Note: Descriptions are shown in the official language in which they were submitted.
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TRANSPORTERS AND ION CHANNELS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of
transporters and ion
channels and to the use of these sequences in the diagnosis, treatment, and
prevention of transport,
neurological, muscle, immunological and cell proliferative disorders, and in
the assessment of the
effects of exogenous compounds on the expression of nucleic acid and amino
acid sequences of
transporters and ion channels.
l0 BACKGROUND OF THE INVENTION
Eukaryotic cells are surrounded and subdivided into functionally distinct
organelles by
hydrophobic lipid bilayer membranes which are highly impermeable to most.polar
molecules. Cells and
organelles require transport proteins to import and export essential nutrients
and metal ions including
K+, NH4+, P;, SO42-, sugars, and vitamins, as well as various metabolic waste
products. Transport
proteins also 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). Transport
can occur by a passive concentration-dependent mechanism, or can be linked to
an energy source
such as ATP hydrolysis or an ion gradient. Proteins that function in transport
include carrier proteins,
which bind to a specific solute and undergo a conformational change that
translocates the bound solute
across the membrane, and channel proteins, which form hydrophilic pores that
allow specific solutes to
diffuse through the membrane down an electrochemical solute gradient.
Carner 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
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 driven by the sodium gradient that exists across the plasma
membrane. Sodium
moves into the cell down its electrochemical gradient and brings the solute
into the cell with it. The
sodium gradient that provides the driving force for solute uptake is
maintained by the ubiquitous
Na+/K+ ATPase system. Sodium-coupled transporters include the mammalian
glucose transporter
(SGLT1), iodide transporter (NIS), and multivitamin transporter (SMUT). All
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
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and for specific targeting of radioisotopes to the thyroid 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).
One of the largest families of transporters is the major facilitator
superfamily (MFS), also
called the uniporter-symporter-antiporter family. MFS transporters are single
polypeptide Garners that
transport small solutes in response to ion gradients. Members of the MFS are
found in all classes of
living organisms, and include transporters for sugars, oligosaccharides,
phosphates, nitrates,
nucleosides, monocarboxylates, and drugs. MFS transporters found in eukaryotes
all have a structure
comprising 12 transmembrane segments (Pao, S.S. et al. (1998) Microbiol.
Molec. Biol. Rev. 62:1-34).
The largest family of MFS transporters is the sugar transporter family, which
includes the seven
glucose transporters (GLUT1-GLUT7) found in humans that are required for the
transport of glucose
and other hexose sugars. These glucose transport proteins have unique tissue
distributions and
physiological functions. GLUTI provides many cell types with their basal
glucose requirements and
transports glucose across epithelial and endothelial barrier tissues; GLUT2
facilitates glucose uptake
or efflux from the liver; GLUT3 regulates glucose supply to neurons; GLUT4 is
responsible for insulin-
regulated glucose disposal; and GLUTS regulates fructose uptake into skeletal
muscle. Defects in
glucose transporters are involved in a recently identified neurological
syndrome causing infantile
seizures and developmental delay, as well as glycogen storage disease, Fanconi-
Bickel syndrome, and
non-insulin-dependent diabetes mellitus (Mueckler, M. (1994) Eur. J. Biochem.
219:713-725; Longo,
N. and L.J. Elsas (1998) Adv. Pediatr. 45:293-313).
Monocarboxylate anion transporters are proton-coupled symporters with a broad
substrate
specificity that includes L-lactate, pyruvate, and the ketone bodies acetate,
acetoacetate, and
beta-hydroxybutyrate. At least seven isoforms have been identified to date.
The isoforms are
predicted to have twelve transmembrane (TM) helical domains with a large
intracellular loop between
TM6 and TM7, and play a critical role in maintaining intracellular pH by
removing the protons that are
produced stoichiometrically with lactate during glycolysis. The best
characterized
H+-monocarboxylate transporter is that of the erythrocyte membrane, which
transports L-lactate and a
wide range of other aliphatic monocarboxylates. Other cells possess H+-linked
monocarboxylate
transporters with differing substrate and inhibitor selectivities. In
particular, cardiac muscle and tumor
cells have transporters that differ in their K", values for certain
substrates, including stereoselectivity
for L- over D-lactate, and in their sensitivity to inhibitors. There are Na+-
monocarboxylate
cotransporters on the luminal surface of intestinal and kidney epithelia,
which allow the uptake of
lactate, pyruvate, and ketone bodies in these tissues. In addition, there are
specific and selective
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transporters for organic cations and organic anions in organs including the
kidney, intestine and liver.
Organic anion transporters are selective for hydrophobic, charged molecules
with electron-attracting
side groups. Organic cation transporters, such as the ammonium transporter,
mediate the secretion of
a variety of drugs and endogenous metabolites, and contribute to the
maintenance of intercellular pH
(Poole, R.C. and A.P. Halestrap (1993) Am. J. Physiol. 264:C761-C782; Price,
N.T. et al. (1998)
Biochem. J. 329:321-328; and Martinelle, K. and I. Haggstrom (1993) J.
Biotechnol. 30:339-350).
ATP-binding cassette (ABC) transporters are members of a superfamily of
membrane
proteins that transport substances ranging from small molecules such as ions,
sugars, amino acids,
peptides, and phospholipids, to lipopeptides, large proteins, and complex
hydrophobic drugs. ABC
transporters consist of four modules: two nucleotide-binding domains (NBD),
which hydrolyze ATP to
supply the energy required for transport, and two membrane-spanning domains
(MSD), each
containing six putative transmembrane segments. These four modules may be
encoded by a single
gene, as is the case for the cystic fibrosis transmembrane regulator (CFTR),
or by separate genes.
When encoded by separate genes, each gene product 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. Several genetic
diseases are attributed to
defects in ABC transporters, such as the following diseases and their
corresponding proteins: cystic
fibrosis (CFTR, an ion channel), adrenoleukodystrophy (adrenoleukodystrophy
protein, ALDP),
Zellweger syndrome (peroxisomal membrane protein-70, PMP70), and
hyperinsulinemic hypoglycemia
(sulfonylurea receptor, SUR). Overexpression of the multidrug resistance (MDR)
protein, another
ABC transporter, in human cancer cells makes the cells resistant to a variety
of cytotoxic drugs used
in chemotherapy (Taglicht, D. and S. Michaelis (1998) Meth. Enzymol. 292:130-
162).
A number of metal ions such as iron, zinc, copper, cobalt, manganese,
molybdenum, selenium,
nickel, and chromium are important as cofactors for a number of enzymes. For
example, copper is
involved in hemoglobin synthesis, connective tissue metabolism, and bone
development, by acting as a
cofactor in oxidoreductases such as superoxide dismutase, ferroxidase
(ceruloplasmin), and lysyl
oxidase. Copper and other metal ions must be provided in the diet, and are
absorbed by transporters in
the gastrointestinal tract. Plasma proteins transport the metal ions to the
liver and other target organs,
where specific transporters move the ions into cells and cellular organelles
as needed. Imbalances in
metal ion metabolism have been associated with a number of disease states
(banks, D.M. (1986) J.
Med. Genet. 23:99-106).
P-type ATPases comprise a class of canon-transporting transmembrane proteins.
They are
integral membrane proteins which use an aspartyl phosphate intermediate to
move cations across a
membrane. Features of P-type ATPases include: (i) a cation channel; (ii) a
stalk, formed by
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extensions of the transmembrane a-helices into the cytoplasm; (iii) an ATP
binding domain; (iv) a
phosphorylated aspartic acid; (v) an adjacent transduction domain; (vi) a
phosphatase domain, which
removes the phosphate from the aspartic acid as part of the reaction cycle;
and (vii) six or more
transmembrane domains. Included in this class are heavy metal-transporting
ATPases as well as
aminophospholipid transporters.
The transport of phosphatidylserine and phosphatidylethanolamine by
aminophospholipid
translocase results in the movement of these molecules from one side of a
bilayer to another. This
transport is conducted by a newly identified subfamily of P-type ATPases which
are proposed to be
amphipath transporters. Amphipath transporters move molecules having both a
hydrophilic and a
hydrophobic region. As many as seventeen different genes belong to this P-type
ATPases subfamily,
being grouped into several distinct classes and subclasses (Halleck, M.S. et
al., (1999) Physiol.
Genomics 1:139-150; Vulpe,C. et al., (1993) Nat. Genet. 3:7-13).
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 uptake of long-chain
fatty acids (Hui, T.Y. et
al. (1998) J. Biol. Chem. 273:27420-27429).
The lipocalin superfamily constitutes a phylogenetically conserved group of
more than forty
proteins that function as extracellular ligand-binding proteins which bind and
transport small
hydrophobic molecules. Members of this family function as Garners of
retinoids, odorants,
chromophores, pheromones, allergens, and sterols, and in a variety of
processes including nutrient
transport, cell growth regulation, immune response, and prostaglandin
synthesis. A subset of these
proteins may be multifunctional, serving as either a biosynthetic enzyme or as
a specific enzyme
inhibitor. (Tanaka, T. et al. (1997) J. Biol. Chem. 272:15789-15795; and van't
Hof, W. et al. (1997) J.
Biol. Chem. 272:1837-1841.)
Members of the lipocalin family display unusually low levels of overall
sequence conservation.
Pairwise sequence identity often falls below 20%. Sequence similarity between
family members is
limited to conserved cysteines which form disulfide bonds and three motifs
which form a juxtaposed
cluster that functions as a target cell recognition site. The lipocalins share
an eight stranded, anti-
parallel beta-sheet which folds back on itself to form a continuously hydrogen-
bonded beta-barrel.
The pocket formed by the barrel functions as an internal ligand binding site.
Seven loops (L1 to L7)
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h ' a::::~ !~. ~ .!::= ~:_-,.-.;e--::e-: --
form short beta-hairpins, except loop L1 which is a large omega loop that
forms a lid to partially close
the internal ligand-binding site (Flower (1996) Biochem. J. 318:1-14).
