arg Met Arg Pro Asn Asp Ile Val
140 145 150
Tyr Asp Cys Leu Pro Leu Tyr His Ser Ala Gly Asn Ile Val Gly
155 160 165
Ile Gly Gln Cys Leu Leu His Gly Met Thr Val Val Ile Arg Lys
170 175 180
Lys Phe Ser Ala Ser Arg Phe Trp Asp Asp Cys Ile Lys Tyr Asn
185 190 195
Cys Thr Ile Val Gln Tyr Ile Gly Glu Leu Cys Arg Tyr Leu Leu
200 205 210
Asn Gln Pro Pro Arg Glu Ala Glu Asn Gln His Gln Val Arg Met
215 220 225
Ala Leu Gly Asn Gly Leu Arg Gln Ser Ile Trp Thr Asn Phe Ser
230 235 240
Ser Arg Phe His Ile Pro Gln Val Ala Glu Phe Tyr Gly Ala Thr
245 250 255
Glu Cys Asn Cys Ser Leu Gly Asn Phe Asp Ser Gln Val Gly Ala
260 265 270
Cys Gly Phe Asn Ser Arg Ile Leu Ser Ser Val Tyr Pro Ile Arg
275 280 285
Leu Val Arg Val Asn Glu Asp Thr Met Glu Leu Ile Arg Gly Pro
290 295 300
Asp Gly Val Cys Ile Pro Cys Gln Pro Gly Glu Pro Gly Gln Leu
305 310 315
Val Gly Arg IIe Ile Gln Lys Asp Pro Leu Arg Arg Phe Asp Gly
320 325 330
Tyr Leu Asn Gln Gly Ala Asn Asn Lys Lys Ile Ala Lys Asp Val
CA 02349818 2001-05-03
WO 00/26245 PCTNS99/26048
335 340 . 345
Phe Lys Lys Gly Asp Gln Ala Tyr Leu Thr Gly Asp Val Leu Val_
350 355 360
Met Asp Glu Leu Gly Tyr Leu Tyr Phe Arg Asp Arg Thr Gly Asp
365 3?0 375
Thr Phe Arg Trp Lys Gly Glu Asn Val Sex Thr Thr Glu Val Glu
380 385 390
Gly Thr Leu Ser Arg Leu Leu Asp Met Ala Asp Val Ala Val Tyr
395 400 405
Gly Val Glu Val Pro Gly Thr Glu Gly Arg Ala Gly Met Ala Ala
410 415 420
Val Ala Ser Pro Thr Gly Asn Cys Asp Leu Glu Arg Phe Ala Gln
425 430 435
Val Leu Glu Lys Glu Leu Pro Leu Tyr Ala Arg Pro Ile Phe Leu
440 445 450
Arg Leu Leu Pro Glu Leu His Lys Thr Gly Thr Tyr Lys Phe Gln
455 460 465
Lys Thr Glu Leu Arg Lys Glu Gly Phe Asp Pro Ala Ile Val Lys
470 475 480
Asp Pro Leu Phe Tyr Leu Asp Ala Gln Lys Gly Arg Tyr Val Pro
485 490 495
Leu Asp Gln Glu Ala Tyr Sex Arg Ile Gln Ala Gly Glu Glu,Lys
500 505 510
Leu
<210> 4
<211> 718
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2274290CD1
<400> 4
Met Leu Val His Leu Phe Arg Val Gly Ile Arg Gly Gly Pro Phe
5 10 15
1
Pro Gly Arg Leu Leu Pro Pro Leu Arg Phe Gln Thr Phe Ser Ala
20 25 30
Val Arg Tyr Ser Asp Gly Tyr Arg Ser Ser Ser Leu Leu Arg Ala
35 40 45
Val Ala His Leu Arg Ser Gln Leu Trp Ala His Leu Pro Arg Ala
50 55 60
Pro Leu Ala Pro Arg Trp Ser Pro Ser Ala Trp Cys Trp Val Gly
65 70 75
Gly Ala Leu Leu Gly Pro Met Val Leu Ser Lys His Pro His Leu
80 85 90
Cys Leu Val Ala Leu Cys Glu Ala Glu Glu Ala Pro Pro Ala Ser
g5 100 105
Ser Thr Pro His Val Val Gly Ser Arg Phe Asn Trp Lys Leu Phe
110 115 120
Trp Gln Phe Leu His Pro His Leu Leu Val Leu Gly Val Ala Val
125 130 135
Val Leu Ala Leu Gly Ala Ala Leu Val Asn Val Gln Ile Pro Leu
6
CA 02349818 2001-05-03
PCTlUS99/26048
WO 00/26245
140 145 . 150
Leu Leu Gly Gln Leu Val Glu Val Val Ala Lys Tyr Thr Arg Asg
155 160 165
His Val Gly Ser Phe Met Thr Glu Ser Gln Asn Leu Ser Thr His
170 175 180
Leu Leu Ile Leu Tyr Gly Val Gln Gly Leu Leu Thr Phe Gly Tyr
185 190 195
Leu Val Leu Leu Ser His Val Gly Glu Arg Met Ala Val Asp Met
200 205 210
Arg Arg Ala Leu Phe Ser Ser Leu Leu Arg Gln Asp Ile Thr Phe
215 220 225
Phe Asp Ala Asn Lys Thr Gly Gln Leu Val Ser Arg Leu Thr Thr
230 235 240
Asp Val Gln Glu Phe Lys Ser Ser Phe Lys Leu Val Ile Ser Gln
245 250 255
Gly Leu Arg Sex Cys Thr Gln Val Ala Gly Cys Leu Val Ser Leu
260 265 270
Ser Met Leu Ser Thr Arg Leu Thr Leu Leu Leu Met Val Ala Thr
275 280 285
Pro Ala Leu Met Gly Val Gly Thr Leu Met Gly Ser Gly Leu Arg
290 295 300
Lys Leu Ser Arg Gln Cys Gln Glu Gln Ile Ala Arg Ala Met Gly
305 310 315
Val Ala Asp Glu Ala Leu Gly Asn Val Arg Thr Val Arg Ala Phe
320 325 330
Ala Met Glu Gln Arg Glu Glu Glu Arg Tyr Gly Ala Glu Leu Glu
335 340 345
Ala Cys Arg Cys Arg Ala Glu Glu Leu Gly Arg Gly Ile Ala Leu
350 355 360
Phe Gln Gly Leu Ser Asn Ile Ala Phe Asn Cys Met Val Leu Gly
365 370 375
Thr Leu Phe Ile Gly Gly Ser Leu Val Ala Gly Gln Gln Leu Thr
380 385 390
Gly Gly Asp Leu Met Ser Phe Leu Val Ala Ser Gln Thr Val Gln
395 400 405
Arg Ser Met Ala Asn Leu Ser Val Leu Phe Gly Gln Val Val Arg
410 415 420
Gly Leu Ser Ala Gly Ala Arg Val Phe Glu Tyr Met Ala Leu Asn
425 430 435
Pro Cys Ile Pro Leu Ser Gly Gly Cys Cys Val Pro Lys Glu Gln
440 445 450
Leu Arg Gly Ser Val Thr Phe Gln Asn Val Cys Phe Ser Tyr Pro
455 460 465
Cys Arg Pro Gly Phe Glu Val Leu Lys Asp Phe Thr Leu Thr Leu
470 475 480
Pro Pro Gly Lys Ile Val Ala Leu Val Gly Gln Ser Gly Gly Gly
485 490 495
Lys Thr Thr Val Ala Ser Leu Leu Glu Arg Phe Tyr Asp Pro Thr
500 505 510
Ala Gly Val Val Met Leu Asp Gly Arg Asp Leu Arg Thr Leu Asp
515 520 525
Pro Ser Trp Leu Arg Gly Gln Val Val G1y Phe Ile Ser Gln Glu
530 535 540
Pro VaI Leu Phe Gly Thr Thr ile Met Glu Asn Ile Arg Phe Gly
545 550 555
7
CA 02349818 2001-05-03
WO OOI26245 PCT/US99/26048
Lys Leu Glu Ala Ser Asp Glu Glu Val Tyr Thr Ala Ala Arg Glu
560 565 570-
Ala Asn Ala His Glu Phe Ile Thr Ser Phe Pro Glu Gly Tyr Asn
575 580 585
Thr Val Val Gly Glu Arg Gly Thr Thr Leu Ser Gly Gly Gln Lys
590 595 600
Gln Arg Leu Ala Ile Ala Arg Ala Leu Ile Lys Gln Pro Thr Val
605 610 615
Leu Ile Leu Asp Glu Ala Thr Ser Ala Leu Asp Ala Glu Ser Glu
620 625 630
Arg Val Val Gln Glu Ala Leu Asp Arg Ala Ser Ala Gly Arg Thr
635 640 645
Val Leu Val Ile Ala His Arg Leu Ser Thr Val Arg Gly Ala His
650 655 660
Cys Ile Val Val Met Ala Asp Gly Arg Val Trp Glu Ala Gly Thr
665 670 675
His Glu Glu Leu Leu Lys Lys Gly Gly Leu Tyr Ala Glu Leu Ile
680 685 690
Arg Arg Gln Ala Leu Asp Ala Pro Arg Thr Ala Ala Pro Pro Pro
695 700 705
Lys Lys Pro Glu Gly Pro Arg Ser His Gln His Lys Ser
710 715
<210> 5
<211> 635
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2740029CD1
<400> 5
Met Ser Val Gly Val Ser Thr Ser Ala Pro Leu Ser Pro Thr Ser
5 10 15
1
Gly Thr Ser Val Gly Met Ser Thr Phe Ser Ile Met Asp Tyr Val
20 25 30
Val Phe Val Leu Leu Leu Val Leu Ser Leu Ala Ile Gly Leu Tyr
35 40 45
His Ala Cys Arg Gly Trp Gly Arg His Thr Val Gly Glu Leu Leu
50 55 60
Met Ala Asp Arg Lys Met Gly Cys Leu Pro Val Ala Leu Ser Leu
65 70 75
Leu Ala Thr Phe Gln Ser Ala Val Ala Ile Leu Gly Val Pro Ser
80 85 90
Glu Ile Tyr Arg Phe Gly Thr Gln Tyr Trp Phe Leu Gly Cys Cys
g5 100 105
Tyr Phe Leu Gly Leu Leu Ile Pro Ala His Ile Phe Ile Pro Val
110 115 120
Phe Tyr Arg Leu His Leu Thr Ser Ala Tyr Glu Tyr Leu Glu Leu
125 130 135
Arg Phe Asn Lys Thr Val Arg Val Cys Gly Thr Val Thr Phe Ile
140 145 150
Phe Gln Met Val Ile Tyr Met Gly Val Val Leu Tyr Ala Pro Ser
g
CA 02349818 2001-05-03
WO 00/26245 PCT/US99/26048
155 160 165
Leu Ala Leu Asn Ala Val Thr Gly Phe Asp Leu Trp Leu Ser Val,.
170 175 180
Leu Ala Leu Gly Ile Val Cys Thr Val Tyr Thr Ala Leu G1y Gly
185 190 195
Leu Lys Ala Val Ile Trp Thr Asp Val Phe Gln Thr Leu Val Met
200 205 210
Phe Leu Gly Gln Leu Ala Val Ile Ile Val Gly Ser Ala Lys Val
215 220 225
Gly Gly Leu Gly Arg Val Trp Ala Val Ala Ser Gln His Gly Arg
230 235 240
Ile Ser Gly Phe Glu Leu Asp Pro Asp Pro Phe Val Arg His Thr
245 250 255
Phe Trp Thr Leu Ala Phe Gly Gly Val Phe Met Met Leu Ser Leu
260 265 270
Tyr Gly Val Asn Gln Ala Gln Val Gln Arg Tyr Leu Ser Ser Arg
275 280 285
Thr Glu Lys Ala Ala Val Leu Ser Cys Tyr Ala Val Phe Pro Phe
290 295 300
Gln Gln Val Ser Leu Cys Val Gly Cys Leu Ile Gly Leu Val Met
305 310 315
Phe Ala Tyr Tyr Gln Glu Tyr Pro Met Ser Ile Gln Gln Ala Gln
320 325 330
Ala Ala Pro Asp Gln Phe Val Leu Tyr Phe Val Met Asp Leu Leu
335 340 345
Lys Gly Leu Pro Gly Leu Pro Gly Leu Phe Ile Ala Cys Leu Phe
350 355 360
Ser Gly Ser Leu Ser Thr Ile Ser Ser Ala Phe Asn Ser Leu Ala
365 370 375
Thr Val Thr Met Glu Asp Leu Ile Arg Pro Trp Phe Pro Glu Phe
380 385 390
Ser Glu Ala Arg Ala Ile Met Leu Ser Arg Gly Leu Ala Phe Gly
395 400 405
Tyr Gly Leu Leu Cys Leu Gly Met Ala Tyr Ile Ser Ser Gln Met
410 415 420
Gly Pro Val Leu Gln Ala Ala Ile Ser Ile Phe Gly Met Val Gly
425 430 435
Gly Pro Leu Leu Gly Leu Phe Cys Leu Gly Met Phe Phe Pro Cys
440 445 450
Ala Asn Pro Pro Gly Ala Val Val Gly Leu Leu Ala Gly Leu Val
455 460 465
Met Ala Phe Trp Ile Gly Ile Gly Ser Ile Val Thr Ser Met Gly
470 475 480
Ser Ser Met Pro Pro Ser Pro Ser Asn Gly Ser Ser Phe Ser Leu
485 490 495
Pro Thr Asn Leu Thr Val Ala Thr Val Thr Thr Leu Met Pro Leu
500 505 510
Thr Thr Phe Ser Lys Pro Thr Gly Leu Gln Arg Phe Tyr Ser Leu
515 520 525
Ser Tyr Leu Trp Tyr Ser Ala His Asn Ser Thr Thr Val Ile Val
530 535 540
Val Gly Leu Ile Val Ser Leu Leu Thr Gly Arg Met Arg Gly Arg
545 550 555
Ser Leu Asn Pro Ala Thr Ile Tyr Pro Val Leu Pro Lys Leu Leu
560 565 570
9
CA 02349818 2001-05-03
WO 00/26245 PCT/US99/26048
Ser Leu Leu Pro Leu Ser Cys Gln Lys Arg Leu His Cys Arg Ser
575 580 58~.
Tyr Gly Gin Asp His Leu Asp Thr Gly Leu Phe Pro Glu Lys Pro
590 595 600
Arg Asn Gly Val Leu Gly Asp Ser Arg Asp Lys Glu Ala Met Ala
605 610 615
Leu Asp Gly Thr Ala Tyr Gln Gly Ser Ser Ser Thr Cys Ile Leu
620 625 630
Gln Glu Thr Ser Leu
635
<210> 6
<211> 535
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2414415CD1
<400> 6
Met Glu Glu Gly Ala Arg His Arg Asn Asn Thr Glu Lys Lys His
1 5 10 15
Pro Gly Gly Gly Glu Ser Asp Ala Ser Pro Glu Ala Gly Ser Gly
20 25 30
Gly Gly Gly Val Ala Leu Lys Lys Glu Ile Gly Leu Val Ser Ala
35 40 45
Cys Gly Ile Ile Val Gly Asn Ile Ile Gly Ser Gly Ile Phe Val
50 55 60
Ser Pro Lys Gly Val Leu Glu Asn Ala Gly Ser Val Gly Leu Ala
65 70 75
Leu Ile Val Trp Ile Val Thr Gly Phe Ile Thr Val Val Gly Ala
80 85 90
Leu Cys Tyr Ala Glu Leu Gly Val Thr Ile Pro Lys Ser Gly Gly
95 100 105
Asp Tyr Ser Tyr Val Lys Asp Ile Phe Gly Gly Leu Ala Gly Phe
110 115 120
Leu Arg Leu Trp Ile Ala Val Leu Val Ile Tyr Pro Thr Asn Gln
125 130 135
Ala Val Ile Ala Leu Thr Phe Ser Asn Tyr Val Leu Gln Pro Leu
140 145 150
Phe Pro Thr Cys Phe Pro Pro Glu Ser Gly Leu Arg Leu Leu Ala
155 160 165
Ala Ile Cys Leu Leu Leu Leu Thr Trp Val Asn Cys Ser Ser Val
170 175 180
Arg Trp Ala Thr Arg Val Gln Asp Ile Phe Thr Ala Gly Lys Leu
185 190 195
Leu Ala Leu Ala Leu Ile Ile Ile Met Gly Ile Val Gln Ile Cys
200 205 210
Lys Gly Glu Tyr Phe Trp Leu Glu Pro Lys Asn Ala Phe Glu Asn
215 220 225
Phe Gln Glu Pro Asp Ile Gly Leu Val Ala Leu Ala Phe Leu Gln
230 235 240
Gly Ser Phe Ala Tyr Gly Gly Trp Asn Phe Leu Asn Tyr Val Thr
CA 02349818 2001-05-03
WO 00/26245 PCT/US99/26048
245 250 , 255
Glu Glu Leu Val Asp Pro Tyr Lys Asn Leu Pro Arg Ala Ile Phe
260 265 270
Ile Ser Ile Pro Leu Val Thr Phe Val Tyr Val Phe Ala Asn Val
275 280 285
Ala Tyr Val Thr Ala Met Ser Pro Gln Glu Leu Leu Ala Ser Asn
290 295 300
Ala Val Ala Val Thr Phe Gly Glu Lys Leu Leu Gly Val Met Ala
305 310 315
Trp Ile Met Pro Ile Ser Val Ala Leu Ser Thr Phe Gly Gly Val
320 325 330
Asn Gly Ser Leu Phe Thr Ser Ser Arg Leu Phe Phe Ala Gly Ala
335 340 345
Arg Glu Gly His Leu Pro Ser Val Leu Ala Met Ile His Val Lys
350 355 360
Arg Cys Thr Pro Ile Pro Ala Leu Leu Phe Thr Cys Ile Ser Thr
365 370 375
Leu Leu Met Leu Val Thr Ser Asp Met Tyr Thr Leu Ile Asn Tyr
380 385 390
Val Gly Phe Ile Asn Tyr Leu Phe Tyr Gly Val Thr Val Ala Gly
395 400 405
Gln Ile Val Leu Arg Trp Lys Lys Pro Asp Ile Pro Arg Pro Ile
410 415 420
Lys Ile Asn Leu Leu Phe Pro Ile Ile Tyr Leu Leu Phe Trp Ala
425 430 435
Phe Leu Leu Val Phe Ser Leu Trp Ser Glu Pro Val Val Cys Gly
440 445 450
Ile Gly Leu Ala Ile Met Leu Thr Gly Val Pro Val Tyr Phe Leu
455 460 465
Gly Val Tyr Trp Gln His Lys Pro Lys Cys Phe Ser Asp Phe Ile
470 475 480
Glu Leu Leu Thr Leu Val Ser Gln Lys Met Cys Val Val Val Tyr
485 490 495
Pro Glu Val Glu Arg Gly Ser Gly Thr Glu Glu Ala Asn Glu Asp
500 505 510
Met Glu Glu Gln Gln Gln Pro Met Tyr Gln Pro Thr Pro Thr Lys
515 520 525
Asp Lys Asp Val Ala Gly Gln Pro Gln Pro
530 535
<210> 7
<211> 456
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2466714CD1
<400> 7
Met Glu Ala Ser Trp Gly Ser Phe Asn Ala Glu Arg Gly Trp Tyr
1 5 10 15
Val Ser Val Gln Gln Pro Glu Glu Ala Glu Ala Glu Glu Leu Ser
20 25 30
11
CA 02349818 2001-05-03
WO 00/26245 PCTNS99/26048
Pro Leu Leu Ser Asn Glu Leu His Arg Gln Arg Ser Pro Gly Val
35 40 45
Ser Phe Gly Leu Ser Val Phe Asn Leu Met Asn Ala Ile Met Gly
50 55 60
Ser Gly Ile Leu Gly Leu Ala Tyr Val Met Ala Asn Thr Gly Val
65 70 75
Phe Gly Phe Ser Phe Leu Leu Leu Thr Val Ala Leu Leu Ala Ser
80 85 90
Tyr Ser Val His Leu Leu Leu Ser Met Cys Ile Gln Thr Ala Val
95 100 105
Thr Ser Tyr Glu Asp Leu Gly Leu Phe Ala Phe Gly Leu Pro Gly
110 115 120
Lys Leu Val Val Ala Gly Thr Ile Ile Ile Gln Asn Ile Gly Ala
125 130 135
Met Ser Ser Tyr Leu Leu Ile Ile Lys Thr Glu Leu Pro Ala Ala
140 145 150
Ile Ala Glu Phe Leu Thr Gly Asp Tyr Asn Arg Tyr Trp Tyr Leu
155 160 165
Asp Gly Gln Thr Leu Leu Ile Ile Ile Cys Val Gly Ile Val Phe
170 175 180
Pro Leu Ala Leu Leu Pro Lys Ile Gly Phe Leu Gly Tyr Thr Ser
185 190 195
Ser Leu Ser Phe Phe Phe Met Met Phe Phe Ala Leu Val Val Ile
200 205 210
Ile Lys Lys Trp Ser Ile Pro Cys Pro Leu Thr Leu Asn Tyr Val
215 220 225
Glu Lys Gly Phe Gln Ile Ser Asn Val Thr Asp Asp Cys Lys Pro
230 235 240
Lys Leu Phe His Phe Ser Lys Glu Ser Ala Tyr Ala Leu Pro Thr
245 250 255
Met Ala Phe Sex Phe Leu Cys His Thr Ser Ile Leu Pro Ile Tyr
260 265 270
Cys Glu Leu Gln Ser Pro Ser Lys Lys Arg Met Gln Asn Val Thr
275 280 285
Asn Thr Ala Ile Ala Leu Ser Phe Leu Ile Tyr Phe Ile Ser Ala
290 295 300
Leu Phe Gly Tyr Leu Thr Phe Tyr Asp Lys Val Glu Ser Glu Leu
305 310 315
Leu Lys Gly Tyr Ser Lys Tyr Leu Ser His Asp Val Val Val Met
320 325 330
Thr Val Lys Leu Cys Ile Leu Phe Ala Val Leu Leu Thr Val Pro
335 340 345
Leu Ile His Phe Pro Ala Arg Lys Ala Val Thr Met Met Phe Phe
350 355 360
Ser Asn Phe Pro Phe Ser Trp Ile Arg His Phe Leu Ile Thr Leu
365 370 375
Ala Leu Asn Ile Ile Ile Val Leu Leu Ala Ile Tyr Val Pro Asp
380 385 390
Ile Arg Asn Val Phe Gly Val Val Gly Ala Ser Thr Ser Thr Cys
395 400 405
Leu Ile Phe Ile Phe Pro Gly Leu Phe Tyr Leu Lys Leu Ser Arg
410 415 420
Glu Asp Phe Leu Ser Trp Lys Lys Leu Gly Ala Phe Val Leu Leu
425 430 435
Ile Phe Gly Ile Leu Val Gly Asn Phe Ser Leu Ala Leu Ile Ile
12.
CA 02349818 2001-05-03
WO 00/26245 PCT/US99/26048
440 445 450
Phe Asp Trp Ile Asn Lys
455
<210> 8
<211> 325
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2617942CD1
<400> 8
Met Phe Ala Asn Leu Lys Tyr Val Ser Leu Gly Ile Leu Val Phe
1 5 10 15
Gln Thr Thr Ser Leu Val Leu Thr Met Arg Tyr Ser Arg Thr Leu
20 25 30
Lys Glu Glu Gly Pro Arg Tyr Leu Ser Ser Thr Ala Val Val Val
35 40 45
Ala Glu Leu Leu Lys Ile Met Ala Cys Ile Leu Leu Val Tyr Lys
50 55 60
Asp Ser Lys Cys Ser Leu Arg Ala Leu Asn Arg Val Leu His Asp
65 70 75
Glu Ile Leu Asn Lys Pro Met Glu Thr Leu Lys Leu Ala Ile Pro
80 85 90
Ser Gly Ile Tyr Thr Leu Gln Asn Asn Leu Leu Tyr Val Ala Leu
95 100 105
Ser Asn Leu Asp Ala Ala Thr Tyr Gln Val Thr Tyr Gln Leu Lys
110 115 120
Ile Leu Thr Thr Ala Leu Phe Ser Val Ser Met Leu Ser Lys Lys
125 130 135
Leu Gly Val Tyr Gln Trp Leu Ser Leu VaI Ile Leu Met Thr Gly
140 145 150
Val Ala Phe Val Gln Trp Pro Ser Asp Ser Gln Leu Asp Ser Lys
155 160 165
Glu Leu Ser Ala Gly Ser Gln Phe Val Gly Leu Met Ala Val Leu
170 175 180
Thr Ala Cys Phe Ser Ser Gly Phe Ala Gly Val Tyr Phe Glu Lys
185 190 195
Ile Leu Lys Glu Thr Lys Gln Ser Val Trp Ile Arg Asn Ile Gln
200 205 210
Leu Gly Phe Phe Gly Ser Ile Phe Gly Leu Met Gly Val Tyr Ile
215 220 225
Tyr Asp Gly Glu Leu Val Ser Lys Asn Gly Phe Phe Gln Gly Tyr
230 235 240
Asn Arg Leu Thr Trp Ile Val Val Val Leu Gln Ala Leu Gly Gly
245 250 255
Leu Val Ile Ala Ala Val Ile Lys Tyr Ala Asp Asn Ile Leu Lys
260 265 270
Gly Phe Ala Thr Ser Leu Ser Ile Ile Leu Ser Thr Leu Ile Ser
275 280 285
Tyr Phe Trp Leu Gln Asp Phe Val Pro Thr Ser Val Phe Phe Leu
290 295 300
13
CA 02349818 2001-05-03
WO 00/26245 PCT/US99/26048
Gly Ala Ile Leu Val Ile Thr Ala Thr Phe Leu Tyr Gly Tyr Asp
305 310 315
Pro Lys Pro Ala Gly Asn Pro Thr Lys Ala
320 325
<210> 9
<211> 178
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2945431CD1
<400> 9
Met Ser Leu Ser Pro Arg Ser Gln Leu Ala Ile Ile Pro Gln Glu
1 5 10 15
Pro Phe Leu Phe Ser Gly Thr Val Arg Glu Asn Leu Asp Pro Gln
20 25 30
Gly Leu His Lys Asp Arg Ala Leu Trp Gln Ala Leu Lys Gln Cys
35 40 45
His Leu Ser Glu Val Ile Thr Ser Met Gly Gly Leu Asp Gly Glu
50 55 60
Leu Gly Glu Gly Gly Arg Ser Leu Ser Leu Gly Gln Arg Gln Leu
65 70 75
Leu Cys Leu Ala Arg Ala Leu Leu Thr Asp Ala Lys Ile Leu Cys
80 85 90
Ile Asp Glu Ala Thr Ala Ser Val Asp GIn Lys Thr Asp Gln Leu
95 100 105
Leu Gln Gln Thr Ile Cys Lys Arg Phe Ala Asn Lys Thr Val Leu
110 115 120
Thr Ile Ala His Arg Leu Asn Thr Ile Leu Asn Ser Asp Arg Val
125 130 135
Leu Val Leu Gln Ala Gly Arg Val Val Glu Leu Asp Ser Pro Ala
140 145 150
Thr Leu Arg Asn Gln Pro His Ser Leu Phe Gln GIn Leu Leu Gln
155 160 165
Ser Ser Gln Gln Gly Val Pro Ala Ser Leu Gly Gly Pro
170 175
<210> 10
<211> 255
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4074113CD1
<400> 10
Met Glu Arg Glu Met Glu Gly Arg Pro Leu His Asn Glu Gly Trp
1 5 10 15
Ile Asp Arg Ser Arg Val Gln Gln Lys Asp Leu Pro Asn Lys Cys
14
CA 02349818 2001-05-03
WO 00/26245 PCT/US99/26048
20 25 30
Pro Gln Thr Leu Trp Ser Glu Gln Ala Phe Pro Pro Asn Pro Gly
35 40 45
Gln Val Gly Ile Val Gly Arg Thr Gly Ala Gly Lys Ser Ser Leu
50 55 60
Ala Ser Gly Leu Leu Arg Leu Pro Glu Ala Ala Glu Gly Gly Ile
65 70 75
Trp Ile Asp Gly Val Pro Ile Ala His Val Gly Leu His Thr Leu
80 85 90
Arg Ser Arg Ile Ser Ile Ile Pro Gln Asp Pro Ile Leu Phe Pro
95 100 105
Gly Ser Leu Arg Met Asn Leu Asp Leu Leu Gln Glu His Ser Asp
110 115 120
Glu Ala Ile Trp Ala Ala Leu Glu Thr Val Gln Leu Lys Ala Leu
125 130 135
Val Ala Ser Leu Pro Gly Gln Leu Gln Tyr Lys Cys Ala Asp Arg
140 145 150
Gly Glu Asp Leu Ser Val Gly Gln Lys Gln Leu Leu Cys Leu Ala
155 160 165
Arg Ala Leu Leu Arg Lys Thr Gln Ile Leu Ile Leu Asp Glu Ala
170 175 180
Thr Ala Ala Val Asp Pro Gly Thr Glu Leu Gln Met Gln Ala Met
185 290 195
Leu Gly Ser Trp Phe Ala Gln Cys Thr Val Leu Leu Ile Ala His
200 205 210
Arg Leu Arg Ser Val Met Asp Cys Ala Arg Val Leu Val Met Asp
215 220 225
Lys Gly Gln Val Ala Glu Ser Gly Ser Pro Ala Gln Leu Leu Ala
230 235 240
Gln Lys Gly Leu Phe Tyr Arg Leu Ala Gln Glu Ser Gly Leu Val
245 250 255
<210> 11
<211> 462
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1413743CD1
<400> 11
Met Ala Gln Val Ser Ile Asn Asn Asp Tyr Ser Glu Trp Asp Leu
1 5 10 15
Ser Thr Asp Ala Gly Glu Arg Ala Arg Leu Leu Gln Ser Pro Cys
20 25 30
Val Asp Thr Ala Pro Lys Ser Glu Trp Glu Ala Ser Pro Gly Gly
35 40 45
Leu Asp Arg Gly Thr Thr Ser Thr Leu Gly Ala Ile Phe Ile Val
50 55 60
Val Asn Ala Cys Leu Gly Ala Gly Leu Leu Asn Phe Pro Ala Ala
65 70 75
Phe Ser Thr Ala Gly Gly Val Ala Ala Gly Ile Ala Leu Gln Met
80 85 90
15
CA 02349818 2001-05-03
WO 00/26245 PCT/US99/26048
Gly Met Leu Val Phe Ile Ile Ser Gly Leu Val Ile Leu Ala Tyr
95 100 105
Cys Ser Gln Ala Ser Asn Glu Arg Thr Tyr Gln Glu VaI Val Trp
110 115 120
Ala Val Cys Gly Lys Leu Thr Gly Val Leu Cys Glu Val Ala Ile
125 130 135
Ala Val Tyr Thr Phe Gly Thr Cys Ile Ala Phe Leu Ile Ile Ile
140 145 150
Gly Asp Gln Gln Asp Lys Ile Ile Ala Val Met Ala Lys Glu Pro
155 160 165
Glu Gly Ala Ser Gly Pro Trp Tyr Thr Asp Arg Lys Phe Thr Ile
170 175 180
Ser Leu Thr Ala Phe Leu Phe Ile Leu Pro Leu Ser Ile Pro Arg
185 190 195
Glu Ile Gly Phe Gln Lys Tyr Ala Ser Phe Leu Ser Val Val Gly
200 205 210
Thr Trp Tyr Val Thr Ala Ile Val Ile Ile Lys Tyr Ile Trp Pro
215 220 225
Asp Lys Glu Met Thr Pro Gly Asn Ile Leu Thr Arg Pro Ala Ser
230 235 240
Trp Met Ala Val Phe Asn Ala Met Pro Thr Ile Cys Phe Gly Phe
245 250 255
Gln Cys His Val Ser Ser Val Pro Val Phe Asn Ser Met Gln Gln
260 265 270
Pro Glu Val Lys Thr Trp Gly Gly Val Val Thr Ala Ala Met Val
275 280 285
Ile Ala Leu Ala Val Tyr Met Gly Thr Gly Ile Cys Gly Phe Leu
290 295 300
Thr Phe GIy Ala Ala Val Asp Pro Asp Val Leu Leu Ser Tyr Pro
305 310 315
Ser Glu Asp Met Ala Val Ala Val Ala Arg Ala Phe Ile Ile Leu
320 325 330
Ser Val Leu Thr Ser Tyr Pro Ile Leu His Phe Cys Gly Arg Ala
335 340 345
Val Val Glu Gly Leu Trp Leu Arg Tyr Gln Gly Val Pro Val Glu
350 355 360
Glu Asp Val Gly Arg Glu Arg Arg Arg Arg Val Leu Gln Thr Leu
365 370 375
Val Trp Phe Leu Leu Thr Leu Leu Leu Ala Leu Phe Ile Pro Asp
380 385 390
Ile Gly Lys Val Ile Ser Val Ile Gly Gly Leu Ala Ala Cys Phe
395 400 405
Ile Phe Val Phe Pro Gly Leu Cys Leu Ile Gln Ala Lys Leu Ser
410 415 420
Glu Met Glu Glu Val Lys Pro Ala Ser Trp Trp Val Leu Val Ser
425 430 435
Tyr Gly Val Leu Leu Val Thr Leu Gly Ala Phe Ile Phe Gly Gln
440 445 450
Thr Thr Ala Asn Ala Ile Phe Val Asp Leu Leu Ala
455 460
<210> 12
<211> 758
<212> PRT
16
CA 02349818 2001-05-03
WO 00/26245 PCTNS99/26048
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1733477CD1
<400> 12
Met Gly Leu Ala Asp Ala Ser Gly Pro Arg Asp Thr Gln Ala Leu
1 5 10 15
Leu Ser Ala Thr Gln Ala Met Asp Leu Arg Arg Arg Asp Tyr His
20 25 30
Met Glu Arg Pro Leu Leu Asn Gln Glu His Leu Glu Glu Leu Gly
35 40 45
Arg Trp Gly Ser Ala Pro Arg Thr His Gln Trp Arg Thr Trp Leu
50 55 60
Gln Cys Ser Arg Ala Arg Ala Tyr Ala Leu Leu Leu Gln His Leu
65 70 75
Pro Val Leu Val Trp Leu Pro Arg Tyr Pro Val Arg Asp Trp Leu
80 85 90
Leu Gly Asp Leu Leu Ser Gly Leu Ser Val Ala Ile Met Gln Leu
95 100 105
Pro Gln Gly Leu Ala Tyr Ala Leu Leu Ala Gly Leu Pro Pro Val
110 115 120
Phe Gly Leu Tyr Ser Ser Phe Tyr Pro Val Phe Ile Tyr Phe Leu
125 130 135
Phe Gly Thr Ser Arg His Ile Ser Val Gly Thr Phe Ala Val Met
140 145 150
Ser Val Met Val Gly Gly Val Thr Glu Ser Leu Ala Pro Gln Ala
155 160 165
Leu Asn Asp Ser Met Ile Asn Glu Thr Ala Arg Asp Ala Ala Arg
170 1?5 180
Val Gln Val Ala Ser Thr Leu Ser Val Leu Val Gly Leu Phe Gln
185 190 195
Val Gly Leu Gly Leu Ile His Phe Gly Phe Val Val Thr Tyr Leu
200 205 210
Ser Glu Pro Leu Val Arg Gly Tyr Thr Thr Ala Ala Ala Val Gln
215 220 225
Val Phe Val Ser Gln Leu Lys Tyr Val Phe Gly Leu His Leu Ser
230 235 240
Ser His Ser Gly Pro Leu Ser Leu Ile Tyr Thr Val Leu Glu Val
245 250 255
Cys Trp Lys Leu Pro Gln Ser Lys Val Gly Thr Val VaI Thr Ala
260 265 270
Ala Val Ala Gly Val Val Leu Val Val Val Lys Leu Leu Asn Asp
275 280 285
Lys Leu Gln Gln Gln Leu Pro Met Pro Ile Pro Gly Glu Leu Leu
290 295 300
Thr Leu Ile Gly Ala Thr Gly Ile Ser Tyr Gly Met Gly Leu Lys
305 310 315
His Arg Phe Glu Val Asp Val Val Gly Asn Ile Pro Ala Gly Leu
320 325 330
Val Pro Pro Val Ala Pro Asn Thr Gln Leu Phe Ser Lys Leu Val
335 340 345
Gly Ser Ala Phe Thr Ile Ala Val Val Gly Phe Ala Ile Ala Ile
350 355 360
17
CA 02349818 2001-05-03
WO 00/26245 PCT/US99/26048
Ser Leu Gly Lys Ile Phe Ala Leu Arg His Gly Tyr Arg Val Asp
365 370 375_
Ser Asn Gln Glu Leu Val Ala Leu Gly Leu Ser Asn Leu Ile Gly
380 385 390
Gly Ile Phe Gln Cys Phe Pro Val Ser Cys Ser Met Ser Arg Ser
395 400 405
Leu Val Gln Glu Ser Thr Gly Gly Asn Sex Gln Val Ala Gly Ala
410 415 420
Ile Ser Ser Leu Phe Ile Leu Leu Ile Ile Val Lys Leu Gly Glu
425 430 435
Leu Phe His Asp Leu Pro Lys Ala Val Leu Ala Ala Ile Ile Ile
440 445 450
Val Asn Leu Lys Gly Met Leu Arg Gln Leu Ser Asp Met Arg Ser
455 460 465
Leu Trp Lys Ala Asn Arg Ala Asp Leu Leu Ile Trp Leu Val Thr
470 475 480
Phe Thr Ala Thr Ile Leu Leu Asn Leu Asp Leu Gly Leu Val Val
485 490 495
Ala Val Ile Phe Ser Leu Leu Leu Val Val Val Arg Thr Gln Met
500 505 510
Pro His Tyr Ser Val Leu Gly Gln Val Pro Asp Thr Asp Ile Tyr
515 520 525
Arg Asp Val Ala Glu Tyr Ser Glu Ala Lys Glu Val Arg Gly Val
530 535 540
Lys VaI Phe Arg Ser Ser Ala Thr Val Tyr Phe Ala Asn Ala Glu
545 550 555
Phe Tyr Ser Asp Ala Leu Lys Gln Arg Cys Gly Val Asp Val Asp
560 565 570
Phe Leu Ile Ser Gln Lys Lys Lys Leu Leu Lys Lys Gln Glu Gln
575 580 585
Leu Lys Leu Lys Gln Leu Gln Lys Glu Glu Lys Leu Arg Lys Gln
590 595 600
Ala Ala Ser Pro Lys Gly Ala Ser Val Ser Ile Asn Val Asn Thr
605 610 615
Ser Leu Glu Asp Met Arg Ser Asn Asn Val Glu Asp Cys Lys Met
620 625 630
Met Val Ser Ser Gly Asp Lys Met Glu Asp Ala Thr Ala Asn Gly
635 640 645
Gln Glu Asp Ser Lys Ala Pro Asp Gly Ser Thr Leu Lys Ala Leu
650 655 660
Gly Leu Pro Gln Pro Asp Phe His Ser Leu Ile Leu Asp Leu Gly
665 670 675
Ala Leu Ser Phe Val Asp Thr Val Cys Leu Lys Ser Leu Lys Asn
680 685 690
Ile Phe His Asp Phe Arg Glu Ile Glu Val Glu Val Tyr Met Ala
695 700 705
Ala Cys His Ser Pro Val Val Ser Gln Leu Glu Ala Gly His Phe
710 715 720
Phe Asp Ala Ser Ile Thr Lys Lys His Leu Phe Ala Ser Val His
725 730 735
Asp Ala Val Thr Phe Ala Leu Gln His Pro Arg Pro Val Pro Asp
740 745 750
Ser Pro Val Ser Val Thr Arg Leu
755
1 g.