Lipocalins are important transport molecules. Each lipocalin associates with a
particular
ligand and delivers that ligand to appropriate target sites within the
organism. Retinol-binding protein
(RBP), one of the best characterized lipocalins, transports retinol from
stores within the liver to target
tissues. Apolipoprotein D (apo D), a component of high density lipoproteins
(HDLs) and low density
lipoproteins (LDLs), functions in the targeted collection and delivery of
cholesterol throughout the
body. Lipocalins are also involved in cell regulatory processes. Apo D, which
is identical to gross-
cystic-disease-fluid protein (GCDFP)-24, is a progesterone/pregnenolone-
binding protein expressed at
high levels in breast cyst fluid. Secretion of apo D in certain human breast
cancer cell lines is
accompanied by reduced cell proliferation and progression of cells to a more
differentiated phenotype.
Similarly, apo D and another lipocalin, a,-acid glycoprotein (AGP), are
involved in nerve cell
regeneration. AGP is also involved in anti-inflammatory and immunosuppressive
activities. AGP is
one of the positive acute-phase proteins (APP); circulating levels of AGP
increase in response to
stress and inflammatory stimulation. AGP accumulates at sites of inflammation
where it inhibits
platelet and neutrophil activation and inhibits phagocytosis. The
immunomodulatory properties of AGP
are due to glycosylation. AGP is 40% carbohydrate, making it unusually acidic
and soluble. The
glycosylation pattern of AGP changes during acute-phase response, and
deglycosylated AGP has no
immunosuppressive activity (Flower (1994) FEBS Lett. 354:7-11; Flower (1996)
supra).
The lipocalin superfamily also includes several animal allergens, including
the mouse major
urinary protein (mMUP), the rat a-2-microgloobulin (rA2U), the bovine (3-
lactoglobulin ((31g), the
cockroach allergen (Bla g4), bovine dander allergen (Bos d2), and the major
horse allergen, designated
Equus caballus allergen 1 (Equ c 1 ). Equ c 1 is a powerful allergen
responsible for about 80% of anti-
horse IgE antibody response in patients who are chronically exposed to horse
allergens. It appears
that lipocalins may contain a common structure that is able to induce the IgE
response (Gregoire, C. et
al., (1996) J. Biol. Chem. 271:32951-32959).
Lipocalins are used as diagnostic and prognostic markers in a variety of
disease states. The
plasma level of AGP is monitored during pregnancy and in diagnosis and
prognosis of conditions
including cancer chemotherapy, renal disfunction, myocardial infarction,
arthritis, and multiple
sclerosis. RBP is used clinically as a marker of tubular reabsorption in the
kidney, and apo D is a
marker in gross cystic breast disease (Flower (1996) supra). Additionally, the
use of lipocalin animal
allergens may help in the diagnosis of allergic reactions to horses (Gregoire
supra), pigs, cockroaches,
mice and rats.
Mitochondrial carrier proteins are transmembrane-spanning proteins which
transport ions and
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charged metabolites between the cytosol and the mitochondrial matrix. Examples
include the ADP,
ATP Garner protein; the 2-oxoglutarate/malate carrier; the phosphate carrier
protein; the pyruvate
Garner; the dicarboxylate carrier which transports malate, succinate,
fumarate, and phosphate; the
tricarboxylate carrier which transports citrate and malate; and the Grave's
disease carrier protein, a
protein recognized by IgG in patients with active Grave's disease, an
autoimmune disorder resulting in
hyperthyroidism. Proteins in this family consist of three tandem repeats of an
approximately 100
amino acid domain, each of which contains two transmembrane regions (Stryer,
L. (1995)
Biochemistry, W.H. Freeman and Company, New York NY, p. 551; PROSITE PDOC00189
Mitochondrial energy transfer proteins signature; Online Mendelian Inheritance
in Man (OMIM)
l0 *275000 Graves Disease).
This class of transporters also includes the mitochondrial uncoupling
proteins, which create
proton leaks across the inner mitochondrial membrane, thus uncoupling
oxidative phosphorylation from
ATP synthesis. The result is energy dissipation in the form of heat.
Mitochondrial uncoupling proteins
have been implicated as modulators of thermoregulation and metabolic rate, and
have been proposed
as potential targets for drugs against metabolic diseases such as obesity
(Ricquier, D. et al. (1999) J.
Int. Med. 245:637-642).
Ion Channels
The electrical potential of a cell is generated and maintained by controlling
the movement of
ions across the plasma membrane. The movement of ions requires ion channels,
which form ion-
selective pores within the membrane. There are two basic types of ion
channels, ion transporters and
gated ion channels. Ion transporters utilize the energy obtained from ATP
hydrolysis to actively
transport an ion against the ion's concentration gradient. Gated ion channels
allow passive flow of an
ion down the ion's electrochemical gradient under restricted conditions.
Together, these types of ion
channels generate, maintain, and utilize an electrochemical gradient that is
used in 1) electrical impulse
conduction down the axon of a nerve cell, 2) transport of molecules into cells
against concentration
gradients, 3) initiation of muscle contraction, and 4) endocrine cell
secretion.
Ion Transporters
Ion transporters generate and maintain the resting electrical potential of a
cell. Utilizing the
energy derived from ATP hydrolysis, they transport ions against the ion's
concentration gradient.
These transmembrane ATPases are divided into three families. The
phosphorylated (P) class ion
transporters, including Na+-K+ ATPase, Caz+-ATPase, and H+-ATPase, are
activated by a
phosphorylation event. P-class ion transporters are responsible for
maintaining resting potential
distributions such that cytosolic concentrations of Na+ and Ca2+ are low and
cytosolic concentration of
K+ is high. The vacuolar (V) class of ion transporters includes H+ pumps on
intracellular organelles,
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such as lysosomes and Golgi. V-class ion transporters are responsible for
generating the low pH
within the lumen of these organelles that is required for function. The
coupling factor (F) class
consists of H+ pumps in the mitochondria. F-class ion transporters utilize a
proton gradient to generate
ATP from ADP and inorganic phosphate (P;).
The P-ATPases are hexamers of a 100 kD subunit with ten transmembrane domains
and
several large cytoplasmic regions that may play a role in ion binding
(Scarborough, G.A. (1999) Curr.
Opin. Cell Biol. 11:517-522). P-type ATPases use an aspartyl phosphate
intermediate to move cations
across a membrane. Features of P-type ATPases include: (i) a cation channel;
(ii) a stalk, formed by
extensions of the transmembrane a-helices into the cytoplasm; (iii) an ATP
binding domain; (iv) a
phosphorylated aspartic acid; (v) an adjacent transduction domain; (vi) a
phosphatase domain, which
removes the phosphate from the aspartic acid as part of the reaction cycle;
and (vii) six or more
transmembrane domains. Included in this class are heavy metal-transporting
ATPases as well as
aminophospholipid transporters. The FIC1 gene encodes a P-type ATPase that is
mutated in two
forms of hereditary cholestasis. The protein product of FIC1 is likely to play
an essential role in bile
acid circulation in the liver (Bull, L.N. et al. (1998) Nat. Genet. 18:219-
224). The V-ATPases are
composed of two functional domains: the V, domain, a peripheral complex
responsible for ATP
hydrolysis; and the Vo domain, an integral complex responsible for proton
translocation across the
membrane. The F-ATPases are structurally and evolutionarily related to the V-
ATPases. The F-
ATPase Fo domain contains 12 copies of the c subunit, a highly hydrophobic
protein composed of two
transmembrane domains and containing a single buried carboxyl group in TM2
that is essential for
proton transport. The V-ATPase Vo domain contains three types of homologous c
subunits with four
or five transmembrane domains and the essential carboxyl group in TM4 or TM3.
Both types of
complex also contain a single a subunit that may be involved in regulating the
pH dependence of
activity (Forgac, M. (1999) J. Biol. Chem. 274:12951-12954).
The resting potential of the cell is utilized in many processes involving
carrier proteins and
gated ion channels. Carner proteins utilize the resting potential to transport
molecules into and out of
the cell. Amino acid and glucose transport into many cells is linked to sodium
ion co-transport
(symport) so that the movement of Na+ down an electrochemical gradient drives
transport of the other
molecule up a concentration gradient. Similarly, cardiac muscle links transfer
of Ca2+ out of the cell
with transport of Na+ into the cell (antiport).
Gated Ion Channels
Gated ion channels control ion flow by regulating the opening and closing of
pores. The ability
to control ion flux through various gating mechanisms allows ion channels to
mediate such diverse
signaling and homeostatic functions as neuronal and endocrine signaling,
muscle contraction,
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fertilization, and regulation of ion and pH balance. Gated ion channels are
categorized according to
the manner of regulating the gating function. Mechanically-gated channels open
their pores in
response to mechanical stress; voltage-gated channels (e.g., Na+, K+, Ca2+,
and CI-channels) open
their pores in response to changes in membrane potential; and ligand-gated
channels (e.g.,
acetylcholine-, serotonin-, and glutamate-gated cation channels, and GABA- and
glycine-gated
chloride channels) open their pores in the presence of a specific ion,
nucleotide, or neurotransmitter.
The gating properties of a particular ion channel (i.e., its threshold for and
duration of opening and
closing) are sometimes modulated by association with auxiliary channel
proteins and/or post
translational modifications, such as phosphorylation.
l0 Mechanically-gated or mechanosensitive ion channels act as transducers for
the senses of
touch, hearing, and balance, and also play important roles in cell volume
regulation, smooth muscle
contraction, and cardiac rhythm generation. A stretch-inactivated channel
(SIC) was recently cloned
from rat kidney. The SIC channel belongs to a group of channels which are
activated by pressure or
stress on the cell membrane and conduct both Caz+ and Na+ (Suzuki, M. et al.
(1999) J. Biol. Chem.
274:6330-6335).
The pore-forming subunits of the voltage-gated cation channels form a
superfamily of ion
channel proteins. The characteristic domain of these channel proteins
comprises six transmembrane
domains (S1-S6), a pore-forming region (P) located between SS and S6, and
intracellular amino and
carboxy termini. In the Na+ and Ca2+ subfamilies, this domain is repeated four
times, while in the K+
channel subfamily, each channel is formed from a tetramer of either identical
or dissimilar subunits.
The P region contains information specifying the ion selectivity for the
channel. In the case of K+
channels, a GYG tripeptide is involved in this selectivity (Ishii, T.M. et al.
(1997) Proc. Natl. Acad.
Sci. USA 94:11651-11656).
Voltage-gated Na+ and K+ channels are necessary for the function of
electrically excitable
cells, such as nerve and muscle cells. Action potentials, which lead to
neurotransmitter release and
muscle contraction, arise from large, transient changes in the permeability of
the membrane to Na+
and K+ ions. Depolarization of the membrane beyond the threshold level opens
voltage-gated Na+
channels. Sodium ions flow into the cell, further depolarizing the membrane
and opening more
voltage-gated Na+ channels, which propagates the depolarization down the
length of the cell.
Depolarization also opens voltage-gated potassium channels. Consequently,
potassium ions flow
outward, which leads to repolarization of the membrane. Voltage-gated channels
utilize charged
residues in the fourth transmembrane segment (S4) to sense voltage change. The
open state lasts
only about 1 millisecond, at which time the channel spontaneously converts
into an inactive state that
cannot be opened irrespective of the membrane potential. Inactivation is
mediated by the channel's
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N-terminus, which acts as a plug that closes the pore. The transition from an
inactive to a closed state
requires a return to resting potential.
Voltage-gated Na+ channels are heterotrimeric complexes composed of a 260 kDa
pore-
forming a subunit that associates with two smaller auxiliary subunits, ail and
~i2. The (32 subunit is a
integral membrane glycoprotein that contains an extracellular Ig domain, and
its association with a and
ail subunits correlates with increased functional expression of the channel, a
change in its gating
properties, as well as an increase in whole cell capacitance due to an
increase in membrane surface
area (Isom, L.L. et al. (1995) Cell 83:433-442).
Non voltage-gated Na+ channels include the members of the amiloride-sensitive
Na+
channel/degenerin (NaC/DEG) family. Channel subunits of this family are
thought to consist of two
transmembrane domains flanking a long extracellular loop, with the amino and
carboxyl termini located
within the cell. The NaC/DEG family includes the epithelial Na+ channel (ENaC)
involved in Na+
reabsorption in epithelia including the airway, distal colon, cortical
collecting duct of the kidney, and
exocrine duct glands. Mutations in ENaC result in pseudohypoaldosteronism type
1 and Liddle's
syndrome (pseudohyperaldosteronism). The NaC/DEG family also includes the
recently characterized
H+-gated canon channels or acid-sensing ion channels (ASIC). ASIC subunits are
expressed in the
brain and form heteromultimeric Na+-permeable channels. These channels require
acid pH
fluctuations for activation. ASIC subunits show homology to the degenerins, a
family of mechanically-
gated channels originally isolated from C. elegans. Mutations in the
degenerins cause
neurodegeneration. ASIC subunits may also have a role in neuronal function, or
in pain perception,
since tissue acidosis causes pain (Waldmann, R. and M. Lazdunski (1998) Curr.
Opin. Neurobiol.
8:418-424; Eglen, R.M. et al. (1999) Trends Pharmacol. Sci. 20:337-342).
K' channels are located in all cell types, and may be regulated by voltage,
ATP concentration,
or second messengers such as Caz+ and cAMP. In non-excitable tissue, K+
channels are involved in
protein synthesis, control of endocrine secretions, and the maintenance of
osmotic equilibrium across
membranes. In neurons and other excitable cells, in addition to regulating
action potentials and
repolarizing membranes, K+ channels are responsible for setting the resting
membrane potential. The
cytosol contains non-diffusible anions and, to balance this,net negative
charge, the cell contains a Na+-
K+ pump and ion channels that provide the redistribution of Na+, K+, and Cl-.
The pump actively
transports Na+ out of the cell and K+ into the cell in a 3:2 ratio. Ion
channels in the plasma membrane
allow K+ and CI - to flow by passive diffusion. Because of the high negative
charge within the cytosol,
Cl- flows out of the cell. The flow of K+ is balanced by an electromotive
force pulling K+ into the cell,
and a K+ concentration gradient pushing K+ out of the cell. Thus, the resting
membrane potential is
primarily regulated by K+flow (Salkoff, L. and T. Jegla (1995) Neuron 15:489-
492).
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Potassium channel subunits of the Shaker-like superfamily all have the
characteristic six
transmembrane/1 pore domain structure. Four subunits combine as homo- or
heterotetramers to form
functional K channels. These pore-forming subunits also associate with various
cytoplasmic ~3
subunits that alter channel inactivation kinetics. The Shaker-like channel
family includes the voltage-
s gated K+ channels as well as the delayed rectifier type channels such as the
human ether-a-go-go
related gene (HERG) associated with long QT, a cardiac dysrythmia syndrome
(Curran, M.E. (1998)
Curr. Opin. Biotechnol. 9:565-572; Kaczorowski, G.J. and M.L. Garcia (1999)
Curr. Opin. Chem.
Biol. 3:448-458).
A second superfamily of K* channels is composed of the inward rectifying
channels (Kir).
Kir channels have the property of preferentially conducting K+ currents in the
inward direction. These
proteins consist of a single potassium selective pore domain and two
transmembrane domains, which
correspond to the fifth and sixth transmembrane domains of voltage-gated K+
channels. Kir subunits
also associate as tetramers. The Kir family includes ROMK1, mutations in which
lead to Banter
syndrome, a renal tubular disorder. Kir channels are also involved in
regulation of cardiac pacemaker
activity, seizures and epilepsy, and insulin regulation (Doupnik, C.A. et al.
(1995) Curr. Opin.
Neurobiol. 5:268-277; Curran, supra).
The recently recognized TWIK K+ channel family includes the mammalian TWIK-1,
TREK-1
and TASK proteins. Members of this family possess an overall structure with
four transmembrane
domains and two P domains. These proteins are probably involved in controlling
the resting potential
in a large set of cell types (Duprat, F. et al. (1997) EMBO J 16:5464-5471).
The voltage-gated Ca2+ channels have been classified into several subtypes
based upon their
electrophysiological and pharmacological characteristics. L-type Ca 2+
channels are predominantly
expressed in heart and skeletal muscle where they play an essential role in
excitation-contraction
coupling. T-type channels are important for cardiac pacemaker activity, while
N-type and P/Q-type
channels are involved in the control of neurotransmitter release in the
central and peripheral nervous
system. The L-type and N-type voltage-gated Ca 2+ channels have been purified
and, though their
functions differ dramatically, they have similar subunit compositions. The
channels are composed of
three subunits. The a, subunit forms the membrane pore and voltage sensor,
while the a28 and (3
subunits modulate the voltage-dependence, gating properties, and the current
amplitude of the channel.
These subunits are encoded by at least six a,, one a28, and four (3 genes. A
fourth subunit, y, has
been identified in skeletal muscle (Walker, D. et al. (1998) J. Biol. Chem.
273:2361-2367; McCleskey,
E.W. (1994) Curr. Opin. Neurobiol. 4:304-312).
The high-voltage-activated Ca Z+ channels that have been characterized
biochemically include
complexes of a pore-forming alphal subunit of approximately 190-250 kDa; a
transmembrane
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complex of alpha2 and delta subunits; an intracellular beta subunit; and in
some cases a
transmembrane gamma subunit. A variety of alphal subunits, alpha2delta
complexes, beta subunits,
and gamma subunits are known. The Cavl family of alphal subunits conduct L-
type Ca Z+ currents,
which initiate muscle contraction, endocrine secretion, and gene
transcription, and are regulated
primarily by second messenger-activated protein phosphorylation pathways. The
Cav2 family of
alphal subunits conduct N-type, P/Q-type, and R-type Ca Z+ currents, which
initiate rapid synaptic
transmission and are regulated primarily by direct interaction with G proteins
and SNARE proteins and
secondarily by protein phosphorylation. The Cav3 family of alphal subunits
conduct T-type Ca 2+
currents, which are activated and inactivated more rapidly and at more
negative membrane potentials
than other Ca z+ current types. The distinct structures and patterns of
regulation of these three
families of Ca 2+ channels provide an array of Ca 2+ entry pathways in
response to changes in
membrane potential and a range of possibilities for regulation of Ca 2+ entry
by second messenger
pathways and interacting proteins (Catterall, W.A. (2000) Annu. Rev. Cell Dev.
Biol. 16:521-555).
The alpha-2 subunit of the voltage-gated Ca 2+-channel may include one or more
Cache
domains. An extracellular Cache domain may be fused to an intracellular
catalytic domain, such as
the histidine kinase, PP2C phosphatase, GGDEF (a predicted diguanylate
cyclase), HD-GYP (a
predicted phosphodiesterase) or adenylyl cyclase domain, or to a noncatalytic
domain, like the
methyl-accepting, DNA-binding winged helix-turn-helix, GAF, PAS or RAMP (a
domain found in
istidine kinases, denylyl cyclases, ethyl-binding proteins and phosphatases).
Small molecules are bound
via the Cache domain and this signal is converted into diverse outputs
depending on the intracellular
domains (Anantharaman, V. and Aravind, L.(2000) Trends Biochem. Sci. 25:535-
537).
The transient receptor family (Trp) of calcium ion channels are thought to
mediate
capacitative calcium entry (CCE). CCE is the Ca2+ influx into cells to
resupply Caz+ stores depleted
by the action of inositol triphosphate (IP3) and other agents in response to
numerous hormones and
growth factors. Trp and Trp-like were first cloned from Drosophila and have
similarity to voltage
gated Ca 2+ channels in the S3 through S6 regions. This suggests that Trp
and/or related proteins may
form mammalian CCE channels (Zhu, X. et al. (1996) Cell 85:661-671; Boulay, G.
et al. (1997) J. Biol.