CA 02349818 2001-05-03
WO 00/26245 PCT/US99/26048
<220> 13 _
<211> 336
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2641908CD1
<400> 13
Met Met Gly Pro Gly Leu Ala Phe Gly Leu Gly Ser Leu Met Leu
1 5 10 15
Arg Leu Tyr Val Asp Ile Asn Gln Met Pro Glu Gly Gly Ile Ser
20 25 30
Leu Thr Ile Lys Asp Pro Arg Trp Val Gly Ala Trp Trp Leu Gly
35 40 45
Phe Leu Ile Ala Ala Gly Ala Val Ala Leu Ala Ala Ile Pro Tyr
50 55 60
Phe Phe Phe Pro Lys Glu Met Pro Lys Glu Lys Arg Glu Leu Gln
65 70 75
Phe Arg Arg Lys Val Leu Ala Val Thr Asp Ser Pro Ala Arg Lys
80 85 90
Gly Lys Asp Ser Pro Ser Lys Gln Ser Pro Gly Glu Ser Thr Lys
95 100 105
Lys Gln Asp Gly Leu Val Gln Ile Ala Pro Asn Leu Thr Val Ile
110 115 120
Gln Phe Ile Lys Val Phe Pro Arg Val Leu Leu Gln Thr Leu Arg
125 130 135
His Pro IIe Phe Leu Leu Val Val Leu Ser Gln Val Cys Leu Ser
140 145 150
Ser Met Ala Ala Gly Met Ala Thr Phe Leu Pro Lys Phe Leu Glu
155 160 165
Arg Gln Phe Ser Ile Thr Ala Ser Tyr Ala Asn Leu Leu Ile Gly
170 175 180
Cys Leu Ser Phe Pro Ser Val Ile Val Gly Ile Val Val Gly Gly
185 190 195
Val Leu Val Lys Arg Leu His Leu Gly Pro Val Gly Cys Gly Ala
200 205 210
Leu Cys Leu Leu Gly Met Leu Leu Cys Leu Phe Phe Ser Leu Pro
215 220 225
Leu Phe Phe Ile Gly Cys Ser Ser His Gln Ile Ala Gly Ile Thr
230 235 240
His Gln Thr Ser Ala His Pro Gly Leu Glu Leu Sex Pro Ser Cys
245 250 255
Met Glu Ala Cys Ser Cys Pro Leu Asp Gly Phe Asn Pro Val Cys
260 265 270
Asp Pro Ser Thr Arg Val Glu Tyr Ile Thr Pro Cys His Ala Gly
275 280 285
Cys Ser Ser Trp Val Val Gln Asp Ala Leu Asp Asn Ser Gln Ser
290 295 300
Pro Pro Thr Ser His Pro His Ala Gly His Gln His Leu Asn Leu
305 310 315
Arg Leu Leu Gln Gly Glu Thr Trp Ala Ala Leu Ala Gly Ala Glu
320 325 330
Glu Pro Val Asp Gly Ala
19
CA 02349818 2001-05-03
WO 00/26245 PCT/US99/26048
335
<210> 14
<211> 103
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2656554CD1
<400> 14
Met Glu Arg Gln Ser Arg Val Met Ser Glu Lys Asp Glu Tyr Gln
1 5 10 15
Phe Gln His Gln Gly Ala Val Glu Leu Leu Val Phe Asn Phe Leu
20 25 30
Leu Ile Leu Thr Ile Leu Thr Ile Trp Leu Phe Lys Asn His Arg
35 40 45
Phe Arg Phe Leu His Glu Thr Gly Gly Ala Met Val Tyr Asp Lys
50 55 60
Pro Pro Lys Phe Ala Met Ser Arg Glu Gln Met Ser Gln Ser Cys
65 70 75
Ser His Thr Ala His Asn Ala Ser Leu Leu Thr Asp Ala Gly Pro
80 85 90
Leu Ser Cys Gly Glu Ser Arg Ala Ser Cys Leu Phe Leu
95 100
<210> 15
<211> 123
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2719228CD1
<400> 15
Met Gln Gly Met Gly Leu Gly Leu Ser Ser Val Phe Ala Leu Cys
1 5 10 15
Leu Gly His Thr Ser Ser Phe Cys Glu Ser Val Val Phe Ala Ser
20 25 30
Ala Ser Ile Gly Leu Gln Thr Phe Asn His Ser Gly Ile Ser Val
35 40 45
Asn Ile Gln Asp Leu Ala Pro Ser Cys Ala Gly Phe Leu Phe Gly
50 55 60
Val Ala Asn Thr Ala Gly Ala Leu Ala Gly Val Val Gly Val Cys
65 70 75
Leu Gly Gly Tyr Leu Met Glu Thr Thr Gly Ser Trp Thr Cys Leu
80 85 90
Phe Asn Leu Val Ala Ile Ile Ser Asn Leu Gly Leu Cys Thr Phe
95 100 105
Leu Val Phe Gly Gln Ala Gln Arg Val Asp Leu Ser Ser Thr His
110 115 120
CA 02349818 2001-05-03
WO 00/26245 PCT/US99/26048
Glu Asp Leu
<210> 16 '
<211> 222
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3657824CD1
<400> 16
Met Lys Gln Glu Ser Ala Ala Pro Asn Thr Pro Pro Thr Ser Gln
1 5 10 15
Ser Pro Thr Pro Ser Ala Gln Phe Pro Arg Asn Asp Gly Asp Pro
20 25 30
Gln Ala Leu Trp Ile Phe Gly Tyr Gly Ser Leu Val Trp Arg Pro
35 40 45
Asp Phe Ala Tyr Ser Asp Ser Arg Val Gly Phe Val Arg Gly Tyr
50 55 60
Ser Arg Arg Phe Trp Gln Gly Asp Thr Phe His Arg Gly Ser Asp
65 70 75
Lys Met Pro Gly Arg Val Val Thr Leu Leu Glu Asp His Glu Gly
80 85 90
Cys Thr Trp Gly Val Ala Tyr Gln Val Gln Gly Glu Gln Val Ser
95 100 105
Lys Ala Leu Lys Tyr Leu Asn Val Arg Glu Ala Val Leu Gly Gly
110 ~ 115 120
Tyr Asp Thr Lys Glu Val Thr Phe Tyr Pro Gln Asp Ala Pro Asp
125 130 . 135
Gln Pro Leu Lys Ala Leu Ala Tyr Val Ala Thr Pro Gln Asn Pro
140 145 150
Gly Tyr Leu Gly Pro Ala Pro Glu Glu Ala Ile Ala Thr Gln Ile
155 160 165
Leu Ala Cys Arg Gly Phe Ser Gly His Asn Leu Glu Tyr Leu Leu
170 175 180
Arg Leu Ala Asp Phe Met Gln Leu Cys Gly Pro Gln Ala Gln Asp
185 190 195
Glu His Leu Ala Ala Ile Val Asp Ala Val Gly Thr Met Leu Pro
200 205 210
Cys Phe Cys Pro Thr Glu Gln Ala Leu Ala Leu Val
215 220
<210> 17
<211> 111
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5378485CD1
<400> 17
Met Leu Ser Ala Leu Pro Gly Trp Gly Pro Ala His Leu Gln Arg
21
CA 02349818 2001-05-03
WO 00/26245 PGT/US99/Z6048
1 5 10 15
Pro Leu Leu Gly Pro Ala Ser Cys Leu Gly Ile Leu Arg Pro Ala
20 25 30'
Met Thr Ala His Ser Phe Ala Leu Pro Val Ile Ile Phe Thr Thr
35 40 45
Phe Trp Gly Leu Val Gly Ile Ala Gly Pro Trp Phe Val Pro Lys
50 55 60
Gly Pro Asn Arg Gly Val Ile Ile Thr Met Leu Val Ala Thr Ala
65 70 75
Val Cys Cys Tyr Leu Phe Trp Leu Ile Ala Ile Leu Ala Gln Leu
80 85 90
Asn Pro Leu Phe Gly Pro Gln Leu Lys Asn Glu Thr Ile Trp Tyr
95 100 105
Val Arg Phe Leu Trp Glu
110
<210> 18
<211> 1303
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 961344CB1
<400> 18
cccacgcgtc cgcccacgcg tccgcaagat atggatgcta acaggcgata aactcgagac 60
agctacctgc attgccaaaa gttcacatct cgtgtctaga acacaagata ttcatatttt 120
cagacaggta accagtcggg gagaggcaca tttggagctg aatgcatttc gaaggaagca 180
tgattgtgca ctagtcatat ctggggactc tctggaggtt tgtctaaagt actacgagca 240
tgaatttgtg gagctggcct gccagtgccc tgccgtggtt tgctgccgct gctcacccac 300
ccagaaggcc cgcattgtga cactgctgca gcagcacaca gggagacgca cctgcgccat 360
cggtgatgga ggaaatgatg tcagcatgat tcaggcagca gactgtggga ttgggattga 420
gggaaaggag ggtaaacagg cctcgctggc ggccgacttc tccatcacgc agttccggca 480
cataggcagg ctgctcatgg tgcacgggcg gaacagctac aagaggtcgg cggcactcgg 540
ccagttcgtc atgcacaggg gccttatcat ctccaccatg caggctgtgt tttcctcagt 600
cttctacttc gcatccgtcc ctttgtatca gggcttcctc atggtggggt atgccaccat 660
atacaccatg ttcccagtgt tctccttagt gctggaccag gacgtgaagc cagagatggc 720
gatgctctac ccggagctgt acaaggacct caccaaggga agatccttgt ccttcaaaac 780
cttcctcatc tgggttttaa taagtattta ccaaggcggc atcctcatgt atggggccct 840
ggtgctcttc gagtctgagt tcgtccacgt ggtggccatc tccttcaccg cactgatcct 900
gaccgagctg ctgatggtgg cgctgaccgt ccgcacgtgg cactggctga tggtggtggc 960
cgagttcctc agcttaggct gctacgtgtc ctcactcgct tttctcaatg aatattttgg 1020
tataggcaga gtgtcttttg gagctttctt agatgttgcc tttatcacca ccgtgacctt 1080
cctgtggaaa gtgtcggcga tcaccgtggt cagctgcctc ccgctgtatg tcctcaagta 1140
cctgaggcgc aagctctctc ctcccagcta ctgcaagctg gcctcctaag gggctgtgca 1200
cccccagcgg gctggcccca gcaccttctg cccttcccag caccttgtgc ccttgccagt 1260
gaacgcaggg tttgccattg ctaccaagca agcaccacaa gaa 1303
<210> 19
<211> 3395
<212> DNA
<213> Homo Sapiens
22
CA 02349818 2001-05-03
WO 00/Z6245 PCTNS99/26048
<220>
<221> misc_feature
<223> Incyte ID No: 3128782CB1
<400> 19
cggaaatagc accgggcgcc gccacagtag ctgta~ctgc caccgcgatg ccgaaggcgc 60
ccaagcagca gccgccggag cccgagtgga tcggggacgg agagagcacg agcccatcag 120
acaaagtggt gaagaaaggg aagaaggaca agaagatcaa aaaaacgttc tttgaagagc 180
tggcagtaga agataaacag gctggggaag aagagaaagt gctcaaggag aaggagcagc 240
agcagcagca acagcaacag cagcagcaaa aaaaaaagcg agatacccga aaaggcaggc 300
ggaagaagga tgtggatgat gatggagaag agaaagagct catggagcgt cttaagaagc 360
tctcagtgcc aaccagtgat gaggaggatg aagtacccgc cccaaaaccc cgcggaggga 420
agaaaaccaa gggtggtaat gtttttgcag ccctgattca ggatcagagt gaggaagagg 480
aggaggaaga aaaacatcct cctaagcctg ccaagccgga gaagaatcgg atcaataagg 540
ccgtatctga ggaacagcag cctgcactca agggcaaaaa gggaaaggaa gagaagtcaa 600
aagggaaggc taagcctcaa aataaattcg ctgctctgga caatgaagag gaggataaag 660
aagaagaaat tataaaggaa aaggagcctc ccaaacaagg gaaggagaag gccaagaagg 720
cagagcaggg ttcagaggaa gaaggagaag gggaagaaga ggaggaggaa ggaggagagt 780
ctaaggcaga tgatccctat gctcatctta gcaaaaagga gaagaaaaag ctgaaaaaac 840
agatggagta tgagcgccaa gtggcttcat taaaagcagc caatgcagct gaaaatgact 900
tctccgtgtc ccaggcggag atgtcctccc gccaagccat gttagaaaat gcatctgaca 960
tcaagctgga gaagttcagc atctccgctc atggcaagga gctgttcgtc aatgcagacc 1020
tgtacattgt agccggccgc cgctacgggc tggtaggacc caatggcaag ggcaagacca 1080
cactcctcaa gcacattgcc aaccgagccc tgagcatccc tcccaacatt gatgtgttgc 1140
tgtgtgagca ggaggtggta gcagatgaga caccagcagt ccaggctgtt cttcgagctg 1200
acaccaagcg attgaagctg ctggaagagg agcggcggct tcagggacag ctggaacaag 1260
gggatgacac agctgctgag aggctagaga aggtgtatga ggaattgcgg gccactgggg 1320
cggcagctgc agaggccaaa gcacggcgga tcctggctgg cctgggcttt gaccctgaaa 1380
tgcagaatcg acccacacag aagttctcag ggggctggcg catgcgtgtc tccctggcca 1440
gggcactgtt catggagccc acactgctga tgctggatga gcccaccaac cacctggacc 1500
tcaacgctgt catctggctt aataactacc tccagggctg gcggaagacc ttgctgatcg 1560
tctcccatga ccagggcttc ttggatgatg tctgcactga tatcatccac ctcgatgccc 1620
agcggctcca ctactatagg ggcaattaca tgaccttcaa aaagatgtac cagcagaagc 1680
agaaagaact gctgaaacag tatgagaagc aagagaaaaa gctgaaggag ctgaaggcag 1740
gcgggaagtc caccaagcag gcggaaaaac aaacgaagga agccctgact cggaagcagc 1800
agaaatgccg acggaaaaac caagatgagg aatcccagga ggcccctgag ctcctgaagc 1860
gccctaagga gtacactgtg cgcttcactt ttccagaccc cccaccactc agccctccag 1920
tgctgggtct gcatggtgtg acattcggct accagggaca gaaaccactc tttaagaact 1980
tggattttgg catcgacatg gattcaagga tttgcattgt gggccctaat ggtgtgggga 2040
agagtacgct actcctgctg ctgactggca agctgacacc gacccatggg gaaatgagaa 2100
agaaccaccg gctgaaaatt ggcttcttca accagcagta tgeagagcag ctgcgcatgg 2160
aggagacgcc cactgagtac ctgcagcggg gcttcaacct gccctaccag gatgcccgca 2220
agtgcctggg ccgcttcggc ctggagagtc acgcccacac catccagatc tgcaaactct 2280
ctggtggtca gaaggcgcga gttgtgtttg ctgagctggc ctgtcgggaa cctgatgtcc 2340
tcatcttgga cgagccaacc aataacctgg acatagagtc tattgatgct ctaggggagg 2400
ccatcaatga atacaagggt gctgtgatcg ttgtcagcca tgatgcccga ctcatcacag 2460
aaaccaattg ccagctgtgg gtggtggagg agcagagtgt tagccaaatc gatggtgact 2520
ttgaagacta caagcgggag gtgttggagg ccctgggtga agtcatggtc agccggcccc 2580
gagagtgaag ctttccttcc cagaagtctc ccgagagaca tatttgtgtg gcctagaagt 2640
cctctgtggt ctcccctcct ctgaagactg cctctggcct gcagctgacc tggcaaccat 2700
tcaggcacat gaaggtggag tgtgaccttg atgtgaccgg gatcccactc tgattgcatc 2760
catttctctg aaagacttgt ttgttctgct tctcttcata taactgagct ggccttatcc 2820
ttggcatccc cctaaacaaa caagaggtga ccaccttatt gtgaggttcc atccagccaa 2880
gtttatgtgg cctattgtct caggactctc atcactcaga agcctgcctc tgatttaccc 2940
tacagcttca ggcccagctg ccccccagtc tttgggtggt gctgttcttt tctggtggat 3000
23
CA 02349818 2001-05-03
WO 00/26245 PCT/US99/26048
ttaatgctga ctcactggta caaacagctg ttgaagctca gagctggagg tgagcttctg 3060
aggcctttgc cattatccag cccaagattt ggtgcctgca gcctcttgtc tggt_tgagga 3120
cttggggcag gaaaggaatg ctgctgaact tgaatttccc tttacaaggg gaagaaataa 3180
aggaaaggag ttgctgccga cctgtcactg tttggagatt gatgggagtt ggaactgttc 3240
tcagtcttga tttgctttat tcagttttct agcagctttt aatagtcccc tcttccccac 3300
taaatggatc ttgtttacag tattactgac agtgtttact gtttaaggat cataggattc 3360
cttaacccca accattcccg caaggaataa gcaat 3395
<210> 20
<211> 2549
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1720440CB1
<400> 20
acacgtccgc atgcaagacc atcaggcgcg atatctttgg cgcctggtcc tcctgaaggt 60
gaaggcaaag gtgcgacagt gcctgcagga gcggcggaca gtgcccattt tgtttgcctc 120
taccgttcgg cgccaccccg acaagacggc cctgatcttc gagggcacag atacccactg 180
gaccttccgc cagctggatg agtactcaag cagtgtagcc aacttcctgc aggcccgggg 240
ctgaccatcg gcgatgtggc tgccatcttc atggagaacc gcaatgagtt cgtgggccta 300
tggctgggca tggccaagct cggtgtggag gcagccctca tcaacaccaa cctgcggcgg 360
gatgctctgc tccactgcct caccacctcg cgcgcacggg cccttgtctt tggcagcgaa 420