Chem. 272:29672-29680). Melastatin is a gene isolated in both the mouse and
human, whose
expression in melanoma cells is inversely correlated with melanoma
aggressiveness in vivo. The
human cDNA transcript corresponds to a 1533-amino acid protein having homology
to members of the
Trp family. It has been proposed that the combined use of malastatin mRNA
expression status and
tumor thickness might allow for the determination of subgroups of patients at
both low and high risk
for developing metastatic disease (Duncan, L.M. et al (2001) J. Clin. Oncol.
19:568-576).
Chloride channels are necessary in endocrine secretion and in regulation of
cytosolic and
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organelle pH. In secretory epithelial cells, Cl- enters the cell across a
basolateral membrane through
an Na+, K+/Cl- cotransporter, accumulating in the cell above its
electrochemical equilibrium
concentration. Secretion of Cl- from the apical surface, in response to
hormonal stimulation, leads to
flow of Na+ and water into the secretory lumen. The cystic fibrosis
transmembrane conductance
S regulator (CFTR) is a chloride channel encoded by the gene for cystic
fibrosis, a common fatal genetic
disorder in humans. CFTR is a member of the ABC transporter family, and is
composed of two
domains each consisting of six transmembrane domains followed by a nucleotide-
binding site. Loss of
CFTR function decreases transepithelial water secretion and, as a result, the
layers of mucus that coat
the respiratory tree, pancreatic ducts, and intestine are dehydrated and
difficult to clear. The resulting
blockage of these sites leads to pancreatic insufficiency, "meconium ileus",
and devastating "chronic
obstructive pulmonary disease" (Al-Awqati, Q. et al. (1992) J. Exp. Biol.
172:245-266).
The voltage-gated chloride channels (CLC) are characterized by 10-12
transmembrane
domains, as well as two small globular domains known as CBS domains. The CLC
subunits probably
function as homotetramers. CLC proteins are involved in regulation of cell
volume, membrane
potential stabilization, signal transduction, and transepithelial transport.
Mutations in CLC-1, expressed
predominantly in skeletal muscle, are responsible for autosomal recessive
generalized myotonia and
autosomal dominant myotonia congenita, while mutations in the kidney channel
CLC-5 lead to kidney
stones (Jentsch, T.J. (1996) Curr. Opin. Neurobiol. 6:303-310).
Ligand-gated channels open their pores when an extracellular or intracellular
mediator binds to
the channel. Neurotransmitter-gated channels are channels that open when a
neurotransmitter binds
to their extracellular domain. These channels exist in the postsynaptic
membrane of nerve or muscle
cells. There are two types of neurotransmitter-gated channels. Sodium channels
open in response to
excitatory neurotransmitters, such as acetylcholine, glutamate, and serotonin.
This opening causes an
influx of Na+ and produces the initial localized depolarization that activates
the voltage-gated channels
and starts the action potential. Chloride channels open in response to
inhibitory neurotransmitters,
such as y-aminobutyric acid (GABA) and glycine, leading to hyperpolarization
of the membrane and
the subsequent generation of an action potential. Neurotransmitter-gated ion
channels have four
transmembrane domains and probably function as pentamers (Jentsch, supra).
Amino acids in the
second transmembrane domain appear to be important in determining channel
permeation and
selectivity (Sather, W.A. et al. (1994) Curr. Opin. Neurobiol. 4:313-323).
Ligand-gated channels can be regulated by intracellular second messengers. For
example,
calcium-activated K+ channels are gated by internal calcium ions. In nerve
cells, an influx of calcium
during depolarization opens K+ channels to modulate the magnitude of the
action potential (Ishi et al.,
supra). The large conductance (BK) channel has been purified from brain and
its subunit composition
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determined. The a subunit of the BK channel has seven rather than six
transmembrane domains in
contrast to voltage-gated K+ channels. The extra transmembrane domain is
located at the subunit N-
terminus. A 28-amino-acid stretch in the C-terminal region of the subunit (the
"calcium bowl" region)
contains many negatively charged residues and is thought to be the region
responsible for calcium
binding. The (3 subunit consists of two transmembrane domains connected by a
glycosylated
extracellular loop, with intracellular N- and C-termini (Kaczorowski, su ra;
Vergara, C. et al. (1998)
Curr. Opin. Neurobiol. 8:321-329).
Cyclic nucleotide-gated (CNG) channels are gated by cytosolic cyclic
nucleotides. The best
examples of these are the cAMP-gated Na+ channels involved in olfaction and
the cGMP-gated
cation channels involved in vision. Both systems involve ligand-mediated
activation of a G-protein
coupled receptor which then alters the level of cyclic nucleotide within the
cell. CNG channels also
represent a major pathway for Ca2+ entry into neurons, and play roles in
neuronal development and
plasticity. CNG channels are tetramers containing at least two types of
subunits, an a subunit which
can form functional homomeric channels, and a ~i subunit, which modulates the
channel properties.
All CNG subunits have six transmembrane domains and a pore forming region
between the fifth and
sixth transmembrane domains, similar to voltage-gated K+ channels. A large C-
terminal domain
contains a cyclic nucleotide binding domain, while the N-terminal domain
confers variation among
channel subtypes (Zufall, F. et al. (1997) Curr. Opin. Neurobiol. 7:404-412).
The activity of other types of ion channel proteins may also be modulated by a
variety of
intracellular signalling proteins. Many channels have sites for
phosphorylation by one or more protein
kinases including protein kinase A, protein kinase C, tyrosine kinase, and
casein kinase II, all of which
regulate ion channel activity in cells. Kir channels are activated by the
binding of the G(3y subunits of
heterotrimeric G-proteins (Reimann, F. and F.M. Ashcroft (1999) Curr. Opin.
Cell. Biol. 11:503-508).
Other proteins are involved in the localization of ion channels to specific
sites in the cell membrane.
Such proteins include the PDZ domain proteins known as MAGUKs (membrane-
associated guanylate
kinases) which regulate the clustering of ion channels at neuronal synapses
(Craven, S.E. and D.S.
Bredt (1998) Cell 93:495-498).
Disease Correlation
The etiology of numerous human diseases and disorders can be attributed to
defects in the
transport of molecules across membranes. Defects in the trafficking of
membrane-bound transporters
and ion channels are associated with several disorders, e.g., cystic fibrosis,
glucose-galactose
malabsorption syndrome, hypercholesterolemia, von Gierke disease, and certain
forms of diabetes
mellitus. Single-gene defect diseases resulting in an inability to transport
small molecules across
membranes include, e.g., cystinuria, iminoglycinuria, Hartup disease, and
Fanconi disease (van't Hoff,
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W.G. (1996) Exp. Nephrol. 4:253-262; Talente, G.M. et al. (1994) Ann. Intern.
Med. 120:218-226;
and Chillon, M. et al. (1995) New Engl. J. Med. 332:1475-1480).
Human diseases caused by mutations in ion channel genes include disorders of
skeletal
muscle, cardiac muscle, and the central nervous system. Mutations in the pore-
forming subunits of
sodium and chloride channels cause myotonia, a muscle disorder in which
relaxation after voluntary
contraction is delayed. Sodium channel myotonias have been treated with
channel blockers.
Mutations in muscle sodium and calcium channels cause forms of periodic
paralysis, while mutations in
the sarcoplasmic calcium release channel, T-tubule calcium channel, and muscle
sodium channel
cause malignant hyperthermia. Cardiac arrythmia disorders such as the long QT
syndromes and
idiopathic ventricular fibrillation are caused by mutations in potassium and
sodium channels (Cooper,
E.C. and L.Y. Jan (1998) Proc. Natl. Acad. Sci. USA 96:4759-4766). All four
known human
idiopathic epilepsy genes code for ion channel proteins (Berkovic, S.F. and
LE. Scheffer (1999) Curr.
Opin. Neurology 12:177-182). Other neurological disorders such as ataxias,
hemiplegic migraine and
hereditary deafness can also result from mutations in ion channel genes (Jen,
J. (1999) Curr. Opin.
Neurobiol. 9:274-280; Cooper, supra).
Ion channels have been the target for many drug therapies. Neurotransmitter-
gated channels
have been targeted in therapies for treatment of insomnia, anxiety,
depression, and schizophrenia.
Voltage-gated channels have been targeted in therapies for arrhythmia,
ischemic stroke, head trauma,
and neurodegenerative disease (Taylor, C.P. and L.S. Narasimhan (1997) Adv.
Pharmacol. 39:47-98).
Various classes of ion channels also play an important role in the perception
of pain, and thus are
potential targets for new analgesics. These include the vanilloid-gated ion
channels, which are
activated by the vanilloid capsaicin, as well as by noxious heat. Local
anesthetics such as lidocaine
and mexiletine which blockade voltage-gated Na+ channels have been useful in
the treatment of
neuropathic pain (Eglen, supra).
Ion channels in the immune system have recently been suggested as targets for
immunomodulation. T-cell activation depends upon calcium signaling, and a
diverse set of T-cell
specific ion channels has been characterized that affect this signaling
process. Channel blocking
agents can inhibit secretion of lymphokines, cell proliferation, and killing
of target cells. A peptide
antagonist of the T-cell potassium channel Kvl.3 was found to suppress delayed-
type hypersensitivity
and allogenic responses in pigs, validating the idea of channel blockers as
safe and efficacious
immunosuppressants (Cahalan, M.D. and K.G. Chandy (1997) Curr. Opin.
Biotechnol. 8:749-756).
Senescence
Most normal eukaryotic cells, after a certain number of divisions, enter a
state of senescence
in which cells remain viable and metabolically active but no longer replicate.