atggcctcag ccatctgtga ggtccatgcc agcctggacc cctcgctcag cctcttctgc 480
tctggctcct gggagcccgg tgcggtgcct ccaagcacag aacacctgga ccctctgctg 540
aaagatgctc ccaagcacct tcccagttgc cctgacaagg gcttcacaga taaactgttc 600
tacatctaca catccggcac cacagggctg cccaaggccg ccatcgtggt gcacagcagg 660
tattaccgca tggctgccct ggtgtactat ggattccgca tgcggcccaa cgacatcgtc 720
tatgactgcc tccccctcta ccactcagca ggaaacatcg tgggaatcgg ccagtgcctg 780
ctgcatggca tgacggtggt gattcggaag aagttctcag cctcccggtt ctgggacgat 840
tgtatcaagt acaactgcac gattgtgcag tacattggtg aactgtgccg ctacctcctg 900
aaccagccac cgcgggaggc agaaaaccag caccaggttc gcatggcact aggcaatggc 960
ctccggcagt ccatctggac caacttttcc agccgcttcc acatacccca ggtggctgag 1020
ttctacgggg ccacagagtg caactgtagc ctgggcaact tcgacagcca ggtgggggcc 1080
tgtggtttca atagccgcat cctgtcctcc gtgtacccca tccggttggt acgtgtcaac 1140
gaggacacca tggagctgat ccgggggccc gacggcgtct gcattccctg ccagccaggt 1200
gagccgggcc agctggtggg ccgcatcatc cagaaagacc ccctgcgccg cttcgatggc 1260
tacctcaacc agggcgccaa caacaagaag attgccaagg atgtcttcaa gaagggggac 1320
caggcctacc ttactggtga tgtgctggtg atggacgagc tgggctacct gtacttccga 1380
gaccgcactg gggacacgtt ccgctggaaa ggtgagaacg tgtccaccac cgaggtggaa 1440
ggcacactca gccgcctgct ggacatggct gacgtggccg tgtatggtgt cgaggtgcca 1500
ggaaccgagg gccgggccgg aatggctgct gtggccagcc ccactggcaa ctgtgacctg 1560
gagcgctttg ctcaggtctt ggagaaggaa ctgcccctgt atgcgcgccc catcttcctg 1620
cgcctcctgc ctgagctgca caaaacagga acctacaagt tccagaagac agagctacgg 1680
aaggagggct ttgacccggc tattgtgaaa gacccgctgt tctatctaga tgcccagaag 1740
ggccgctacg tcccgctgga ccaagaggcc tacagccgca tccaggcagg cgaggagaag 1800
ctgtgattcc ccccatccct ctgagggccg gcggatgctg gatccggagc cccaggttcc 1860
gccccagagc ggtcctggac aaggccagac caaagcaagc agggcctggc acctccatcc 1920
tgaggtgctg cccctccatc caaaactgcc aagtgactca ttgccttccc aacccttcca 1980
gaggctttct gtgaaagtct catgtccaag ttccgtcttc tgggctgggc aggccctctg 2040
gttcccaggc tgagactgac gggttttctc aggatgatgt cttgggtgag ggtagggaga 2100
ggacaagggg tcaccgagcc cttcccagag agcagggagc ttataaatgg aaccagagca 2160
24,
CA 02349818 2001-05-03
WO 00/26245 PCTNS99/26048
gaagtcccca gactcaggaa gtcaacagag tgggcaggga cagtggtagc atccatctgg 2220
tggccaaaga gaatcgtagc cccagagctg cccaagttca ctgggctcca cccc_cacctc 2280
caggagggga ggagaggacc tgacatctgt aggtggcccc tgatgcccca tctacagcag 2340
gaggtcagga ccacgcccct ggcctctccc cactccccca tcctcctccc tgggtggctg 2400
cctgattatc cctcaggcag ggcctctcag tccttgtggg tctgtgtcac ctccatctca 2460
gtcttggcct ggctatgagg ggaggaggaa tgggagaggg ggctcagggg ccaataaact 2520
ctgccttgag tcctcctaaa aaaaaaaaa 2549
<210> 21
<211> 2562
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2274290CB1
<400> 21
gcgggagcca acatagagcc ctcagtggga tgagggtgaa actgctattg ccggcggctc 60
ctgttttacc gcgtcagcat gctggtgcat ttatttcggg tcgggattcg gggtggccca 120
ttcccaggca ggctgctacc gcccctccgc ttccagacat tctcagctgt caggtactct 180
gatggctacc gcagctcctc cctcctccgg gccgtggccc acctgcggtc ccagctctgg 240
gcccacctcc ctcgagcccc cctagctccc agatggagcc cctctgcctg gtgctgggtt 300
gggggagccc tgctaggccc catggtactg agtaagcatc cccacctctg ccttgtggcc 360
ctgtgtgagg cagaagaggc ccctcctgcc agctccacac cccatgtcgt ggggtctcgc 420
tttaactgga agctcttctg gcagtttctg cacccccacc tgctggtcct gggggtagcc 480
gtcgtgctgg ccttgggtgc ggcactcgtg aatgtacaga tccccctgct cctgggccag 540
ctggtagagg tcgtggccaa gtacacaagg gaccacgtag ggagtttcat gactgagtcc 600
cagaatctca gcacccacct gcttatcctc tatggtgtcc agggactgct gaccttcggg 660
tacctggtgc tgctgtccca cgttggcgag cgcatggctg tggacatgcg gagggccctc 720
ttcagctccc tgctccgaca agacatcacc ttctttgacg ccaataagac agggcagctg 780
gtgagccgct tgacaactga cgtgcaggag tttaagtcat ccttcaagct tgtcatctcc 840
caggggctgc gaagctgcac ccaggtggca ggctgcctgg tgtccctgtc catgctgtcg 900
acacgcctca cgctgctgct gatggtggcc acaccagccc tgatgggagt gggcaccctg 960
atgggctcag gcctccgaaa attgtctcgc cagtgtcagg agcagatcgc cagggcaatg 1020
ggcgtagcag acgaggccct gggcaatgtg cggactgtgc gtgccttcgc catggagcaa 1080
cgggaagagg agcgctatgg ggcagagctg gaagcctgcc gctgccgggc agaggagctg 1140
ggccgcggca tcgccttgtt ccaagggctt tccaacatcg ccttcaactg catggtcttg 1200
ggtaccctat ttattggggg ctcccttgtg gccggacagc agctgacagg gggagacctc 1260
atgtccttcc tggtggcctc ccagacagtg caaaggtcca tggccaacct ctctgtcctg 1320
tttgggcagg tggtccgggg gctgagtgca ggtgcccggg tctttgagta catggccctg 1380
aacccctgca tcccactgtc tgggggctgc tgcgtcccca aagagcagct gcgtggctcc 1440
gttacatttc agaacgtctg cttcagctac ccctgccgcc ccggcttcga ggtgctgaaa 1500
gacttcaccc tgacgctgcc ccctggcaag atcgtggccc tcgtgggcca gtctggcgga 1560
ggaaagacca ccgtggcttc cctgctggag cgcttctacg accccacggc aggcgtggtg 1620
atgctggatg ggcgggacct gcgcaccctt gacccctcct ggctccgggg ccaggttgtc 1680
ggcttcatca gccaggagcc cgtcctgttt gggacgacca tcatggaaaa catccgcttt 1740
gggaagctgg aagcttccga tgaagaggtg tacacagccg cccgggaagc gaatgctcac 1800
gagttcatca ccagcttccc cgagggctac aacacggtcg tcggtgaacg gggcactacc 1860
ctgtctgggg gccagaagca gcgcctggcc atcgcccgag cccttatcaa gcagcccacg 1920
gtgctgatac tggatgaagc taccagcgcg ctggatgcag agtccgagcg ggttgtacag 1980
gaggccctgg accgggccag tgcaggccgc acggtgctgg taattgccca ccggctcagc 2040
actgtccgtg gggcccactg cattgtcgtc atggccgatg gccgtgtctg ggaggctggg 2100
acacatgaag agctcctgaa gaaaggcggg ctatacgccg agctcatccg gaggcaggcc 2160
25
CA 02349818 2001-05-03
PCT/US99/26048
WO 00/26245
ctggatgccc cgaggacagc ggcccccccg cccaaaaagc cagaaggccc caggagccac 2220
cagcacaagt cctgagaagg gccccctgag gtgtggtcgc tgccaagcat cagtgttagg 2280
ctc agcctggggg agcctactgg ggactgagcc cccaggaggg ccagcatgtg 2340
gctgggg
gagagtcgct gcggct9ctc ctgctcacaa taaagccggg gccgagcagc tggcagggga 2400
ggccaatccc tccctcccct ccccagtcct gccggctgcc tccctcccac cagagtctgc 2460
cagagtcatt gggctgcaat gggcagagac agagttccac gagacacctc cactctattc 2520
tccctttgcc cagacccctc cagacctctc aagagacgtt ct 2562
<210> 22
<211> 2314
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2740029CB1
<400> 22
cgctggtctt catgcgccct agccctcttt cggggatact ggccgacccc ctcttccttt 60
tcccctttag tgaaggcctc ccccgtcgcc gcgcggcttc ccggagccga ctgcagactc 120
cctcagcccg gtgttccccg cgtccggacg ccgaggtcgc ggcttcgcag aaactcgggc 180
ccctccatcc gccctcagaa aagggagcga tgttgatctc aggaagcaca aagggacctt 240
cctagctctg actgaaccac ggagctcacc ttggacagta tcactccgtg gaggaagact 300
gtgagactgt ggctggaagc cagattgtag ccacacatcc gcccctgccc taccccagag 360
ccctggagca gcaactggct gcagatcaca gacacagtga ggatatgagt gtaggggtga 420
gcacctcagc ccctctttcc ccaacctcgg gcacaagcgt gggcatgtct accttctcca 480
tcatggacta tgtggtgttc gtcctgctgc tggttctctc tcttgccatt gggctctacc 540
atgcttgtcg tggctggggc cggcatactg ttggtgagct gctgatggcg gaccgcaaaa 600
tgggctgcct tccggtggca ctgtccctgc tggccacctt ccagtcagcc gtggccatcc 660
tgggtgtgcc gtcagagatc taccgatttg ggacccaata ttggttcctg ggctgctgct 720
actttctggg gctgctgata cctgcacaca tcttcatccc cgttttctac cgcctgcatc 780
tcaccagtgc ctatgagtac ctggagcttc gattcaataa aactgtgcga gtgtgtggaa 840
ctgtgacctt catctttcag atggtgatct acatgggagt tgtgctctat gctccgtcat 900
tggctctcaa tgcagtgact ggctttgatc tgtggctgtc cgtgctggcc ctgggcattg 960
tctgtaccgt ctatacagct ctgggtgggc tgaaggccgt catctggaca gatgtgttcc 1020
agacactggt catgttcctc gggcagctgg cagttatcat cgtggggtca gccaaggtgg 1080
gcggcttggg gcgtgtgtgg gccgtggctt cccagcacgg ccgcatctct gggtttgagc 1140
tggatccaga cccctttgtg cggcacacct tctggacctt ggccttcggg ggtgtcttca 1200
tgatgctctc cttatacggg gtgaaccagg ctcaggtgca gcggtacctc agttcccgca 1260
cggagaaggc tgctgtgctc tcctgttatg cagtgttccc cttccagcag gtgtccctct 1320
gcgtgggctg cctcattggc ctggtcatgt tcgcgtatta ccaggagtat cccatgagca 1380
ttcagcaggc tcaggcagcc ccagaccagt tcgtcctgta ctttgtgatg gatctcctga 1440
agggcctgcc aggcctgcca gggctcttca ttgcctgcct cttcagcggc tctctcagca 1500
ctatatcctc tgcttttaat tcattggcaa ctgttacgat ggaagacctg attcgacctt 1560
ggttccctga gttctctgaa gcccgggcca tcatgctttc cagaggcctt gcctttggct 1620
atgggctgct ttgtctagga atggcctata tttcctccca gatgggacct gtgctgcagg 1680
cagcaatcag catctttggc atggttgggg gaccgctgct gggactcttc tgccttggaa 1740
tgttctttcc atgtgctaac cctcctggtg ctgttgtggg cctgttggct gggctcgtca 1800
tggccttctg gattggcatc gggagcatcg tgaccagcat gggctccagc atgccaccct 1860
ctccctctaa tgggtccagc ttctccctgc ccaccaatct aaccgttgcc actgtgacca 1920
cactgatgcc cttgactacc ttctccaagc ccacagggct gcagcggttc tattccttgt 1980
cttacttatg gtacagtgct cacaactcca ccacagtgat tgtggtgggc ctgattgtca 2040
gtctactcac tgggagaatg cgaggccggt ccctgaaccc tgcaaccatt tacccagtgt 2100
tgccaaagct cctgtccctc cttccgttgt cctgtcagaa gcggctccac tgcaggagct 2160
26
CA 02349818 2001-05-03
PCTNS99/26048
WO 00/26245
acggccagga ccacctcgac actggcctgt ttcctgagaa gccgaggaat ggtgtgctgg 2220
gggacagcag agacaaggag gccatggccc tggatggcac agcctatcag gggagcagct 2280
ccacctgcat cctccaggag acctccctgt gatg 2314
<210> 23
<211> 2155
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2414415CB1
<400> 23
gtggttccta tttcggaaaa ggacgttcta attcaaagct ctctcccaat atatttacac 60
gaatacgcat ttagaaaggg aggcagcttt tgaggttgca atcctactga gaaggatgga 120
agaaggagcc aggcaccgaa acaacaccga aaagaaacac ccaggtgggg gcgagtcgga 180
cgccagcccc gaggctggtt ccggaggggg cggagtagcc ctgaagaaag agatcggatt 240
ggtcagtgcc tgtggtatca tcgtagggaa catcatcggc tctggaatct ttgtctcgcc 300
aaagggagtg ctggagaatg ctggttctgt gggccttgct ctcatcgtct ggattgtgac 360
gggcttcatc acagttgtgg gagccctctg ctatgctgaa ctcggggtca ccatccccaa 420
atctggaggt gactactcct atgtcaagga catcttcgga ggactggctg ggttcctgag 480
gctgtggatt gctgtgctgg tgatctaccc caccaaccag gctgtcatcg ccctcacctt 540
ctccaactac gtgctgcagc cgctcttccc cacctgcttc cccccagagt ctggccttcg 600
gctcctggct gccatctgct tattgctcct cacatgggtc aactgttcca gtgtgcggtg 660
ggccacccgg gttcaagaca tcttcacagc tgggaagctc ctggccttgg ccctgattat 720
catcatgggg attgtacaga tatgcaaagg agagtacttc tggctggagc caaagaatgc 780
atttgagaat ttccaggaac ctgacatcgg cctcgtcgca ctggctttcc ttcagggctc 840
ctttgcctat ggaggctgga actttctgaa ttacgtgact gaggagcttg ttgatcccta 900
caagaacctt cccagagcca tcttcatctc catcccactg gtcacatttg tgtatgtctt 960
tgccaatgtc gcttatgtca ctgcaatgtc cccccaggag ctgctggcat ccaacgccgt 1020
cgctgtgact tttggagaga agctcctagg agtcatggcc tggatcatgc ccatttctgt 1080
tgccctgtcc acatttggag gagttaatgg gtctctcttc acctcctctc ggctgttctt 1140
cgctggagcc cgagagggcc accttcccag tgtgttggcc atgatccacg tgaagcgctg 1200
caccccaatc ccagccctgc tcttcacatg catctccacc ctgctgatgc tggtcaccag 1260
cgacatgtac acactcatca actacgtggg cttcatcaac tacctcttct atggggtcac 1320
ggttgctgga cagatagtcc ttcgctggaa gaagcctgat atcccccgcc ccatcaagat 1380
caacctgctg ttccccatca tctacttgct gttctgggcc ttcctgctgg tcttcagcct 1440
gtggtcagag ccggtggtgt gtggcattgg cctggccatc atgctgacag gagtgcctgt 1500
ctatttcctg ggtgtttact ggcaacacaa gcccaagtgt ttcagtgact tcattgagct 1560
gctaaccctg gtgagccaga agatgtgtgt ggtcgtgtac cccgaggtgg agcggggctc 1620
agggacagag gaggctaatg aggacatgga ggagcagcag cagcccatgt accaacccac 1680
tcccacgaag gacaaggacg tggcggggca gccccagccc tgaggaccac cattccctgg 1740
ctactctctc cttcctcccc cttttatcct acctccctgc cttggtcccg ccaacacatg 1800
cgagtacaca cacacccctc tctctgcttt tgtcaggcag tggtaggact ttggtgtggg 1860
tggtgagaaa ttgtaaacaa aaactgacat tcatacccaa agaaccagcc tctcacccca 1920
gggtccatgt cccaggcccc actccagtgc tgcccacact cccagctgct ggaggagagg 1980
ggagatgcca aggtgccctg caggacctcc ctccgggcca caccctcagc tgcctcttca 2040
ggaaccggag ctcattactg ccttccctcc cagggaggcc ccttcagaga ggagaggcca 2100
caggagctgc attgtggggg gacaggctca agcaattctg tccccatcaa ggggt 2155
<210> 24
<2I1> 1475
27
CA 02349818 2001-05-03
WO 00/26245
pCT/US99/26048
<212> DNA
<213> Homo sapiens '
<220>
<221> misc_feature
<223> Incyte ID No: 2466714CB1
<400> 24
ggagcgcagg gcaggggtag aggctcgtag atggaactgg tagtcagctg gagagcagca 60
tggaggcgtc ctgggggagc ttcaacgctg agcggggctg gtatgtctct gtccagcagc 120
ctgaagaagc ggaggccgaa gagttgagtc cgttgctaag caacgaactt cacagacagc 180
gatccccagg tgtttcattt ggtttatcag tgtttaattt gatgaatgcc atcatgggaa 240
gtggcatcct tggcttagct tatgttatgg ctaataccgg tgtctttgga tttagcttct 300
tgctgctgac agttgctctc ctggcttctt actcagtcca tcttctgctt agtatgtgta 360
ttcagacagc tgtaacatct tatgaagatc ttggactctt tgcatttgga ttacctggaa 420
agttggtggt ggcaggcacc ataataattc agaatattgg agctatgtca tcttatcttt 480
taattattaa aacagagctt cctgctgcta ttgcagaatt tttgactgga gactataata 540
gatattggta tcttgatgga caaacactac taataatcat atgtgttggc attgtgttcc 600
ctcttgcact tcttcccaaa ataggctttc ttggctacac aagtagttta tcatttttct 660
ttatgatgtt ctttgctctt gtggtaataa ttaaaaaatg gtccatccct tgtcctctga 720
cattaaatta tgtagagaaa ggcttccaga tttcaaatgt tacagatgat tgtaagccaa 780
agctctttca tttctccaaa gagagtgctt atgccttacc aaccatggct ttttcatttc 840
tctgccatac ctcaatattg cccatatact gtgaacttca aagtccttca aagaaaagaa 900
tgcagaatgt taccaataca gcaattgctt t.aagttttct catttatttt atatctgcac 960
tctttgggta cctcactttt tatgacaaag tggagtcaga attactaaaa ggttatagta 1020
aatacttatc acatgatgtt gttgtcatga ctgtgaagtt atgcatacta tttgctgtgc 1080
ttttgacagt ccctctaatc cacttccctg ccagaaaagc tgtaacaatg atgtttttct 1140
ccaattttcc attctcatgg attcgccatt ttttgatcac tctagcactc aatattatca 1200
tcgttttact tgcaatatat gttcctgaca ttagaaatgt atttggtgta gttggtgcca 1260
gtacatcaac atgtttgatt tttatattcc caggactatt ttatcttaaa cttagcagag 1320
aggattttct gtcatggaaa aagcttgggg cattcgtttt gctcatcttt ggaattttgg 1380
ttgggaattt tagtttagca ctcatcattt ttgattggat taataaataa aagaaatatt 1440
ttcctacttc ttacaagaat aataaaaaaa aaaaa 1475
<210> 25
<211> 1793
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2617942CB1
<400> 25
gcggtggcta cggcgacggg agccggcggc gctgcgggtc agcggtcgcg taggacccag 60
cggactcggc agcctggggc gcccggcgga gctgaaccgc ggcccccggt ggtgggctca 120
gccggtcgag ctgcgcggga ggcaaatgaa gataaaacaa tgttcgccaa cctaaaatac 180
gtttccctgg gaattttggt ctttcagact accagtttgg ttctaacaat gcgttattcc 240
agaactttaa aagaagaagg acctcgttat ctatcttcta cagcagtggt tgttgctgaa 300
cttttgaaga taatggcctg cattttattg gtctacaaag acagcaaatg tagtctaaga 360
gcactgaatc gagtactaca tgatgaaatt cttaataaac ctatggaaac acttaaactt 420
gctattccat cagggatcta tactcttcag aataatttac tgtatgtggc actatcaaat 480
ctagatgcag ctacttatca ggtcacgtat cagttaaaaa ttcttacaac agcattattt 540
tctgtgtcta tgcttagtaa aaaattgggt gtataccagt ggctgtccct agtaattttg 600
28
CA 02349818 2001-05-03
pCT/US99/26048
WO 00/26245
atgacaggag ttgcttttgt acagtggccc tcagattctc agcttgattc taaggaactt 660
tcagctggtt ctcaatttgt aggactcatg gcagttctca cagcatgttt ttcaagtggc 720
tttgctgggg tttactttga gaaaatctta aaagaaacaa aacaatcagt gtggataaga 780
aatattcagc ttggtttctt tggaagtata tttggattaa tgggtgtata catttatgat 840
ggagaactgg tatcaaagaa tggatttttt cagggatata accgactgac ctggatagta 900
gttgttcttc aggcacttgg aggccttgta atagctgctg ttattaagta tgcagataat 960
attttaaaag gatttgcaac ctctttatcg ataatattat caacattgat ctcctatttt 1020
tggcttcaag attttgtgcc aaccagtgtc tttttccttg gagccatcct tgtaataaca 1080
gctacttttt tgtatggtta tgatcccaaa cctgcaggaa atcccactaa agcatagttg 1140
tatactatct ttaactggtt tttcacgatg gggcactagg aatctcgaca ttaatcttgc 1200
acagaggact tctacagagt ctgagaagat atcatcatgc tgaatctgat catactgttt 1260
tttaaaagtt taaggataag acatgtgtat atgtaacaaa acacattgca tctagaaatc 1320
aaaacttgaa agtatttcca gggattagga ttagaaggaa tattagagga aacttgaaat 1380
ctgagtttaa aaagatttta cctttttgat tgctgcagaa atgtcctatg cactctttgc 1440
aagagcacac aacaaatgtc agataccaat ttttgcaaat tagatttaat cttattaaat 1500
gtttttatct tactctttct gtacagatat atcaaatcac atgaaatatt taaagtttga 1560
aaattataat tacctataaa gctgtgaaaa atagaagtat aatttgaaaa aacatttcac 1620
ttatcagaga tttttatatt tatacaaaag attacttaat gaaggattgc taaatgtttt 1680
tgggtcaatt accttaagat taatattccg ggtctgatct gtcagggaat aaatatcaaa 1740
tctaaatttt aatgtggggg ttcatactat ttctcccata agaattttag ggt 1793
<210> 26
<211> 1141
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2945431CB1
<400> 26
ctggtttgtg tgtgtgcacc tccgtgaaat gtaggcacct tgaggacaga gtccagcctt 60
tggtttcttt ggtattgctc atagcactgg cacagttcta ggtacccagc tactaacaga 120
tcatttggtg gggatggggt ggggagcaga gtggggttat gttcaggtct catacccagg 180
ctttcatgga ggtgctagcc ctgtagtcag aaactgagct gggagcagaa gtggctacat 240
ctccaaccac tagactccat gtcattgtcc cccagatccc agttggctat catcccccag 300
gagccctttt tgttcagtgg gactgttcgg gaaaacctgg acccccaggg cctacataag 360
gacagggcct tgtggcaggc cctgaagcag tgccacctga gtgaggtgat tacatccatg 420
ggtggtctgg atggtgagct gggtgagggg ggccggagct tatctcttgg gcagaggcag 480
ctgttgtgtt tggccagggc tctcctcaca gatgccaaga tcctgtgtat cgatgaggcc 540
acagcaagtg tggaccagaa gacagaccag ctgctccagc agaccatctg caaacgcttt 600
gccaacaaga cagtgctgac cattgcccat aggctcaaca cgatcctgaa ctcagaccgg 660
gtgctggtgc tacaagcggg gagagtggta gagctggact ccccggccac cctgcgcaac 720
cagccccact ccctgttcca gcagctgctg cagagcagcc agcagggagt ccctgcctca 780
ctcggaggtc cctgagccca atcccacacc ctgcagagtt ctcccctctc tctgatccag 840
gccgggccta tacagaggtg ctggctgctt gtttacattc tcctctgggg ctctacctct 900
ccacacttcc ccagaaggga aaagggcacc ctggattact ctttggaaat cactccttgg 960
tgggcagcat cctgaggctt ccccagaacc aggcctctgc tctggccctc ttgcatctgg 1020
aacgccaggt gggtttttct ggcataggag cccacttgca ttttcatagt tttatttgat 1080
aaaattccat cttacattct gtgtattaaa aaaataatat ttctggtgtg agaaaaaaaa 1140
1141
a
<210> 27
29
CA 02349818 2001-05-03
PCT/US99/26048
WO 00/26245
<211> 1371 .
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4074113CB1
<400> 27
gactggatgg attgatgggt ggatatatag aaaggtagac agatggaaag agagatggaa 60
ggtagacctt tacacaatga gggatggata gacagatctc gggtacagca gaaagatctc 120
cccaataaat gcccacaaac cctctggtca gagcaggcct ttcctcccaa ccccgggcag 180
gtgggcatcg ttggcaggac cggggcaggg aagtcctccc tggccagtgg gctgctgcgg 240
ctcccagagg cagctgaggg tgggatctgg atcgacgggg tccccattgc ccacgtgggg 300
ctgcacacac tgcgctccag gatcagcatc atcccccagg accccatcct gttccctggc 360
tctctgcgga tgaacctcga cctgctgcag gagcactcgg acgaggctat ctgggcagcc 420
ctggagacgg tgcagctcaa agccttggtg gccagcctgc ccggccagct gcagtacaag 480
tgtgctgacc gaggcgagga cctgagcgtg ggccagaaac agctcctgtg tctggcacgt 540
gcccttctcc ggaagaccca gatcctcatc ctggacgagg ctactgctgc cgtggaccct 600
ggcacggagc tgcagatgca ggccatgctc gggagctggt ttgcacagtg cactgtgctg 660
ctcattgccc accgcctgcg ctccgtgatg gactgtgccc gggttctggt catggacaag 720
gggcaggtgg cagagagcgg cagcccggcc cagctgctgg cccagaaggg cctgttttac 780
agactggccc aggagtcagg cctggtctga gccaggaccc tcaaccgtac cccagttgga 840
ccagcccgca cagcctgcag tgctggagat ggaagtgacc cgtggtcatc gatagctcca 900
cacgatattg agtctagacc tgtgtttgct ctctgggagg aaaatggcag agaaagtggc 960
caattatcac agagcatcag agccggaagg acctagcaat acacaggtct gcccgggcag 1020
ggcccatctc gccctgtcca ccctgcagcc aatgtcaaca gcgactctca gccccgctgt 1080
actctggact cacctggggg cctcaagcac atgcccaggc tcccggctag acccttaaat 1140
cagaatctct gaggctggga actgccatgc tgtgtgtact ttttacaaat taacactttt 1200
attttgggat aatcccagac tcacatgcag ttaaagaaac aataatatag agagattcgt 1260
gtacttggta ccccatttca cccaatggta acatcttgca aaactctagg ataaagcatc 1320
acagccaggg tgttgacatt gacacaacaa tcttgctcgg atgtccgcga g 1371
<210> 28
<211> 2752
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1413743CB1
<400> 28
gggaaagaat cccccaagct ccatttcatg agtaagcgtg agagccgctc agtttcctcc 60
agctctgctg aagccagcac agaagtagcc caaactcttc cctctgctga cagcaaattt 120
taggcaaagt cttgagaaag aagaaattgg gtccagaaag ggaagtgagg agaatcagat 180
cccagacctt tggggagaag gagcaaccgc ctctggcaca gcccatcagg gagaaagagc 240
aggttgagaa gagtcctaag ctaacagccc caaacaggtg ggtgttgctc agctccctga 300
ggcatgtggt tgtaaggcag aacccacaga ccttgcagga agaaggctct cggggccatg 360
gcccaggtca gcatcaacaa tgactacagc gagtgggact tgagcacgga tgccggggag 420
cgggctcggc tgctgcagag tccctgtgtg gacacagccc ccaagagtga gtgggaagcc 480
tctcctgggg gtctggacag aggcaccact tccacacttg gggccatctt catcgtcgtc 540
aacgcgtgcc tgggtgcagg gttactcaac ttcccagcag ccttcagcac tgcggggggc 600
gtggcagcag gcatcgcact gcagatgggt atgctggttt tcatcatcag tggccttgtc 660
30
CA 02349818 2001-05-03
PCTNS99/26048
WO 00/26245
atcctggcct actgctccca ggccagcaat gagaggacct accaggaggt ggtatgggct 720
gtgtgtggca agctgacagg tgtgctatgt gaggtggcca tcgctgtcta cacctttggc 780
acctgcattg ccttcctaat catcattggc gaccagcagg acaagattat agctgtgatg 840
gcgaaagagc cggagggggc cagcggccct tggtacacag accgcaagtt caccatcagc 900
ctcactgcct tcctcttcat cctgcccctc tccatcccca gggagattgg tttccagaaa 960
tatgccagct tcctgagcgt cgtgggtacc tggtacgtca cagccatcgt tatcatcaag 1020
tacatctggc cagataaaga gatgacccca gggaacatcc tgaccaggcc ggcttcctgg 1080
atggctgtgt tcaatgccat gcccaccatc tgcttcggat ttcagtgcca cgtcagcagt 1140
gtgcccgtct tcaacagcat gcagcagcct gaagtgaaga cctggggtgg agtggtgaca 1200
gctgccatgg tcatagccct cgctgtctac atggggacag gcatctgtgg cttcctgacc 1260
tttggagctg ctgtggatcc tgacgtgctc ctgtcctatc cctcggagga catggccgtg 1320
gccgttgccc gagccttcat catcctgagc gtgctcacct cctaccctat cctgcacttc 1380
tgtgggcggg cggtggtgga aggcctgtgg ctgcgctacc agggg9tgcc agtggaggag 1440
gacgtggggc gggagcggcg gcggcgagtg ctgcagacgc tggtctggtt cctgctcacc 1500
ctgctgctgg cgctcttcat ccctgacatc ggcaaggtga tctcagtcat tggaggcctg 1560
gccgcctgct tcatcttcgt cttcccaggg ctgtgcctca ttcaagccaa actctctgag 1620
atggaagagg tcaaaccagc cagctggtgg gtgctggtca gctacggagt cctcttggtc 1680
accctgggag ccttcatctt cggccagacc acagccaacg ccatctttgt ggatctcttg 1740
gcataaccac tgcctcccag ggaacacaag gcctttgcca ttggtcgcag gaacccatct 1800
cttagagcta tggggccatt cttagtccac gatcattcca actggtggga tgacatccgg 1860
acatcctctt ccagggactg gggcaaactc aggccccaca cctctggaca gctcaaatcc 1920
agtccccttc ctgctcccca gtcctggcag tgccgtggat ggcggcagga agtctcacat 1980
catagaggac ccctcctcct ctcccagttc tcaacttctc catgcctgga atccacgggt 2040
gaagagagtc ggtagatctc ataagaaaga atccagtctg acttccctct ggagaatgac 2100
tatggacaga aggccaccat cctccacaga gcaccctgtc ctgagtaggg gttgtgctca 2160
ttaccccagg ccagtggtag cttcctcagg agcctggcca cttccaacgg tagcactgaa 2220
gtcatgcaaa tgcatagtca ggtagattca gaccttgtcc acaccttcct gggcaacccc 2280
caccatgaac ctgtcagcct ctttcccata gctaatagac atttcccagg ccttgagggg 2340
ccccaccctg tctctttcat caaacctgat ggtccaggct gggcatccct ctcctcctcc 2400
atccccagac atcaccaggt ctaatgttta caaacggtgc cagcccggct ctgaagccaa 2460
gggccgtccc gtgccacggt gctgtgagta ttcctccgtt agctttcccc ataaggttgg 2520
gagtatctgc ttttgtgtct gagatgggcc cctcttttca gaggccgcag ggtgggtgat 2580
ggagaaggct gagaaccttt cagaccctct gtgtgggctg ggctggtcag aatcagggtg 2640
tacctccccg acaccttctt tttcagtgat gttttctctt ctccctgcct ttcctctgcc 2700
tcctcccctg ccagccctag cgtgactacc cagagacaaa aaaaaaaaaa as 2752
<210> 29
<211> 2580
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1733477CB1
<400> 29
ggagcagccc gcaccggaca acttgcgagc catggggctg gcggatgcgt cgggaccgag 60
ggacacacag gcactgctgt ctgcaacaca agcaatggac ctgcggaggc gagactacca 120
catggaacgg ccgctgctga accaggagca tttggaggag ctggggcgct ggggctcagc 180
acctaggacc caccagtggc ggacctggtt gcagtgctcc cgtgctcggg cctatgccct 240
tctgctccaa cacctcccgg ttttggtctg gttaccccgg tatcctgtgc gtgactggct 300
cctgggtgac ctgttatccg gcctgagtgt ggccatcatg cagcttccgc agggcttggc 360
ctacgccctc ctggctggat tgccccccgt gtttggcctc tatagctcct tctaccctgt 420
cttcatctac ttcctgtttg gcacttcccg gcacatctcc gtggggacct ttgctgtcat 480
31
CA 02349818 2001-05-03
PCTNS99/Z6048