A number of phenotypic
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changes such as increased cell size and pH-dependent beta-galactosidase
activity, and molecular
changes such as the upregulation of particular genes, occur in senescent cells
(Shelton (1999) Current
Biology 9:939-945). When senescent cells are exposed to mitogens, a number of
genes are
upregulated, but the cells do not proliferate. Evidence indicates that
senescent cells accumulate with
age in vivo, contributing to the aging of an organism. In addition, senescence
suppresses
tumorigenesis, and many genes necessary for senescence also function as tumor
suppressor genes,
such as p53 and the retinoblastoma susceptibility gene. Most tumors contain
cells that have surpassed
their replicative limit, i.e. they are immortalized. Many oncogenes
immortalize cells as a first step
toward tumor formation.
A variety of challenges, such as oxidative stress, radiation, activated
oncoproteins, and cell
cycle inhibitors, induce a senescent phenotype, indicating that senescence is
influenced by a number of
proliferative and anti-proliferative signals (Shelton supra). Senescence is
correlated with the
progressive shortening of telomeres that occurs with each cell division.
Expression of the catalytic
component of telomerase in cells prevents telomere shortening and immortalizes
cells such as
fibroblasts and epithelial cells, but not other types of cells, such as CD8+ T
cells (Migliaccio et al.
(2000) J. Immunol. 165:4978-4984). Thus, senescence is controlled by telomere
shortening as well as
other mechanisms depending on the type of cell.
A number of genes that are differentially expressed between senescent and
presenescent
cells have been identified as part of ongoing studies to understand the role
of senescence in aging and
tumorigenesis. Most senescent cells are growth arrested in the G1 stage of the
cell cycle. While
expression of many cell cycle genes is similar in senescent and presenescent
cells (Cristofalo (1992)
Ann. N. Y. Acad. Sci. 663:187-194), expression of others genes such as cyclin-
dependent kinases p21
and p16, which inhibit proliferation, and cyclins D1 and E is elevated in
senescent cells. Other genes
that are not directly involved in the cell cycle are also upregulated such as
extracellular matrix proteins
fibronectin, procollagen, and osteonectin; and proteases such as collagenase,
stromelysin, and
cathepsin B (Chen (2000) Ann. N.Y. Acad. Sci. 908:111-125). Genes
underexpressed in senescent
cells include those that encode heat shock proteins, c-fos, and cdc-2 (Chen s
bra).
P-glycoprotein is a member of the ABC transporter family that is expressed on
cells of the
immune system and plays a role in the secretion of cytokines and cytotoxic
molecules. P-glycoprotein
expression and function were found to be increased in aging lymphocytes. These
differences may
play a role in the changes in immune response, including increased frequency
of infections and
autoimmune phenomena, associated with human aging (Aggrawal, S. et al. (1997)
J. Clin. Immunol.
17:448-454).
The discovery of new transporters and ion channels, and the polynucleotides
encoding them,
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satisfies a need in the art by providing new compositions which are useful in
the diagnosis, prevention,
and treatment of transport, neurological, muscle, immunological and cell
proliferative disorders, and in
the assessment of the effects of exogenous compounds on the expression of
nucleic acid and amino
acid sequences of transporters and ion channels.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, transporters and ion channels,
referred to
collectively as "TRICH" and individually as "TRICH-1," "TRICH-2," "TRICH-3,"
"TRICH-4,"
"TRICH-5," "TRICH-6," "TRICH-7," "TRICH-8," "TRICH-9," "TRICH-10," "TRICH-11,"
"TRICH-12," "TRICH-13," "TRICH-14," "TRICH-15," "TRICH-16," "TRICH-17," "TRICH-
18,"
"TRICH-19," and "TRICH-20." In one aspect, the invention provides an isolated
polypeptide
selected from the group consisting of a) a polypeptide comprising an amino
acid sequence selected
from the group consisting of SEQ )D NO:1-20, b) a polypeptide comprising a
naturally occurring
amino acid sequence at least 90% identical to an amino acid sequence selected
from the group
consisting of SEQ 1D NO:1-20, c) a biologically active fragment of a
polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID NO:1-20, and d) an
immunogenic fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1-20.
In one alternative, the invention provides an isolated polypeptide comprising
the amino acid sequence
of SEQ )D NO:1-20.
The invention further provides an isolated polynucleotide encoding a
polypeptide selected from
the group consisting of a) a polypeptide comprising an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-20, b) a polypeptide comprising a naturally occurnng
amino acid sequence
at least 90% identical to an amino acid sequence selected from the group
consisting of SEQ ID NO:1-
20, c) a biologically active fragment of a polypeptide having an amino acid
sequence selected from the
group consisting of SEQ >D NO:1-20, and d) an immunogenic fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID NO:1-20. In
one alternative, the
polynucleotide encodes a polypeptide selected from the group consisting of SEQ
)D NO:1-20. In
another alternative, the polynucleotide is selected from the group consisting
of SEQ ID N0:21-40.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide selected
from the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ >D NO:1-20, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ ID NO:1-20, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
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consisting of SEQ ID NO:1-20, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ID NO:1-20. In one
alternative, the
invention provides a cell transformed with the recombinant polynucleotide. In
another alternative, the
invention provides a transgenic organism comprising the recombinant
polynucleotide.
The invention also provides a method for producing a polypeptide selected from
the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ID NO:1-20, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ ID NO:1-20, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-20, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ID NO:1-20. The method
comprises a)
culturing a cell under conditions suitable for expression of the polypeptide,
wherein said cell is
transformed with a recombinant polynucleotide comprising a promoter sequence
operably linked to a
polynucleotide encoding the polypeptide, and b) recovering the polypeptide so
expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid sequence
selected from the group consisting of SEQ ID.NO:I-20, b) a polypeptide
comprising a naturally
occurring amino acid sequence at least 90% identical to an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-20, c) a biologically active fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID NO:1-20, and
d) an immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
ID NO:1-20.
The invention further provides an isolated polynucleotide selected from the
group consisting of
a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of SEQ
ID N0:21-40, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ ID N0:21-40,
c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide complementary to
the polynucleotide of b), and e) an RNA equivalent of a)-d). In one
alternative, the polynucleotide
comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide selected from
the group consisting of
a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of SEQ
ID N0:21-40, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ ID N0:21-40,
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c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide complementary to
the polynucleotide of b), and e) an RNA equivalent of a)-d). The method
comprises a) hybridizing the
sample with a probe comprising at least 20 contiguous nucleotides comprising a
sequence
complementary to said target polynucleotide in the sample, and which probe
specifically hybridizes to
said target polynucleotide, under conditions whereby a hybridization complex
is formed between said
probe and said target polynucleotide or fragments thereof, and b) detecting
the presence or absence of
said hybridization complex, and optionally, if present, the amount thereof. In
one alternative, the probe
comprises at least 60 contiguous nucleotides.
The invention further provides a method for detecting a target polynucleotide
in a sample, said
target polynucleotide having a sequence of a polynucleotide selected from the
group consisting of a) a
polynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:21-40, b) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:21-40, c) a
polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
complementary to the
polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises
a) amplifying said
target polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b)
detecting the presence or absence of said amplified target polynucleotide or
fragment thereof, and,
optionally, if present, the amount thereof.
The invention further provides a composition comprising an effective amount of
a polypeptide
selected from the group consisting of a) a polypeptide comprising an amino
acid sequence selected
from the group consisting of SEQ >D NO:1-20, b) a polypeptide comprising a
naturally occurring
amino acid sequence at least 90% identical to an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-20, c) a biologically active fragment of a
polypeptide having an amino acid
sequence selected from the group consisting of SEQ )D NO:1-20, and d) an
immunogenic fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ m NO:l-20,
and a pharmaceutically acceptable excipient. In one embodiment, the
composition comprises an amino
acid sequence selected from the group consisting of SEQ ID NO:1-20. The
invention additionally
provides a method of treating a disease or condition associated with decreased
expression of
functional TRICH, comprising administering to a patient in need of such
treatment the composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1-20, b) a
polypeptide comprising a
naturally occurnng amino acid sequence at least 90% identical to an amino acid
sequence selected
from the group consisting of SEQ ID NO:1-20, c) a biologically active fragment
of a polypeptide
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having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-20, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:I-20. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting agonist activity in the sample. In
one alternative, the
invention provides a composition comprising an agonist compound identified by
the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with decreased expression of
functional TRICH, comprising
administering to a patient in need of such treatment the composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID NO:1-20, b) a
polypeptide
comprising a naturally occurring amino acid sequence at least 90% identical to
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-20, c) a
biologically active fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1-20,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ II7 NO:l-20. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting antagonist activity in the sample.
In one alternative, the
invention provides a composition comprising an antagonist compound identified
by the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with overexpression of functional
TRICH, comprising
administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that
specifically binds to
a polypeptide selected from the group consisting of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:I-20, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-20, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ )D
NO:1-20, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-20. The method comprises a) combining the
polypeptide with at least one
test compound under suitable conditions, and b) detecting binding of the
polypeptide to the test
compound, thereby identifying a compound that specifically binds to the
polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1-20, b) a
polypeptide comprising a
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naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ )D NO:1-20, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-20, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-20. The method comprises a) combining the
polypeptide with at least one
test compound under conditions permissive for the activity of the polypeptide,
b) assessing the activity
of the polypeptide in the presence of the test compound, and c) comparing the
activity of the
polypeptide in the presence of the test compound with the activity of the
polypeptide in the absence of
the test compound, wherein a change in the activity of the polypeptide in the
presence of the test
compound is indicative of a compound that modulates the activity of the
polypeptide.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
polynucleotide sequence selected from the group consisting of SEQ m N0:21-40,
the method
comprising a) exposing a sample comprising the target polynucleotide to a
compound, b) detecting
altered expression of the target polynucleotide, and c) comparing the
expression of the target
polynucleotide in the presence of varying amounts of the compound and in the
absence of the
compound.