WO 00/26245
gtctgtgatg gtgggcggtg tgacagaatc cctggccccg caggccttga acgactccat 540
gatcaatgag acagccagag atgctgcccg ggtacaggtg gcctccacac tcagtgtcct 600
ggttggcctc ttccaggtgg ggctgggcct gatccacttc ggcttcgtgg tcacctacct 660
gtcagaacct cttgtccgag gctataccac agctgcagct gtgcaggtct tcgtctcaca 720
gctcaagtat gtgtttggcc tccatctgag cagccactct gggccactgt ccctcatcta 780
tacagtgctg gaggtctgct ggaagctgcc ccagagcaag gttggcaccg tggtcactgc 840
agctgtggct g9ggtggtgc tcgtggtggt gaagctgttg aatgacaagc tgcagcagca 900
gctgcccatg ccgatacccg gggagctgct cacgctcatc ggggccacag gcatctccta 960
tggcatgggt ctaaagcaca gatttgaggt agatgtcgtg ggcaacatcc ctgcagggct 1020
ggtgccccca gtggccccca acacccagct gttctcaaag ctcgtgggca gcgccttcac 1080
catcgctgtg gttgggtttg ccattgccat ctcactgggg aagatcttcg ccctgaggca 1140
cggctaccgg gtggacagca accaggagct ggtggccctg ggcctcagta accttatcgg 1200
aggcatcttc cagtgcttcc ccgtgagttg ctctatgtct cggagcctgg tacaggagag 1260
caccgggggc aactcgcagg ttgctggagc catctcttcc cttttcatcc tcctcatcat 1320
tgtcaaactt ggggaactct tccatgacct gcccaaggcg gtcctggcag ccatcatcat 1380
tgtgaacctg aagggcatgc tgaggcagct cagcgacatg cgctccctct ggaaggccaa 1440
tcgggcggat ctgcttatct ggctggtgac cttcacggcc accatcttgc tgaacctgga 1500
ccttggcttg gtggttgcgg tcatcttctc cctgctgctc gtggtggtcc ggacacagat 1560
gccccactac tctgtcctgg ggcaggtgcc agacacggat atttacagag atgtggcaga 1620
gtactcagag gccaaggaag tccggggggt gaaggtcttc cgctcctcgg ccaccgtgta 1680
ctttgccaat gctgagttct acagtgatgc gctgaagcag aggtgtggtg tggatgtcga 1740
cttcctcatc tcccagaaga agaaactgct caagaagcag gagcagctga agctgaagca 1800
actgcagaaa gaggagaagc ttcggaaaca ggctgcctcc cccaagggcg cctcagtttc 1860
cattaatgtc aacaccagcc ttgaagacat gaggagcaac aacgttgagg actgcaagat 1920
gatggtgagc tcaggagata agatggaaga tgcaacagcc aatggtcaag aagactccaa 1980
ggccccagat gggtccacac tgaaggccct gggcctgcct cagccagact tccacagcct 2040
catcctggac ctgggtgccc tctcctttgt ggacactgtg tgcctcaaga gcctgaagaa 2100
tattttccat gacttccggg agattgaggt ggaggtgtac atggcggcct gccacagccc 2160
tgtggtcagc cagcttgagg ctgggcactt cttcgatgca tccatcacca agaagcatct 2220
ctttgcctct gtccatgatg ctgtcacctt tgccctccaa cacccgaggc ctgtccccga 2280
cagccctgtt tcggtcacca gactctgaac atgctacatc ctgcccaaga ctgcacctct 2340
ggaggtgcag ggcacccttg agaagcccct cacccctagg ccgcctccag gtgctaccca 2400
ggagtcccct ccatgtacac acacacaact cagggaagga ggtcctggga ctccaagttc 2460
agcgctccag gtctgggaca gggcctgcat gcagtcaggc tggcagtggc gcggtacagg 2520
gagggaactg gtgcatattt tagcctcagg aataaagatt tgtctgctca aaaaaaaaaa 2580
<210> 30
<211> 1481
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2641908CB1
<400> 30
tgatgattgt gctggtggtg gtgatcatga cagagacaac aataacaatc atcacatcgt 60
gatggtaatg tcgtgactaa atttgtcatt tagtcacaac gatatgggtg atgtgaatga 120
gggtgatatt taagctgaaa ggaatagaaa tgatgatgac agcaactcgc ccctctacct 180
cgggatcctg tttgcagtga ccatgatggg gccaggcctg gcctttgggc tgggcagcct 240
catgctgcgc ctttatgtgg acattaacca gatgccagaa ggtggtatca gcctgaccat 300
aaaggacccc cgatgggtgg gtgcctggtg gctgggtttc ctcatcgctg ccggtgcagt 360
ggccctggct gccatcccct acttcttctt ccccaaggaa atgcccaagg aaaaacgtga 420
gcttcagttt cggcgaaagg tcttagcagt cacagactca cctgccagga agggcaagga 480
32
CA 02349818 2001-05-03
PCTNS99/26048
WO 00/26245
ctctccctct aagcagagcc ctggggagtc cacgaagaag caggatggcc tagtccagat 540
tgcaccaaac ctgactgtga tccagttcat taaagtcttc cccagggtgc tgctgcagac 600
cctacgccac cccatcttcc tgctggtggt cctgtcccag gtatgcttgt catccatggc 660
tgcgggcatg gccaccttcc tgcccaagtt cctggagcgc cagttttcca tcacagcctc 720
ctacgccaac ctgctcatcg gctgcctctc cttcccttcg gtcatcgtgg gcatcgtggt 780
gggtggcgtc ctggtcaagc ggctccacct gggccctgtg ggatgcggtg ccctttgcct 840
gctggggatg ctgctgtgcc tcttcttcag cctgccgctc ttctttatcg gctgctccag 900
ccaccagatt gcgggcatca cacaccagac cagtgcccac cctgggctgg agctgtctcc 960
aagctgcatg gaggcctgct cctgcccatt ggacggcttt aaccctgtct gcgaccccag 1020
cactcgtgtg gaatacatca caccctgcca cgcaggctgc tcaagctggg tggtccagga 1080
tgctctggac aacagccaga gtcctcccac ctcccaccct catgctgggc atcagcatct 1140
aaacctgagg ctcctccagg gagagacctg ggctgcactg gctggtgcag aagaacctgt 1200
tgatggtgca tagtccttca gaagccagcc aggcaccacc tgggcctgag agcccttcca 1260
gagaccccca ggccttggca ggtggagcag tgaactcctg tggatatggg aaccgattca 1320
aatccttctt aggcctctaa ctgactctgt taccttaggc aaattattta actagtgcct 1380
cagtttcttg gtctgtaaaa taggggagat attattaagt gcctactaca gagcaggaat 1440
gtgctgaata aatgctttac ctggatgaaa aaaaaaaaaa a 1481
<210> 31
<211> 667
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2656554CB1
<400> 31
ctaaagtggc agtgtttctt ctgaaattct caggcagtca gactgtctta ggcaaatctt 60
gataaaatag cccttatcca ggtttttatc taaggaatcc caagaagact ggggaatgga 120
gagacagtca agggttatgt cagaaaagga tgagtatcag tttcaacatc agggagcggt 180
ggagctgctt gtcttcaatt ttttgctcat ccttaccatt ttgacaatct ggttatttaa 240
aaatcatcga ttccgcttct tgcatgaaac tggaggagca atggtgtatg acaagccgcc 300
gaaatttgcc atgtcacgag agcaaatgtc acagtcatgt tctcacacgg cacataatgc 360
aagtctgttg acagatgcgg gtccattgtc atgtggggag tcgagggcga gctgtttgtt 420
tttgtaatga tgttgggaag tgatggctct gcagtcacaa agagcagcct tctctcactg 480
gctgcaccga tgaacattac gaagttctag aaaaaacatc acttcaaaat gcctggagta 540
attcctctta tatcaactaa tttcaagaag aaaacttgca gaaactaacc ccacccctct 600
taagagaata ttgtgtccaa gtccttttta tttatacgaa cagtgtctta ttttcttata 660
667
atgaaat
<210> 32
<211> 1635
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2719228CB1
<400> 32
atagctgtct tgagccccaa gcctcttcct cccctgctgc ccctctgcag ccattcggga 60
tgggaccccc tctggggtgt cagcacgaaa gggctaacgg gagccccttc cttggcctcc 120
33
CA 02349818 2001-05-03
PCTNS99l26048
WO 00/26245
ccctgtaggt tacagagcca tcacggtgcg gaagctcatg cagggcatgg gccttggcct 180
ctccagcgtc tttgctctgt gcctgggcca cacctccagc ttctgtgagt ctgtggtctt 240
tgcatcagcc tccatcggcc tccagacctt caaccacagt ggcatttctg ttaa'catcca 300
ggacttggcc ccgtcctgcg ccggctttct gtttggtgtg gccaacacag ccggggcctt 360
ggcaggtgtc gtgggtgtgt gtctaggcgg ctacttgatg gagaccacgg gctcctggac 420
ttgcctgttc aaccttgtgg ccatcatcag caacctgggg ctgtgcacct tcctggtgtt 480
tggacaggct cagagggtgg acctgagctc tacccatgag gacctctagc tcccaacccc 540
acagcctctc caaggaccca ggcgccagca gccccgggac acaggggact cagtgtgtga 600
gacttggtca ctccatgtca gacacacgag cagagaggaa cacaaaccac tgtggagcct 660
gaagctcctt aagaagagtc cacaacagct ggtgggaggg tggggtgggc ctgggtccag 720
accaggctcg ctgctctctg ggcctcagtt tccccacctg ccagcgggct cggccctgtc 780
ctcctcacag gctggtgtgg ccgtcagggt gggtggggtt attgttagta ggcgcagcct 840
cattcccacc acgatctgtt ccgcgtggtt cccgccaaac ctccctcggt cgccgtgttc 900
tccgcaagcc tcctgcagcg cccgcctgcc aatgtgaggc tggcaccagg ctgcagcctc 960
cccaatccca gcccactttg ctgtgtctct ggcgggctgt cctccttggt gggagctgtc 1020
ctgcacactg taggatgctt aaaggtatcc ctggcctcca cccaccccta gccagcagct 1080
cccagtcaga caacagccag aaatgtctcc agactctgcc cagcctcccc aggtagccac 1140
cctcgagaca cgacctcaga gtctctgtgt ctcctagaag cctgacagag acccccaggg 1200
cagtgggtgg gtggcgggct agagaccctt gcctgtgtcc gggaccctgg cgccgctctc 1260
ccctcctgtg gatccctccg cactaacagt gttctcagtg ggcagacgcc tgggcacccc 1320
ttgggccctg cccagcatgg ccatggcgca ggctctcgaa cccgcatggc tttcccaggc 1380
ctggtgattc tgctctccag ggacggttgg caccttcctc gggggcgggc cccacgcacc 1440
ccagaacaca cagacccacc tttctggcgt tctttctacc tcccttttcg ttgcctgagg 1500
agctggtggt ttcatgagtt aatgatacat cttgcaaggt gtacacatag agaaaaaaac 1560
ctaaaaatgt ggaaaagcac gccaaagcct tatttaaata ataactatta aactattcaa 1620
1635
aaagaaaaaa aaaaa
<210> 33
<211> 1447
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3657824CB1
<400> 33
ccgagcggtg ccaggccagg tgtgtgcgtc cgtcggtctt tccgtgccca cgccggagac 60
cagccccgga ggccgcctgg gcctatccct gtgccaggca ccatgaagca ggagtctgca 120
gccccgaaca ccccgcccac ctcgcagtcc cctacgccgt ccgctcagtt cccccgaaac 180
gacggcgacc ctcaagcgct gtggattttc gggtacggct ccctggtgtg gaggcccgac 240
ttcgcctaca gcgacagccg tgtgggcttc gtgcgcggct acagccgccg tttctggcag 300
ggagacacct tccatcgggg cagcgacaag atgcctggcc gtgtggtgac gctccttgaa 360
gatcatgagg gctgcacttg gggcgtggca taccaagtgc aaggggagca ggtaagcaag 420
gccctgaagt acctgaatgt gcgagaggca gtgcttggtg gctacgatac caaggaggtc 480
accttctatc cccaagatgc tcctgaccaa ccactgaagg cattggccta tgtggccacc 540
ccacagaacc ctggttacct gggccctgcg cctgaagagg ccattgccac gcagatcctg 600
gcctgccggg gcttctccgg ccacaacctt gaatacttgc tgcgtctggc agacttcatg 660
cagctctgtg ggcctcaggc gcaggacgag cacctggcag ccatcgtgga cgctgtgggc 720
accatgttgc cctgcttctg ccccaccgag caggctctgg cgctggtgtg aggggctgag 780
cccctgcggg gagtgctcat gtggacatca gggccagaca cccactccag tgcacaagac 840
agacttgcga ccgcttgagc ccactgagca gatatggtgg gtggctggag gcttctcttt 900
ctcagtccct gcctgtctgc cagcctgcag ctctcctgct tgacactgac ttactacttg 960
aaactttatt tattgcacca tgttggtgtg gtgggcaggt ggagggcctg ccctggacac 1020
34
CA 02349818 2001-05-03
PCT/US99/26048
WO 00/26245
aggggccctg ctgagcagtg gccccatcct ggaacttgac cagattcccc ccagtgctgc 1080
tgctaacccc acaccaccca ggcctccacc tccccaggga gtctccaaga gcctcgatcc 1140
tctgctcact cagcccagcc atccatagcc ctgggaattc cacctgccaa ggatoccagc 1200
aggctggatg agggatagta gggcatgagg agaaggagcc ctgtaaggac tgaggccccg 1260
gccagccctt ctcctccacc agttccccag agcagagctg gagctgatgc ctggacacag 1320
ctgctgagcc tggcctgggc ctcttaccca cttggttgtt ttcttgtccc tctgtctgtc 1380
tgtctatcta cttgtctgtc tgggccactc ctgcctgtgt gttggtctat tcctgggaag 1440
1447
ctcatca
<210> 34
<211> 657
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5378485CB1
<400> 34
gactcctgtt gcgcatgctc agcgcgctgc ccggctgggg acccgcgcac ctgcagcgcc 60
cgctgctcgg ccctgcatcc tgcctgggca tcctgcgccc ggccatgacg gcgcactcat 120
tcgccctccc ggtcatcatc ttcaccacgt tctggggcct cgtcggcatc gccgggccct 180
ggttcgtgcc gaagggaccc aaccgcggag tgatcatcac catgctggtc gccaccgccg 240
tctgctgtta cctcttctgg ctcatcgcca tcctggcgca gctgaacccc ctgttcgggc 300
cccagctgaa gaatgagacc atctggtacg tgcgcttcct gtgggagtga cccgccgccc 360
ccgacccagg tgcccagctc tcggaatgac tgtggctcca ctgtccctga caaccccttc 420
gtccggaccc tcccccacac aactatgtct ggtcaccagc tccctcctgc tggcacccag 480
agacccggac ccgcaggccc tgcctggttc ctggaagtct tcccagtctt cccagccagc 540
ccggggccct ggggagccct gggcacagca gcggccgagg ggatgtcctg ctccaatact 600
cgcactgctc tggagtttgc actctttcgc aaggagatgc tgctggggag ctggtat 657
<210> 35
<211> 646
<212> PRT
<213> Mus musculus
<300>
<308> GenBank ID No: 82612939
<400> 35
Met Arg Ala Pro Gly Ala Gly Thr Ala Ser Val Ala Ser Leu Ala
5 10 15
Leu Leu Trp Phe Leu Gly Leu Pro Trp Thr Trp Ser Ala Ala Ala
20 25 30
Ala Phe Cys Val Tyr Val Gly Gly Gly Gly Trp Arg Phe Leu Arg
40 45
35
Ile Val Cys Lys Thr Ala Arg Arg Asp Leu Phe Gly Leu Ser Val
55 60
50
Leu Ile Arg Val Arg Leu Glu Leu Arg Arg His Arg Arg Ala Gly
70 75
65
Asp Thr Ile Pro Cys Ile Phe Gln Ala Val Ala Arg Arg Gln Pro
85 90
80
Glu Arg Leu Ala Leu Val Asp Ala Ser Ser Gly Ile Cys Trp Thr
100 105
95
35
CA 02349818 2001-05-03
PCTNS99/26048
WO 00/26245
Phe Ala Gln Leu Asp Thr Tyr Ser Asn Ala Val Ala Asn Leu Phe
110 115 120
Arg Gln Leu Gly Phe Ala Pro Gly Asp Val Va1 Ala Val Phe Leu
125 130 135
Glu Gly Arg Pro Glu Phe Val Gly Leu Trp Leu Gly Leu Ala Lys
140 145 150
Ala Gly Val Val Ala Ala Leu Leu Asn Val Asn Leu Arg Arg Glu
155 160 165
Pro Leu Ala Phe Cys Leu Gly Thr Ser Ala Ala Lys Ala Leu Ile
170 175 180
Tyr Gly Gly Glu Met Ala Ala Ala Val Ala Glu Val Ser Glu Gln
185 190 195
Leu Gly Lys Ser Leu Leu Lys Phe Cys Ser Gly Asp Leu Gly Pro
200 205 210
Glu Ser Ile Leu Pro Asp Thr Gln Leu Leu Asp Pro Met Leu Ala
215 220 225
Glu Ala Pro Thr Thr Pro Leu Ala Gln Ala Pro Gly Lys Gly Met
230 235 240
Asp Asp Arg Leu Phe Tyr Ile Tyr Thr Ser Gly Thr Thr Gly Leu
245 250 255
Pro Lys Ala Ala Ile Val Val His Ser Arg Tyr Tyr Arg Ile Ala
260 265 270
Ala Phe Gly His His Ser Tyr Ser Met Arg Ala Ala Asp Val Leu
275 280 285
Tyr Asp Cys Leu Pro Leu Tyr His Ser Ala Gly Asn Ile Met Gly
290 295 300
Val Gly Gln Cys Val Ile Tyr Gly Leu Thr Val Val Leu Arg Lys
305 310 315
Lys Phe Ser Ala Ser Arg Phe Trp Asp Asp Cys Val Lys Tyr Asn
320 325 330
Cys Thr Val Val Gln Tyr Ile Gly Glu Ile Cys Arg Tyr Leu Leu
335 340 345
Arg Gln Pro Val Arg Asp Val Glu Gln Arg His Arg Val Arg Leu
350 355 360
Ala Val Gly Asn Gly Leu Arg Pro Ala Ile Trp Glu Glu Phe Thr
365 370 375
Gln Arg Phe Gly Val Pro Gln Ile Gly Glu Phe Tyr Gly Ala Thr
380 385 390
Glu Cys Asn Cys Ser Ile Ala Asn Met Asp Gly Lys Val Gly Ser
395 400 405
Cys Gly Phe Asn Ser Arg Ile Leu Thr His Val Tyr Pro Ile Arg
410 415 420
Leu Val Lys Val Asn Glu Asp Thr Met Glu Pro Leu Arg Asp Ser
425 430 435
Glu Gly Leu Cys Ile Pro Cys Gln Pro Gly Glu Pro Gly Leu Leu
440 445 450
Val Gly Gln Ile Asn Gln Gln Asp Pro Leu Arg Arg Phe Asp Gly
455 460 465
Tyr Val Ser Asp Ser Ala Thr Asn Lys Lys Ile Ala His Ser Val
470 475 480
Phe Arg Lys Gly Asp Ser Ala Tyr Leu Ser Gly Asp Val Leu Val
485 490 495
Met Asp Glu Leu Gly Tyr Met Tyr Phe Arg Asp Arg Ser Gly Asp
500 505 510
Thr Phe Arg Trp Arg Gly Glu Asn Val Ser Thr Thr Glu Val Glu
36
CA 02349818 2001-05-03
pCT/US99l26048
WO 00/26245
515 520 525
Ala Val Leu Ser Arg Leu Leu Gly Gln Thr Asp Val Ala Va1 Tyr
530 535 540
Gly Val Ala Val Pro Gly Val Glu Gly Lys Ala Gly Met Ala Ala
545 550 555
Ile Ala Asp Pro His Ser Gln Leu Asp Pro Asn Ser Met Tyr Gln
560 565 570
Glu Leu Gln Lys Val Leu Ala Ser Tyr Ala Arg Pro Ile Phe Leu
575 580 585
Arg Leu Leu Pro Gln Val Asp Thr Thr Gly Thr Phe Lys Ile Gln
590 595 600
Lys Thr Arg Leu Gln Arg Glu Gly Phe Asp Pro Arg Gln Thr Ser
605 610 615
Asp Arg Leu Phe Phe Leu Asp Leu Lys Gln Gly Arg Tyr Val Pro
620 625 630
Leu Asp Glu Arg Val His Ala Arg Ile Cys Ala Gly Asp Phe Ser
640 645
635
Leu
<210> 36
<211> 691
<212> PRT
<213> Schistosoma mansoni
<300>
<308> GenBank ID No: g425474
<400> 36
Met Phe Ser Ala Leu Cys Arg Arg Gly Phe Leu Thr Asn Lys Val
10 15
Ser Gln Phe Arg Ser Thr Tyr Lys Cys Asp His Tyr Asn Leu Lys
20 25 30
Thr His Ile Lys Pro Leu Lys Cys Ser Ser Ser Leu Arg Leu Thr
35 40 45
Val Gly Thr Gly Leu Phe Ile Ala Leu His Ser Lys Ile Ser Pro
50 55 60
Glu Ser Arg Ile Gln Thr Val Gln Cys Glu Val Asp Ser Tyr Gln
65 70 75
Thr Asp Gln Ile Thr Phe Ala Lys Ser Gly Gly Ile Pro Arg Tyr
80 85 90
Ile Gly Val Leu Ile Leu Pro Asp Cys Val Tyr Leu Phe Gly Ala
95 100 105
Ile Leu Gly Ala Phe Val Ala Ala Val Met Asn Val Tyr Ile Pro
110 115 120
Leu Tyr Leu Gly Asp Phe Val Ser Ser Leu Ser Arg Cys Val Val
125 130 135
Thr His Glu Gly Phe Val Ser Ala Val Tyr Val Pro Thr Leu Arg
140 145 150
Leu Cys Ser Ser Tyr Leu Leu Gln Ser Leu Ser Thr Phe Leu Tyr
155 160 165
Ile Gly Leu Leu Gly Ser Val Gly Glu Arg Met Ala Arg Arg Met
170 175 180
Arg Ile Gln Leu Phe Arg Lys Leu Val Tyr Gln Asp Val Ala Tyr
190 195
185
37
CA 02349818 2001-05-03
PGTNS99/26048
WO 00126245
Phe Asp Val His Ser Ser Gly Lys Leu Val Glu Ile Ile Gly Ser
200 205 210
Asp Val Gln Asn Phe Lys Ser Ser Phe Lys Gln Cys Ile Ser Gln
215 220 225
Gly Leu Arg Asn Gly Ile Gln Val Val Gly Ser Val Phe Ala Leu
230 235 240
Leu Ser Ile Ser Pro Thr Leu Thr Ala Ala Leu Ile Gly Cys Leu
245 250 255
Pro Cys Val Phe Leu Ile Gly Ser Leu Met Gly Thr Glu Leu Arg
260 265 270
His Ile Ser Arg Glu Val Gln Ser Gln Asn Ser Leu Phe Ala Ser
275 280 285
Leu Ile Asp Glu Ala Phe Ser His Ile Arg Thr Val Lys Ser Leu
290 295 300
Ala Met Glu Asp Phe Leu Ile Asn Lys Ile Asn Tyr Asn Val Asp
305 310 315
Lys Ala Lys Met Leu Ser Glu Lys Leu Ser Phe Gly Ile Gly Ser
320 325 330
Phe Gln Gly Leu Ser Asn Leu Thr Leu Asn Gly Val Val Leu Gly
335 340 345
Val Leu Tyr Val Gly Gly His Leu Met Ser Arg Gly Glu Leu Asp
350 355 360
Ala Gly His Leu Met Ser Phe Leu Ala Thr Thr Gln Thr Leu Gln
365 370 3?5
Arg Ser Leu Thr Gln Leu Ser Leu Leu Tyr Gly Gln Val Val Arg
380 385 390
Gly Tyr Thr Ala Leu Lys Arg Ile His Asp Ile Leu Ala Leu Pro
395 400 405
Ser Gly Ile Gly Ser Ile Pro Ser Ser Ser Ser Ser Leu Val Val
410 415 420
Ser Lys Gln His Val Asn Asn Ile Lys Glu Leu Pro Ser Ser Ser
425 430 435
Ile Tyr Ser Ala Pro Ser Ile Glu Phe Ser Asp Val Lys Phe Ala
440 445 450
Tyr Pro Asn Arg Pro Glu Thr Ile Val Leu Asn Glu Leu Ser Met
455 460 465
Phe Leu Pro Gly Gly Lys Val Ile Ala Leu Val Gly Gln Ser Gly
470 4?5 480
Ala Gly Lys Ser Thr Val Val Ser Leu Leu Glu Arg Phe Tyr Asp
485 490 495
Pro Ile Ser Gly Glu Ile Leu Leu Asn Gly Asp Lys Leu Thr Asn
500 505 510
Phe Asn Val Asn Tyr Leu Arg Ser Lys Leu Ile Gly Tyr Ile Ser
515 520 525
Gln Glu Pro Gln Ile Phe Asn Ala Ser Ile Arg Glu Asn Ile Arg
530 535 540
Phe Gly Arg Phe Asp Ala Thr Asp Glu Glu Val Glu Glu Ala Ala
545 550 555
Lys Leu Ala Tyr Ala His Asp Phe Ile Ser Asn Asp Leu Pro Tyr
560 565 570
Gly Tyr Asp Thr Leu Val Gly Gln Gly Thr Gly Thr Ile Ala Gly
575 580 585
Leu Ser Gly Gly Gln Arg Gln Arg Ile Ala Ile Ala Arg Ile Leu
590 595 600
Leu Lys Asri Ala Pro Ile Leu Leu Met Asp Glu Ala Thr Ser Ala
38
CA 02349818 2001-05-03
PCTNS99/26048
WO 00/26245
605 610 615
Leu Asp Thr Glu Ser Glu Ala Lys Val Gln Asn Ala Leu Asn Asn_
620 625 630
A1a Met Lys Gly Arg Thr Val Leu Ile Ile Ala His Arg Leu Ser
635 640 645
Thr Val Arg Lys Ala Asp Leu Ile Leu Val Met Ser Lys Gly Gln
650 655 660
Ile Val Glu Lys Gly Thr His Ser Glu Leu Met Ala Asn His Gly
665 670 675
Tyr Tyr Tyr Asn Leu Val Gln Arg Gln Glu Gly Cys Asp Val Phe
685 690
680
Asp
<210> 37
<211> 634
<212> PRT
<213> Rattus norvegicus
<300>
<308> GenBank ID No: 83015617
<400> 37
Met Thr Val Ala Ser Thr Ala Ala Pro Ser Tyr Thr Thr Ser Asp
5 10 15
Thr Asn Arg Val Ile Ser Thr Phe Ser Val Val Asp Tyr Val Val
20 25 30
Phe Gly Leu Leu Leu Val Leu Ser Leu Val Ile Gly Leu Tyr His
35 40 45
Ala Cys Arg Gly Trp Gly Arg His Thr Val Gly Glu Leu Leu Met
50 55 60
Ala Asp Arg Lys Met Gly Cys Leu Pro Val Ala Leu Ser Leu Leu
65 70 75
Ala Thr Phe Gln Ser Ala Val Ala Ile Leu Gly Gly Pro Ala Glu
80 85 90
Ile Tyr Arg Phe Gly Thr Gln Tyr Trp Phe Leu Gly Cys Ser Tyr
95 100 105
Phe Leu Gly Leu Leu Ile Pro Ala His Ile Phe Ile Pro Val Phe
110 115 120
Tyr Arg Leu His Leu Thr Ser Ala Tyr Glu Tyr Leu Glu Leu Arg
125 130 135
Phe Asn Lys Ala Val Arg Ile Cys Gly Thr Val Thr Phe Ile Phe
140 145 150
Gln Met Val Val Tyr Met Gly Val Ala Leu Tyr Ala Pro Ser Leu
155 160 165
Ala Leu Asn Ala Val Thr Gly Phe Asp Leu Trp Leu Ser Val Leu
170 175 180
Ala Leu Gly Ile Val Cys Asn Ile Tyr Thr Ala Leu Gly Gly Leu
185 190 195
Lys Ala Val Ile Trp Thr Asp Val Phe Gln Thr Leu Ile Met Phe
200 205 210
Leu Gly Gln Leu Va1 Val Ile Ile Val Gly Ala Ala Lys Val Gly
215 220 225
Gly Leu Gly His Val Trp Ala Val Ala Ser Gln His Gly Leu Ile
235 240
230
39
CA 02349818 2001-05-03
pCT/US99/26048
WO 00/26245
Ser Gly Ile Glu Leu Asp Pro Asp Pro Phe Val Arg His Thr Phe
245 250 255
Trp Thr Leu Ala Phe Gly Gly Val Phe Met Met Leu Ser Leu Tyr
260 265 270
Gly Val Asn Gln Ala Gln Val Gln Arg Tyr Leu Ser Ser His Ser
275 280 285
Glu Lys Ala Ala Val Leu Ser Cys Tyr Ala Val Phe Pro Cys Gln
290 295 300
Gln Val Ala Leu Cys Met Sex Cys Leu Ile Gly Leu Val Met Phe
305 310 315
Ala Tyr Tyr Lys Lys Tyr Ser Met Ser Pro Gln Gln Glu Gln Ala
320 325 330
Ala Pro Asp Gln Leu Val Leu Tyr Phe Val Met Asp Leu Leu Lys
335 340 345
Asp Met Pro Gly Leu Pro Gly Leu Phe Val Ala Cys Leu Phe Ser
350 355 360
Gly Ser Leu Ser Thr Ile Ser Ser Ala Phe Asn Ser Leu Ala Thr
365 370 375
Val Thr Met Glu Asp Leu Ile Gln Pro Trp Phe Pro Gln Leu Thr
380 385 390
Glu Thr Arg Ala Ile Met Leu Ser Arg Ser Leu Ala Phe Ala Tyr
395 400 405
Gly Leu Val Cys Leu Gly Met Ala Tyr Val Ser Ser His Leu Gly
410 415 420
Ser Val Leu Gln Ala Ala Leu Ser Ile Phe Gly Met Val Gly Gly
425 430 435
Pro Leu Leu Gly Leu Phe Cys Leu Gly Met Phe Phe Pro Cys Ala
440 445 450
Asn Pro Leu Gly Ala Ile Val Gly Leu Leu Thr Gly Leu Thr Met
455 460 465
Ala Phe Trp Ile Gly Ile Gly Ser Ile Val Ser Arg Met Ser Ser
470 475 480
Ala Ala Ala Ser Pro Pro Leu Asn Gly Ser Ser Ser Phe Leu Pro
485 490 495
Ser Asn Leu Thr Val Ala Thr Val Thr Thr Leu Met Pro Ser Thr
500 505 510
Leu Ser Lys Pro Thr Gly Leu Gln Gln Phe Tyr Ser Leu Ser Tyr
515 520 525
Leu Trp Tyr Ser Ala His Asn Ser Thr Thr Val Ile Ala Val Gly
530 535 540
Leu Ile Val Ser Leu Leu Thr Gly Gly Met Arg Gly Arg Ser Leu
545 550 555
Asn Pro Gly Thr Ile Tyr Pro Val Leu Pro Lys Leu Leu Ala Leu
560 565 570
Leu Pro Leu Ser Cys Gln Lys Arg Leu Cys Trp Arg Ser His Asn
575 580 585
Gln Asp Ile Pro Val Val Thr Asn Leu Phe Pro Glu Lys Met Gly
590 595 600
Asn Gly Ala Leu Gln Asp Ser Arg Asp Lys Glu Arg Met Ala Glu
605 610 615
Asp Gly Leu Val His Gln Pro Cys Ser Pro Thr Tyr Ile Val Gln
625 630
620
Glu Thr Ser Leu
40
CA 02349818 2001-05-03
PCTNS99/Z6048
WO OOI26245
<210> 38
<211> 507
<212> PRT
<213> Homo Sapiens
<300>
<308> GenBank ID No: g3639058
<400> 38
Met Ala Gly Ala Gly Pro Lys Arg Arg Ala Leu Ala Ala Pro Ala
10 15
1 5
Ala Glu Glu Lys Glu Glu Ala Arg Glu Lys Met Leu Ala Ala Lys
20 25 30
Ser Ala Asp Gly Ser Ala Pro Ala Gly Glu Gly Glu Gly Val Thr
35 40 45
Leu Gln Arg Asn Ile Thr Leu Leu Asn Gly Val Ala Ile Ile Val
50 55 60
Gly Thr Ile Ile Gly Ser Gly Ile Phe Val Thr Pro Thr Gly Val
65 70 75
Leu Lys Glu A1a Gly Ser Pro Gly Leu Ala Leu Val Val Trp Ala
80 85 90
Ala Cys Gly Val Phe Ser Ile Val Gly Ala Leu Cys Tyr Ala Glu
95 100 105
Leu Gly Thr Thr Ile Ser Lys Ser Gly Gly Asp Tyr Ala Tyr Met
110 115 120
Leu Glu Val Tyr Gly Ser Leu Pro Ala Phe Leu Lys Leu Trp Ile
125 130 135
Glu Leu Leu Ile Ile Arg Pro Sex Ser Gln Tyr Ile Val Ala Leu
140 145 150
Val Phe Ala Thr Tyr Leu Leu Lys Pro Leu Phe Pro Thr Cys Pro
155 160 165
Val Pro Glu Glu Ala Ala Lys Leu Val Ala Cys Leu Cys Val Leu
170 175 180
Leu Leu Thr Ala Val Asn Cys Tyr Ser Val Lys Ala Ala Thr Arg
185 190 195
Val Gln Asp Ala Phe Ala Ala Ala Lys Leu Leu Ala Leu Ala Leu
200 205 210
Ile Ile Leu Leu Gly Phe Val Gln Ile Gly Lys Gly Asp Val Ser
215 220 225
Asn Leu Asp Pro Lys Phe Ser Phe Glu Gly Thr Lys Leu Asp Val
230 235 240
Gly Asn Ile Val Leu Ala Leu Tyr Ser Gly Leu Phe Ala Tyr Gly
245 250 255
Gly TrP ~n Tyr Leu Asn Phe Val Thr Glu Glu Met Ile Asn Pro
260 265 270
Tyr Arg Asn Leu Pro Leu Ala Ile Ile Ile Ser Leu Pro Ile Val
275 280 285
Thr Leu Val Tyr Val Leu Thr Asn Leu Ala Tyr Phe Thr Thr Leu
290 295 300
Ser Thr Glu Gln Met Leu Ser Ser Glu Ala Val Ala Val Asp Phe
305 310 315
Gly Asn Tyr His Leu Gly Val Met Ser Trp Ile Ile Pro Val Phe
320 325 330
Val Gly Leu Ser Cys Phe Gly Ser Val Asn Gly Ser Leu Phe Thr
41
CA 02349818 2001-05-03
PCTNS99IZ6048
WO OOI26245
335 340 345
Ser Ser Arg Leu Phe Phe Val Gly Ser Arg Glu Gly His Leu Pro
350 355 360
Ser Ile Leu Ser Met Ile His Pro Gln Leu Leu Thr Pro Val Pro
365 370 375
Ser Leu Val Phe Thr Cys Val Met Thr Leu Leu Tyr Ala Phe Ser
380 385 390
Lys Asp Ile Phe Ser Val Ile Asn Phe Phe Ser Phe Phe Asn Trp
395 400 405
Leu Cys Val Ala Leu Ala Ile IIe Gly Met Ile Trp Leu Arg His
410 415 420
Arg Lys Pro Glu Leu Glu Arg Pro Ile Lys Val Asn Leu Ala Leu
425 430 435
Pro Val Phe Phe Ile Leu Ala Cys Leu Phe Leu Ile Ala Val Ser
440 445 450
Phe Trp Lys Thr Pro Val Glu Cys Gly Ile Gly Phe Thr Ile Ile
455 460 465
Leu Ser Gly Leu Pro Val Tyr Phe Phe Gly Val Trp Trp Lys Asn
470 475 480
Lys Pro Lys Trp Leu Leu Gln Gly Ile Phe Ser Thr Thr Val Leu
485 490 495
Cys Gln Lys Leu Met Gln Val Val Pro Gln Glu Thr
500 505
<210> 39
<211> 504
<212> PRT
<213> Homo sapiens
<300>