The invention further provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound; b)
hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide selected from the group consisting
of i) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
)D N0:21-40, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ )D N0:21-40,
iii) a polynucleotide
having a sequence complementary to i), iv) a polynucleotide complementary to
the polynucleotide of
ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions
whereby a specific
hybridization complex is formed between said probe and a target polynucleotide
in the biological
sample, said target polynucleotide selected from the group consisting of i) a
polynucleotide comprising
a polynucleotide sequence selected from the group consisting of SEQ >D N0:21-
40, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ 1D N0:21-40,
iii) a polynucleotide
complementary to the polynucleotide of i), iv) a polynucleotide complementary
to the polynucleotide of
ii), and v) an RNA equivalent of i)-iv). Alternatively, the target
polynucleotide comprises a fragment
of a polynucleotide sequence selected from the group consisting of i)-v)
above; c) quantifying the
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amount of hybridization complex; and d) comparing the amount of hybridization
complex in the treated
biological sample with the amount of hybridization complex in an untreated
biological sample, wherein
a difference in the amount of hybridization complex in the treated biological
sample is indicative of
toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the present invention.
Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog, and the PROTEOME database identification numbers and annotations of
PROTEOME
database homologs, for polypeptides of the invention. The probability scores
for the matches between
each polypeptide and its homolog(s) are also shown.
Table 3 shows structural features of polypeptide sequences of the invention,
including
predicted motifs and domains, along with the methods, algorithms, and
searchable databases used for
analysis of the polypeptides.
Table 4 lists the cDNA and/or genomic DNA fragments which were used to
assemble
polynucleotide sequences of the invention, along with selected fragments of
the polynucleotide
sequences.
Table 5 shows the representative cDNA library for polynucleotides of the
invention.
Table 6 provides an appendix which describes the tissues and vectors used for
construction of
the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which will
be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
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
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forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods are now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing the
cell lines, protocols, reagents and vectors which are reported in the
publications and which might be
used in connection with the invention. Nothing herein is to be construed as an
admission that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
l0 DEFINITIONS
"TRICH" refers to the amino acid sequences of substantially purified TRICH
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
TRICH. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of TRICH either by
directly interacting with
TRICH or by acting on components of the biological pathway in which TRICH
participates.
An "allelic variant" is an alternative form of the gene encoding TRICH.
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 TRICH include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as TRICH or a
polypeptide with at least one functional characteristic of TRICH. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe of
the polynucleotide encoding TRICH, and improper or unexpected hybridization to
allelic variants, with
a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding TRICH.
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 TRICH.
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
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or immunological activity of TRICH is retained. For example, negatively
charged amino acids may
include aspartic acid and glutamic acid, and positively charged amino acids
may include lysine and
arginine. Amino acids with uncharged polar side chains having similar
hydrophilicity values may
include: asparagine and glutamine; and serine and threonine. Amino acids with
uncharged side chains
having similar hydrophilicity values may include: leucine, isoleucine, and
valine; glycine and alanine;
and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurnng or synthetic
molecules. Where "amino acid sequence" is recited to refer to a sequence of a
naturally occurring
l0 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 carned 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 TRICH. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of TRICH either by
directly interacting with TRICH or by acting on components of the biological
pathway in which
TRICH participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')Z, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind TRICH polypeptides can be prepared using intact
polypeptides or using fragments
containing small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide used
to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from
the translation of RNA,
or synthesized chemically, and can be conjugated to a carrier protein if
desired. Commonly used
carriers that are chemically coupled to peptides include bovine serum albumin,
thyroglobulin, and
keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize
the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures on
the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen used
to elicit the immune response) for binding to an antibody.
The term "aptamer" refers to a nucleic acid or oligonucleotide molecule that
binds to a
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specific molecular target. Aptamers are derived from an in vitro evolutionary
process (e.g., SELEX
(Systematic Evolution of Ligands by EXponential Enrichment), described in U.S.
Patent No.
5,270,163), which selects for target-specific aptamer sequences from large
combinatorial libraries.
Aptamer compositions may be double-stranded or single-stranded, and may
include
deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other
nucleotide-like molecules. The
nucleotide components of an aptamer may have modified sugar groups (e.g., the
2'-OH group of a
ribonucleotide may be replaced by 2'-F or 2'-NHZ), which may improve a desired
property, e.g.,
resistance to nucleases or longer lifetime in blood. Aptamers may be
conjugated to other molecules,
e.g., a high molecular weight Garner to slow clearance of the aptamer from the
circulatory system.
Aptamers may be specifically cross-linked to their cognate ligands, e.g., by
photo-activation of a
cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-
13.)
The term "intramer" refers to an aptamer which is expressed in vivo. For
example, a vaccinia
virus-based RNA expression system has been used to express specific RNA
aptamers at high levels
in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl Acad. Sci.
USA 96:3606-3610).
1'S The term "spiegelmer" refers to an aptamer which includes L-DNA, L-RNA, or
other left-
handed nucleotide derivatives or nucleotide-like molecules. Aptamers
containing left-handed
nucleotides are resistant to degradation by naturally occurnng enzymes, which
normally act on
substrates containing right-handed nucleotides.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA; RNA;
peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages
such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
designation "positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurnng molecule. Likewise, "immunologically active"
or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic TRICH, or
of any oligopeptide thereof,
to induce a specific immune response in appropriate animals or cells and to
bind with specific
antibodies.
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"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising a
given amino acid sequence" refer broadly to any composition containing the
given polynucleotide or
amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding TRICH or fragments
of TRICH may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCI), detergents
(e.g., sodium dodecyl sulfate;
SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm
DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to
repeated DNA sequence analysis to resolve uncalled bases, extended using the
XL-PCR kit (Applied
Biosystems, Foster City CA) in the 5' and/or the 3' direction, and
resequenced, or which has been
assembled from one or more overlapping cDNA, EST, or genomic DNA fragments
using a computer
program for fragment assembly, such as the GELVIEW fragment assembly system
(GCG, Madison
WI) or Phrap (University of Washington, Seattle WA). Some sequences have been
both extended
and assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of the
protein is conserved and not significantly changed by such substitutions. The
table below shows amino
acids which may be substituted for an original amino acid in a protein and
which are regarded as
conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
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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, andlor (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide can include, for example,
replacement of hydrogen by an
alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a
polypeptide which retains
at least one biological or immunological function of the natural molecule. A
derivative polypeptide is
one modified by glycosylation, pegylation, or any similar process that retains
at least one biological or
immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
"Differential expression" refers to increased or upregulated; or decreased,
downregulated, or
absent gene or protein expression, determined by comparing at least two
different samples. Such
comparisons may be carried out between, for example, a treated and an
untreated sample, or a
diseased and a normal sample.
"Exon shuffling" refers to the recombination of different coding regions
(exons). Since an
exon may represent a structural or functional domain of the encoded protein,
new proteins may be
assembled through the novel reassortment of stable substructures, thus
allowing acceleration of the
evolution of new protein functions.
A "fragment" is a unique portion of TRICH or the polynucleotide encoding TRICH
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example, a
fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10, 15,
16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
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from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ ID N0:21-40 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ )D N0:21-40, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:21-40 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish SEQ
>D N0:21-40 from related polynucleotide sequences. The precise length of a
fragment of SEQ ID
N0:21-40 and the region of SEQ ID N0:21-40 to which the fragment corresponds
are routinely
determinable by one of ordinary skill in the art based on the intended purpose
for the fragment.
A fragment of SEQ ID NO:1-20 is encoded by a fragment of SEQ ID N0:21-40. A
fragment of SEQ ID NO:1-20 comprises a region of unique amino acid sequence
that specifically
identifies SEQ ID NO:1-20. For example, a fragment of SEQ ID NO:1-20 is useful
as an
immunogenic peptide for the development of antibodies that specifically
recognize SEQ ID NO:1-20.
The precise length of a fragment of SEQ ID NO:1-20 and the region of SEQ ID
NO:1-20 to which
the fragment corresponds are routinely determinable by one of ordinary skill
in the art based on the
intended purpose for the fragment.
A "full length" polynucleotide sequence is one containing at least a
translation initiation codon
(e.g., methionine) followed by an open reading frame and a translation
termination codon. A "full
length" polynucleotide sequence encodes a "full length" polypeptide sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between two
or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide
sequences, refer to
the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps in
the sequences being compared in order to optimize alignment between two
sequences, and therefore
achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
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follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The
"weighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
is provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment
Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410),
which is available from
several sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence analysis
programs including "blastn," that is used to align a known polynucleotide
sequence with other
polynucleotide sequences from a variety of databases. Also available is a tool
called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.govlgorf/bl2.html. The
"BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: -2
Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off.' SO
Expect: 10
Word Size: 11
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ D7 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
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sequences that all encode substantially the same protein.
The phrases "percent identity" and "°Io identity," as applied to
polypeptide sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some alignment
methods take into account conservative amino acid substitutions. Such
conservative substitutions,
explained in more detail above, generally preserve the charge and-
hydrophobicity at the site of
substitution, thus preserving the structure (and therefore function) of the
polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version
2Ø12 (April-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Open Gap: 1l and Extension Gap: 1 penalties
Gap x drop-off.' S0
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ )D number, or may be measured over
a shorter length,
for example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
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The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the
stringency of the hybridization process, with more stringent conditions
allowing less non-specific
binding, i.e., binding between pairs of nucleic acid strands that are not
perfectly matched. Permissive
conditions for annealing of nucleic acid sequences are routinely determinable
by one of ordinary skill in
the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
1 % (w/v) SDS, and about 100 p.g/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook, J. et al. (1989)
Molecular Cloning: A Laboratory Manual, 2°d ed., vol. 1-3, Cold Spring
Harbor Fress, Plainview NY;
specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present invention
include wash conditions of 68°C in the presence of about 0.2 x SSC and
about 0.1% SDS, for 1 hour.
Alternatively, temperatures of about 65°C, 60°C, 55°C, or
42°C may be used. SSC concentration may
be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1 %.
Typically, blocking
reagents are used to block non-specific hybridization. Such blocking reagents
include, for instance,
sheared and denatured salmon sperm DNA at about 100-200 p.g/ml. Organic
solvent, such as
formamide at a concentration of about 35-50% v/v, may also be used under
particular circumstances,
such as for RNA:DNA hybridizations. Useful variations on these wash conditions
will be readily
apparent to those of ordinary skill in the art. Hybridization, particularly
under high stringency
conditions, may be suggestive of evolutionary similarity between the
nucleotides. Such similarity is
strongly indicative of a similar role for the nucleotides and their encoded
polypeptides.
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The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of TRICH
which is
capable of eliciting an immune response when introduced into a living
organism, for example, a
mammal. The term "immunogenic fragment" also includes any polypeptide or
oligopeptide fragment
of TRICH which is useful in any of the antibody production methods disclosed
herein or known in the
art.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides,
polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of TRICH. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other biological,
functional, or immunological properties of TRICH.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
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comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of.
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition. PNAs
preferentially bind complementary single stranded DNA or RNA and stop
transcript elongation, and
may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an TRICH may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in the
art. These processes may occur synthetically or biochemically. Biochemical
modifications will vary
by cell type depending on the enzymatic milieu of TRICH.
"Probe" refers to nucleic acid sequences encoding TRICH, their complements, or
fragments
l0 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 polymerise enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence, e.g., by the polymerise chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual,
2°d ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Biolo~v, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to 5,000
nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection
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programs have incorporated additional features for expanded capabilities. For
example, the PrimOU
primer selection program (available to the public from the Genome Center at
University of Texas
South West Medical Center, Dallas TX) is capable of choosing specific primers
from megabase
sequences and is thus useful for designing primers on a genome-wide scope. The
Primer3 primer
selection program (available to the public from the Whitehead Institute/MIT
Center for Genome
Research, Cambridge MA) allows the user to input a "mispriming library," in
which sequences to
avoid as primer binding sites are user-specified. Primer3 is useful, in
particular, for the selection of
oligonucleotides for microarrays. (The source code for the latter two primer
selection programs may
also be obtained from their respective sources and modified to meet the user's
specific needs.) The
PrimeGen program (available to the public from the UK Human Genome Mapping
Project Resource
Centre, Cambridge UK) designs primers based on multiple sequence alignments,
thereby allowing
selection of primers that hybridize to either the most conserved or least
conserved regions of aligned
nucleic acid sequences. Hence, this program is useful for identification of
both unique and conserved
oligonucleotides and polynucleotide fragments. The oligonucleotides and
polynucleotide fragments
identified by any of the above selection methods are useful in hybridization
technologies, for example,
as PCR or sequencing primers, microarray elements, or specific probes to
identify fully or partially
complementary polynucleotides in a sample of nucleic acids. Methods of
oligonucleotide selection are
not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter
sequence. Such a recombinant nucleic acid may be part of a vector that is
used, for example, to
transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and S' and 3'
untranslated regions
(UTRs). Regulatory elements interact with host or viral proteins which control
transcription,
translation, or RNA stability.
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"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of
the nitrogenous base thymine are replaced with uracil, and the sugar backbone
is composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing TRICH,
l0 nucleic acids encoding TRICH, or fragments thereof may comprise a bodily
fluid; an extract from a
cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic
DNA, RNA, or cDNA,
in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular structure
of the protein, e.g., the antigenic determinant or epitope, recognized by the
binding molecule. For
example, if an antibody is specific for epitope "A," the presence of a
polypeptide comprising the
epitope A, or the presence of free unlabeled A, in a reaction containing free
labeled A and the
antibody will reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 75% free, and most preferably at least 90% free from other
components with
which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides
by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" or "expression profile" refers to the collective pattern
of gene expression
by a particular cell type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into
a recipient
cell. Transformation may occur under natural or artificial conditions
according to various methods
well known in the art, and may rely on any known method for the insertion of
foreign nucleic acid
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sequences into a prokaryotic or eukaryotic host cell. The method for
transformation is selected based
on the type of host cell being transformed and may include, but is not limited
to, bacteriophage or viral
infection, electroporation, heat shock, lipofection, and particle bombardment.
The term "transformed
cells" includes stably transformed cells in which the inserted DNA is capable
of replication either as
an autonomously replicating plasmid or as part of the host chromosome, as well
as transiently
transformed cells which express the inserted DNA or RNA for limited periods of
time.
A "transgenic organism," as used herein, is any organism, including but not
limited to animals
and plants, in which one or more of the cells of the organism contains
heterologous nucleic acid
introduced by way of human intervention, such as by transgenic techniques well
known in the art. The
nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor of the cell,
by way of deliberate genetic manipulation, such as by microinjection or by
infection with a
recombinant virus. The term genetic manipulation does not include classical
cross-breeding, or in vitro
fertilization, but rather is directed to the introduction of a recombinant DNA
molecule. The transgenic
organisms contemplated in accordance with the present invention include
bacteria, cyanobacteria,
fungi, plants and animals. The isolated DNA of the present invention can be
introduced into the host
by methods known in the art, for example infection, transfection,
transformation or transconjugation.
Techniques for transferring the DNA of the present invention into such
organisms are widely known
and provided in references such as Sambrook et al: (1989), supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99% or greater
sequence identity over a certain defined length. A variant may be described
as, for example, an
"allelic" (as defined above), "splice," "species," or "polymorphic" variant. A
splice variant may have
significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternate splicing of exons during mRNA processing. The
corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotide sequences that vary
from one species to
another. The resulting polypeptides will generally 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
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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 91%, at least
92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
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 transporters and ion
channels
(TRICH), the polynucleotides encoding TRICH, and the use of these compositions
for the diagnosis,
treatment, or prevention of transport, neurological, muscle, immunological and
cell proliferative
disorders.
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to a
single Incyte project identification number (Incyte Project ID). Each
polypeptide sequence is denoted
by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:)
and an Incyte
polypeptide sequence number (Incyte Polypeptide ID) as shown. Each
polynucleotide sequence is
denoted by both a polynucleotide sequence identification number
(Polynucleotide SEQ ID NO:) and an
Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as
shown.
Table 2 shows sequences with homology to the polypeptides of the invention as
identified by
BLAST analysis against the GenBank protein (genpept) database and the PROTEOME
database.
Columns 1 and 2 show the polypeptide sequence identification number
(Polypeptide SEQ ID NO:) and
the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID)
for polypeptides of the
invention. Column 3 shows the GenBank identification number (GenBank ID NO:)
of the nearest
GenBank homolog and the PROTEOME database identification numbers (PROTEOME ID
NO:) of
the nearest PROTEOME database homologs. Column 4 shows the probability scores
for the matches
between each polypeptide and its homolog(s). Column 5 shows the annotation of
the GenBank and
PROTEOME database homolog(s) along with relevant citations where applicable,
all of which are
expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the
invention. Columns 1 and
2 show the polypeptide sequence identification number (SEQ ID N0:) and the
corresponding Incyte
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polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of
the invention. Column
3 shows the number of amino acid residues in each polypeptide. Column 4 shows
potential
phosphorylation sites, and column 5 shows potential glycosylation sites, as
determined by the MOTIFS
program of the GCG sequence analysis software package (Genetics Computer
Group, Madison WI).
Column 6 shows amino acid residues comprising signature sequences, domains,
and motifs. Column 7
shows analytical methods for protein structure/function analysis and in some
cases, searchable
databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the
invention, and these
properties establish that the claimed polypeptides are transporters and ion
channels. For example,
SEQ ID N0:3 is 85% identical, from residue M27 to residue N989, to rabbit
anion exchanger 4a
(GenBank ID g11611537) as determined by the Basic Local Alignment Search Tool
(BLAST). (See
Table 2.) The BLAST probability score is 0.0, which indicates the probability
of obtaining the
observed polypeptide sequence alignment by chance. SEQ ID N0:3 also contains a
HC03-
transporter family domain as determined by searching for statistically
significant matches in the hidden
Markov model (HMM)-based PFAM database of conserved protein family domains.
(See Table 3.)
Data from BLIMPS and PROFILESCAN analyses provide further corroborative
evidence that SEQ
ID N0:3 is an anion exchanger.
In another example, SEQ ID N0:6 is 47% identical, from residue S7 to residue
E350, to
hamster Na+ dependent deal bile acid transporter (GenBank ID g455033) as
determined by the Basic
Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability
score is 3.7e-88,
which indicates the probability of obtaining the observed polypeptide sequence
alignment by chance.
SEQ ID N0:6 also contains a sodium bile acid symporter family domain as
determined by searching
for statistically significant matches in the hidden Markov model (HMM)-based
PFAM database of
conserved protein family domains. (See Table 3.) Data from additional BLAST
analyses using the
PRODOM and DOMO databases provide further corroborative evidence that SEQ ID
N0:6 is a
sodium/bile acid symporter.
In another example, SEQ ID N0:9 is 68% identical, from residue E6 to residue
I349, to mouse
Ac39/physophilin, a subunit of the vacuolar ATPase (GenBank ID g1226235) as
determined by the
Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST
probability score is 3.2e-
130, which indicates the probability of obtaining the observed polypeptide
sequence alignment by
chance. SEQ ID N0:9 also contains an ATP synthase (C/AC39) subunit domain as
determined by
searching for statistically significant matches in the hidden Markov model
(HMM)-based PFAM
database of conserved protein family domains. (See Table 3.) Data from
additional BLAST analyses
using the PRODOM and DOMO databases provide further corroborative evidence
that SEQ ID
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N0:9 is a vacuolar ATPase subunit.