<308> GenBank ID No: 81840045
<400> 39
Met Glu Ala Pro Leu Gln Thr Glu Met Val Glu Leu Val Pro Asn
5 10 15
Gly Lys His Ser Glu Gly Leu Leu Pro Val Ile Thr Pro Met Ala
20 25 30
Gly Asn Gln Arg Val Glu Asp Pro Ala Arg Ser Cys Met Glu Gly
35 40 45
Lys Ser Phe Leu Gln Lys Ser Pro Ser Lys Glu Pro His Phe Thr
50 55 60
Asp Phe Glu Gly Lys Thr Ser Phe Gly Met Ser Val Phe Asn Leu
65 70 75
Sex Asn Ala Ile Met Gly Ser Gly Ile Leu Gly Leu Ala Tyr Ala
80 85 90
Met Ala Asn Thr Gly Ile Ile Leu Phe Leu Phe Leu Leu Thr Ala
95 100 105
Val Ala Leu Leu Ser Ser Tyr Ser Ile His Leu Leu Leu Lys Ser
110 115 120
Ser Gly Val Val Gly Ile Arg Ala Tyr Glu Gln Leu Gly Tyr Arg
125 130 135
Ala Phe Gly Thr Pro Gly Lys Leu Ala Ala Ala Leu Ala Ile Thr
145 150
140
42
CA 02349818 2001-05-03
pCT/US99/26048
WO OOI26245
Leu Gln Asn Ile Gly Ala Met Ser Ser Tyr Leu Tyr Ile Ile Lys
155 160 ~ 165
Ser Glu Leu Pro Leu Val Ile Gln Thr Phe Leu Asn Leu Glu Glu
170 175 180
Lys Thr Ser Asp Trp Tyr Met Asn Gly Asn Tyr Leu Val Ile Leu
185 190 195
Val Ser Val Thr Ile Ile Leu Pro Leu Ala Leu Met Arg Gln Leu
200 205 210
Gly Tyr Leu Gly Tyr Ser Ser Gly Phe Ser Leu Ser Cys Met Val
215 220 225
Phe Phe Leu Ile Ala Val Ile Tyr Lys Lys Phe His Val Pro Cys
230 235 240
Pro Leu Pro Pro Asn Phe Asn Asn Thr Thr Gly Asn Phe Ser His
245 250 255
Val GIu Ile Val Lys Glu Lys Val Gln Leu Gln Val Glu Pro Glu
260 265 270
Ala Ser Ala Phe Cys Thr Pro Ser Tyr Phe Thr Leu Asn Ser Gln
275 280 285
Thr Ala Tyr Thr Ile Pro Ile Met Ala Phe Ala Phe Val Cys His
290 295 300
Pro Glu Val Leu Pro Ile Tyr Thr Glu Leu Lys Asp Pro Ser Lys
305 310 315
Lys Lys Met Gln His Ile Ser Asn Leu Ser Ile Ala Val Met Tyr
320 325 330
Ile Met Tyr Phe Leu Ala Ala Leu Phe Gly Tyr Leu Thr Phe Tyr
335 340 345
Asn Gly Val Glu Ser Glu Leu Leu His Thr Tyr Ser Lys Val Asp
350 355 360
Pro Phe Asp Val Leu Ile Leu Cys Val Arg Val Ala Val Leu Thr
365 370 375
Ala Val Thr Leu Thr Val Pro Ile Val Leu Phe Pro Val Arg Arg
380 385 390
Ala Ile Gln Gln Met Leu Phe Pro Asn Gln Glu Phe Ser Trp Leu
395 400 405
Arg His Val Leu Ile Ala Val Gly Leu Leu Thr Cys Ile Asn Leu
410 415 420
Leu Val Ile Phe Ala Pro Asn Ile Leu Gly Ile Phe GIy Val Ile
425 430 435
Gly Ala Thr Ser Ala Pro Phe Leu Ile Phe Ile Phe Pro Ala Ile
440 445 450
Phe Tyr Phe Arg Ile Met Pro Thr Glu Lys Glu Pro Ala Arg Ser
455 460 465
Thr Pro Lys Ile Leu Ala Leu Cys Phe Ala Met Leu Gly Phe Leu
470 475 480
Leu Met Thr Met Ser Leu Ser Phe Ile Ile Ile Asp Trp Ala Ser
490 495
485
Gly Thr Ser Arg His Gly Gly Asn His
500
<210> 40
<211> 393
<212> PRT
<213> Homo sapiens
43
CA 02349818 2001-05-03
PC'fNS99126048
WO 00/26245
<300>
<308> GenBank ID No: g1526438
<400> 40
Met Ala Ala Val Gly Ala Gly Gly Ser Thr Ala Ala Pro Gly Pro
1 5 10 15
Gly Ala Val Ser Ala Gly Ala Leu Glu Pro Gly Thr Ala Ser Ala
20 25 30
Ala His Arg Arg Leu Lys Tyr Ile Ser Leu Ala Val Leu Val Val
35 40 45
Gln Asn Ala Ser Leu Ile Leu Ser Ile Arg Tyr Ala Arg Thr Leu
50 55 60
Pro Gly Asp Arg Phe Phe Ala Thr Thr Ala VaI Val Met Ala Glu
65 70 75
Val Leu Lys Gly Leu Thr Cys Leu Leu Leu Leu Phe Ala Gln Lys
80 85 90
Arg Gly Asn Val Lys His Leu Val Leu Phe Leu His Glu Ala Val
95 100 105
Leu Val Gln Tyr Val Asp Thr Leu Lys Leu Ala Va1 Pro Ser Leu
110 115 120
Ile Tyr Thr Leu Gln Asn Asn Leu Gln Tyr Val Ala Ile Ser Asn
125 130 135
Leu Pro Ala Ala Thr Phe Gln Val Thr Tyr Gln Leu Lys Ile Leu
140 145 150
Thr Thr Ala Leu Phe Ser Val Leu Met Leu Asn Arg Ser Leu Ser
155 160 165
Arg Leu Gln Trp Ala Ser Leu Leu Leu Leu Phe Thr Gly Val Ala
170 175 180
Ile Val Gln Ala Gln Gln Ala Gly Gly Gly Gly Pro Arg Pro Leu
185 190 195
Asp Gln Asn Pro Gly Ala Gly Leu Ala Ala Val Val Ala Ser Cys
200 205 2I0
Leu Ser Ser Gly Phe Ala Gly Val Tyr Phe Glu Lys Ile Leu Lys
215 220 225
Gly Ser Ser Gly Ser Val Trp Leu Arg Asn Leu Gln Leu Gly Leu
230 235 240
Phe Gly Thr Ala Leu Gly Leu Val Gly Leu Trp Trp Ala Glu Gly
245 250 255
Thr Ala Val Ala Thr Arg Gly Phe Phe Phe Gly Tyr Thr Pro Ala
260 265 270
Val Trp Gly Val Val Leu Asn Gln Ala Phe Gly Gly Leu Leu Val
275 280 285
Ala Val Val Val Lys Tyr Ala Asp Asn Ile Leu Lys G1Y Phe Ala
290 295 300
Thr Ser Leu Ser Ile Val Leu Ser Thr Val Ala Ser Ile Arg Leu
305 310 315
Phe Gly Phe His Val Asp Pro Leu Phe Ala Leu Gly Ala Gly Leu
320 325 330
Val Ile Gly Ala Val Tyr Leu Tyr Ser Leu Pro Arg Gly Ala Ala
335 340 345
Lys Ala Ile Ala Sex Ala Ser Ala Ser Ala Ser Gly Pro Cys Val
350 355 360
His Gln Gln Pro Pro Gly Gln Pro Pro Pro Pro Gln Leu Sex Ser
365 370 375
His Arg Gly Asp Leu Ile Thr Glu Pro Phe Leu Pro Lys Ser Val
44
CA 02349818 2001-05-03
PCTIUS99/26048
WO 00/26245
385 390
380
Leu Val Lys '
<210> 41
<211> 893
<212> PRT
<213> Homo Sapiens
<300>
<3pg> GenBank ID No: g3335175
<400> 41
His Val Gln Asp Phe Thr Ala Phe Trp Asp Lys Ala Ser Glu Thr
5 10 15
Pro Thr Leu Gln Gly Leu Ser Phe Thr Val Arg Pro Gly Glu Leu
20 25 30
Leu Ala Val Val Gly Pro Val Gly Ala Gly Lys Ser Ser Leu Leu
35 40 45
Ser Ala Val Leu Gly Glu Leu Ala Pro Ser His Gly Leu Val Ser
50 55 60
Val His Gly Arg Ile Ala Tyr Val Ser Gln Gln Pro Trp Val Phe
65 ?0 75
Ser Gly Thr Leu Arg Ser Asn Ile Leu Phe Gly Lys Lys Tyr Glu
80 85 90
Lys Glu Arg Tyr Glu Lys Val Ile Lys Ala Cys Ala Leu Lys Lys
95 100 105
Asp Leu Gln Leu Leu Glu Asp Gly Asp Leu Thr Val Ile Gly Asp
110 115 120
Arg Gly Thr Thr Leu Ser Gly Gly Gln Lys Ala Arg Val Asn Leu
125 130 135
Ala Arg Ala Val Tyr Gln Asp Ala Asp Ile Tyr Leu Leu Asp Asp
140 145 150
Pro Leu Ser Ala Val Asp Ala Glu Val Ser Arg His Leu Phe Glu
155 160 165
Leu Cys Ile Cys Gln Ile Leu His Glu Lys Ile Thr Ile Leu Val
170 175 180
Thr His Gln Leu Gln Tyr Leu Lys Ala Ala Ser Gln Ile Leu Ile
185 190 195
Leu Lys Asp Gly Lys Met Val Gln Lys Gly Thr Tyr Thr Glu Phe
200 205 210
Leu Lys Ser Gly Ile Asp Phe Gly Ser Leu Leu Lys Lys Asp Asn
215 220 225
Glu Glu Ser Glu Gln Pro Pro Val Pro Gly Thr Pro Thr Leu Arg
230 235 240
Asn Arg Thr Phe Ser Glu Ser Ser Val Trp Ser Gln Gln Ser Ser
245 250 255
Arg Pro Ser Leu Lys Asp Gly Ala Leu Glu Ser Gln Asp Thr Glu
260 265 270
Asn Val Pro Val Thr Leu Ser Glu Glu Asn Arg Ser Glu Gly Lys
275 280 285
Val Gly Phe Gln Ala Tyr Lys Asn Tyr Phe Arg Ala Gly Ala His
290 295 300
Trp Ile Val Phe Ile Phe Leu Ile Leu Leu Asn Thr Ala Ala Gln
45
CA 02349818 2001-05-03
PCT/US99/26048
WO 00/26245
305 310 315
Val Ala Tyr Val Leu Gln Asp Trp Trp Leu Ser Tyr Trp Ala Asn
320 325 336
Lys Gln Ser Met Leu Asn Val Thr Val Asn Gly Gly Gly Asn Val
335 340 345
Thr Glu Lys Leu Asp Leu Asn Trp Tyr Leu Gly Ile Tyr Ser Gly
350 355 360
Leu Thr Val Ala Thr Val Leu Phe Gly Ile Ala Arg Ser Leu Leu
365 370 375
Val Phe Tyr Val Leu Val Asn Ser Ser Gln Thr Leu His Asn Lys
380 385 390
Met Phe GIu Ser Ile Leu Lys Ala Pro Val Leu Phe Phe Asp Arg
395 400 405
Asn Pro Ile Gly Arg Ile Leu Asn Arg Phe Ser Lys Asp Ile Gly
410 415 420
His Leu Asp Asp Leu Leu Pro Leu Thr Phe Leu Asp Phe Ile Glr1
425 430 435
Thr Leu Leu Gln Val Val Gly Val Val Ser Val Ala Val Ala Val
440 445 450
Ile Pro Trp Ile Ala Ile Pro Leu Val Pro Leu Gly Ile Ile Phe
455 460 465
Ile Phe Leu Arg Arg Tyr Phe Leu Glu Thr Ser Arg Asp Val Lys
470 475 480
Arg Leu Glu Sex Thr Thr Arg Ser Pro Val Phe Ser His Leu Ser
485 490 495
Ser Ser Leu Gln Gly Leu Trp Thr Ile Arg Ala Tyr Lys Ala Glu
500 505 510
Glu Arg Cys Gln Glu Leu Phe Asp Ala His Gln Asp Leu His Ser
515 520 525
Glu Ala Trp Phe Leu Phe Leu Thr Thr Ser Arg Trp Phe Ala Val
530 535 540
Arg Leu Asp Ala Ile Cys Ala Met Phe Val Ile Ile Val Ala Phe
545 550 555
Gly Ser Leu Ile Leu Ala Lys Thr Leu Asp Ala Gly Gln Val Gly
560 565 570
Leu Ala Leu Ser Tyr Ala Leu Thr Leu Met Gly Met Phe Gln Trp
575 580 585
Cys Val Arg Gln Ser Ala Glu Val Glu Asn Met Met Ile Ser Val
590 595 600
Glu Arg Val Ile Glu Tyr Thr Asp Leu Glu Lys Glu Ala Pro Trp
605 610 615
Glu Tyr Gln Lys Arg Pro Pro Pro Ala Trp Pro His Glu Gly Val
620 625 630
Ile Ile Phe Asp Asn Val Asn Phe Met Tyr Ser Pro Gly Gly Pro
635 640 645
Leu Val Leu Lys His Leu Thr Ala Leu Ile Lys Ser Gln Glu Lys
650 655 660
Val Gly Ile Val Gly Arg Thr Gly Ala Gly Lys Ser Ser Leu Ile
665 670 675
Ser Ala Leu Phe Arg Leu Ser Glu Pro Glu Gly Lys Ile Trp Ile
680 685 690
Asp Lys Ile Leu Thr Thr Glu Ile Gly Leu His Asp Leu Arg Lys
695 700 705
Lys Met Ser Ile Ile Pro Gln Glu Pro Val Leu Phe Thr Gly Thr
715 720
710
46
CA 02349818 2001-05-03
PCTNS99/26048
WO OOI26245
Met Arg Lys Asn Leu Asp Pro Phe Lys Glu His Thr Asp Glu Glu
725 730 735,
Leu Trp Asn Ala Leu Gln Glu Val Gln Leu Lys Glu Thr Ile Glu
740 745 750
Asp Leu Pro Gly Lys Met Asp Thr Glu Leu Ala Glu Ser Gly Ser
755 760 765
Asn Phe Ser Val Gly Gln Arg Gln Leu Val Cys Leu Ala Arg Ala
770 775 780
Ile Leu Arg Lys Asn Gln Ile Leu Ile Ile Asp Glu Ala Thr Ala
785 790 795
Asn Val Asp Pro Arg Thr Asp G1u Leu Ile Gln Lys Lys Ile Arg
800 805 810
Glu Lys Phe Ala His Cys Thr Val Leu Thr Ile Ala His Arg Leu
815 820 825
Asn Thr Ile Ile Asp Ser Asp Lys Ile Met Val Leu Asp Ser Gly
830 835 840
Arg Leu Lys Glu Tyr Asp Glu Pro Tyr Val Leu Leu Gln Asn Lys
845 850 855
Glu Ser Leu Phe Tyr Lys Met Val Gln Gln Leu Gly Lys Ala Glu
860 865 8?0
Ala Ala Ala Leu Thr Glu Thr Ala Lys Gln Val Ile Leu Gln Lys
880 885
875
Lys Leu Ser Thr Tyr Trp Ser His
890
<210> 42
<211> 453
<212> PRT
<213> Homo Sapiens
<300>
<308> GenBank ID No: g1279457
<400> 42
Met Ala Leu Arg Gly Phe Cys Ser Arg Trp Leu Arg Pro Ala Leu
10 15
Ala Ile Gly Leu Phe Ala Ser Met Ala Ala Val Leu Leu Gly Gly
20 25 30
Ala Arg Ala Ser Arg Leu Leu Phe Gln Arg Leu Leu Trp Asp Val
35 40 45
Val Arg Ser Pro Ile Ser Phe Phe Glu Arg Thr Pro Ile Gly His
50 55 60
Leu Leu Asn Arg Phe Ser Lys Glu Thr Asp Thr Val Asp Val Asp
65 70 75
Ile Pro Asp Lys Leu Arg Ser Leu Leu Met Tyr Ala Phe Gly Leu
80 85 90
Leu Glu Val Ser Leu Val Val Glu Trp Pro Thr Pro Leu Pro Leu
95 100 105
Trp Pro Ser Cys His Cys Phe Ser Ser Thr Leu Gly Phe Arg Trp
110 115 120
Leu Ala Ala Asn Val Glu Leu Leu Gly Asn Gly Leu Val Phe Ala
125 130 135
Ala Ala Thr Cys Ala Val Leu Ser Lys Ala His Leu Ser Ala Gly
145 150
140
47
CA 02349818 2001-05-03
PCTNS99/26048
WO 00/26245
Leu Val Gly Phe Ser Val Ser Ala Ala Leu Gln Val Thr Gln Thrr
155 160 165
Leu Gln Trp Val Val Arg Asn Trp Thr Asp Leu Glu Asn Ser Ile
170 175 180
Val Ser Val Glu Arg Met Gln Asp Tyr Ala Trp Thr Pro Lys Glu
185 190 195
Ala Pro Trp Arg Leu Pro Thr Cys Ala Ala Gln Pro Pro Trp Pro
200 205 210
Gln Gly Gly Gln Ile Glu Phe Arg Asp Phe Gly Leu Arg Tyr Arg
215 220 225
Pro Glu Leu Pro Leu Ala Val Gln Gly Val Ser Phe Lys Ile His
230 235 240
Ala Gly Glu Lys Val Gly Ile Val Gly Arg Thr Gly Ala Gly Lys
245 250 255
Ser Ser Leu Ala Ser Gly Leu Leu Arg Leu Gln Glu Ala Ala Glu
260 265 270
Gly Gly Ile Trp Ile Asp Gly Val Pro Ile Ala His Val Gly Val
275 280 285
His Thr Leu Arg Ser Arg Ile Ser Ile Ile Pro Gln Asp Pro Ile
290 295 300
Leu Phe Pro Gly Ser Leu Arg Met Asn Leu Asp Leu Leu Gln Glu
305 310 315
His Ser Asp Glu Ala Ile Trp Ala Ala Leu Glu Thr Val Gln Leu
320 325 330
Lys Ala Leu Val Ala Cys Leu Pro Gly Gln Leu Gln Tyr Lys Cys
335 340 345
Ala Asp Arg Gly Glu Asp Leu Ser Val Gly Gln Lys Gln Leu Leu
350 355 360
Cys Leu Ala Arg Ala Leu Leu Arg Lys Thr Gln Ile Leu Ile Leu
365 370 375
Asp Glu Ala Thr Ala Ala Val Asp Pro Gly Thr Glu Leu Gln Met
380 385 390
Gln Ala Met Leu Gly Ser Trp Phe Ala Gln Cys Thr Val Leu Leu
395 400 405
Ile Ala His Arg Leu Arg Ser Val Met Asp Cys Ala Arg Val Leu
410 415 420
Val Met Asp Lys Gly Gln Val Ala Glu Ser Gly Ser Pro Ala Gln
430 435
425
Leu Leu Ala Gln Lys Gly Leu Phe Tyr Arg Leu Ala Gln Glu Ser
445 450
440
Gly Leu Val
48