In another example, SEQ ID NO:10 is 83% identical, from residue M154 to
residue 8591, to
murine melastatin (GenBank >D g3047272) as determined by the Basic Local
Alignment Search Tool
(BLAST). (See Table 2.) The BLAST probability score is 8.6e-200, which
indicates the probability
of obtaining the observed polypeptide sequence alignment by chance. SEQ >D
NO:10 also contains a
transient receptor domain as determined by searching for statistically
significant matches in the hidden
Markov model (HMM)-based PFAM database of conserved protein family domains.
(See Table 3.)
Data from BLIMPS, analysis provide further corroborative evidence that SEQ ID
NO:10 is a calcium
ion channel (note that melastatin has homology to members of the "transient
receptor" family of
"calcium channels").
In another example, SEQ )D N0:12 is 51% identical, from residue 6761 to
residue E1326, to
rat multidrug resistance protein MRPS (GenBank ID g6682827) as determined by
the Basic Local
Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is
3.5e-236, which
indicates the probability of obtaining the observed polypeptide sequence
alignment by chance. SEQ
ID N0:12 also contains two ABC transporter transmembrane regions and two ABC
transporter
domains as determined by searching for statistically significant matches in
the hidden Markov model
(HMM)-based PFAM database of conserved protein family domains. (See Table 3.)
Data from
BLIMPS, MOTIFS, and PROFILESCAN analyses provide further corroborative
evidence that SEQ
ID N0:12 is an ABC transporter.
For example, SEQ 1D N0:18 is 76% identical, from residue M1 to residue D597,
to rat renal
osmotic stress-induced Na-Cl organic solute cotransporter (GenBank ID g531469)
as determined by
the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST
probability score is
1.2e-260, which indicates the probability of obtaining the observed
polypeptide sequence alignment by
chance. SEQ 1D N0:18 also contains a sodium:neurotransmitter symporter family
domain as
determined by searching for statistically significant matches in the hidden
Markov model (HMM)-
based PFAM database of conserved protein family domains. (See Table 3.) Data
from BLIMPS and
PROF1LESCAN analyses provide further corroborative evidence that SEQ ID N0:18
is a sodium
dependent organic solute transporter. SEQ ID NO:I-2, SEQ 1D N0:4-5, SEQ m N0:7-
8, SEQ >D
NO:11, SEQ 1D N0:13-17 and SEQ ID N0:19-20 were analyzed and annotated in a
similar manner.
The algorithms and parameters for the analysis of SEQ ID NO:1-20 are described
in Table 7.
As shown in Table 4, the full length polynucleotide sequences of the present
invention were
assembled using cDNA sequences or coding (exon) sequences derived from genomic
DNA, or any
combination of these two types of sequences. Column 1 lists the polynucleotide
sequence
identification number (Polynucleotide SEQ m NO:), the corresponding Incyte
polynucleotide
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consensus sequence number (Incyte 117) for each polynucleotide of the
invention, and the length of
each polynucleotide sequence in basepairs. Column 2 shows the nucleotide start
(5') and stop (3')
positions of the cDNA and/or genomic sequences used to assemble the full
length polynucleotide
sequences of the invention, and of fragments of the polynucleotide sequences
which are useful, for
example, in hybridization or amplification technologies that identify SEQ ID
N0:21-40 or that
distinguish between SEQ )D N0:21-40 and related polynucleotide sequences.
The polynucleotide fragments described in Column 2 of Table 4 may refer
specifically, for
example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from
pooled cDNA
libraries. Alternatively, the polynucleotide fragments described in column 2
may refer to GenBank
cDNAs or ESTs which contributed to the assembly of the full length
polynucleotide sequences. In
addition, the polynucleotide fragments described in column 2 may identify
sequences derived from the
ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences
including the
designation "ENST"). Alternatively, the polynucleotide fragments described in
column 2 may be
derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those
sequences
including the designation "NM" or "NT") or the NCBI RefSeq Protein Sequence
Records (i.e., those
sequences including the designation "NP"). Alternatively, the polynucleotide
fragments described in
column 2 may refer to assemblages of both cDNA and Genscan-predicted exons
brought together by
an "exon stitching" algorithm. For example, a polynucleotide sequence
identified as
FL_XX~XX N, NZ_YYYYY N3 N4 represents a "stitched" sequence in which ~ is the
identification number of the cluster of sequences to which the algorithm was
applied, and YYYYY is the
number of the prediction generated by the algorithm, and N,,2, j..., if
present, represent specific exons
that may have been manually edited during analysis (See Example V).
Alternatively, the
polynucleotide fragments in column 2 may refer to assemblages of exons brought
together by an
"exon-stretching" algorithm. For example, a polynucleotide sequence identified
as
FLX~AAAA~BBBBB_1 N is a "stretched" sequence, with ~ being the Incyte
project identification number, gAAAAA being the GenBank identification number
of the human
genomic sequence to which the "exon-stretching" algorithm was applied, gBBBBB
being the GenBank
identification number or NCBI RefSeq identification number of the nearest
GenBank protein homolog,
and N referring to specific exons (See Example V). In instances where a RefSeq
sequence was used
as a protein homolog for the "exon-stretching" algorithm, a RefSeq identifier
(denoted by "NM,"
"NP," or "NT") may be used in place of the GenBank identifier (i.e., gBBBBB).
Alternatively, a prefix identifies component sequences that were hand-edited,
predicted from
genomic DNA sequences, or derived from a combination of sequence analysis
methods. The
following Table lists examples of component sequence prefixes and
corresponding sequence analysis
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methods associated with the prefixes (see Example IV and Example V).
Prefix Type of analysis and/or examples of programs
GNN, GFG,Exon prediction from genomic sequences using,
for example,
ENST GENSCAN (Stanford University, CA, USA) or
FGENES
(Computer Genomics Group, The Sanger Centre,
Cambridge, UK)
GBI Hand-edited analysis of genomic sequences.
FL Stitched or stretched genomic sequences
(see Example V).
INCY Full length transcript and exon prediction
from mapping of EST
sequences to the genome. Genomic location
and EST composition
data are combined to predict the exons and
resulting transcript.
In some cases, Incyte cDNA coverage redundant with the sequence coverage shown
in
Table 4 was obtained to confirm the final consensus polynucleotide sequence,
but the relevant Incyte
cDNA identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length
polynucleotide
sequences which were assembled using Incyte cDNA sequences. The representative
cDNA library
is the Incyte cDNA library which is most frequently represented by the Incyte
cDNA sequences
which were used to assemble and confirm the above polynucleotide sequences.
The tissues and
vectors which were used to construct the cDNA libraries shown in Table 5 are
described in Table 6.
The invention also encompasses TRICH variants. A preferred TRICH 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 TRICH amino acid sequence, and which contains at
least one functional or
structural characteristic of TRICH.
The invention also encompasses polynucleotides which encode TRICH. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ )D N0:21-40, which encodes TRICH. The
polynucleotide
sequences of SEQ ID N0:21-40, as presented in the Sequence Listing, embrace
the equivalent RNA
sequences, wherein occurrences of the nitrogenous base thymine are replaced
with uracil, and the
sugar backbone is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding
TRICH. In
particular, such a variant polynucleotide sequence will have at least about
70%, or alternatively at least
about 85%, or even at least about 95% polynucleotide sequence identity to the
polynucleotide
sequence encoding TRICH. A particular aspect of the invention encompasses a
variant of a
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polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID N0:21-
40 which has at least about 70%, or alternatively at least about 85%, or even
at least about 95%
polynucleotide sequence identity to a nucleic acid sequence selected from the
group consisting of SEQ
ID N0:21-40. Any one of the polynucleotide variants described above can encode
an amino acid
sequence which contains at least one functional or structural characteristic
of TRICH.
In addition, or in the alternative, a polynucleotide variant of the invention
is a splice variant of a
polynucleotide sequence encoding TRICH. A splice variant may have portions
which have significant
sequence identity to the polynucleotide sequence encoding TRICH, but will
generally have a greater or
lesser number of polynucleotides due to additions or deletions of blocks of
sequence arising from
alternate splicing of exons during mRNA processing. A splice variant may have
less than about 70%,
or alternatively less than about 60%, or alternatively less than about 50%
polynucleotide sequence
identity to the polynucleotide sequence encoding TRICH over its entire length;
however, portions of
the splice variant will have at least about 70%, or alternatively at least
about 85%, or alternatively at
least about 95%, or alternatively 100% polynucleotide sequence identity to
portions of the
polynucleotide sequence encoding TRICH. For example, a polynucleotide
comprising a sequence of
SEQ ID N0:40 is a splice variant of a polynucleotide comprising a sequence of
SEQ >D N0:29. Any
one of the splice variants described above can encode an amino acid sequence
which contains at least
one functional or structural characteristic of TRICH.
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 TRICH, 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 occurnng TRICH, and all such variations
are to be considered as
being specifically disclosed.
Although nucleotide sequences which encode TRICH and its variants are
generally capable of
hybridizing to the nucleotide sequence of the naturally occurring TRICH under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding TRICH or
its derivatives possessing a substantially different codon usage, e.g.,
inclusion of non-naturally
occurnng 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 TRICH and its derivatives without altering the encoded amino
acid sequences
41
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
include the production of RNA transcripts having more desirable properties,
such as a greater half-life,
than transcripts produced from the naturally occurnng sequence.
The invention also encompasses production of DNA sequences which encode TRICH
and
TRICH 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 TRICH 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 1D
N0:21-40 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods
Enzymol. 152:507-
511.) Hybridization conditions, including annealing and wash conditions, are
described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(Applied
Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerases and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
(Applied Biosystems). Sequencing is then carried out using either the ABI 373
or 377 DNA
sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing
system
(Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The
resulting sequences
are analyzed using a variety of algorithms which are well known in the art.
(See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY,
unit 7.7; Meyers,
R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp.
856-853.)
The nucleic acid sequences encoding TRICH 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
42
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
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
legations may be used to insert an engineered double-stranded sequence into a
region of unknown
sequence before performing PCR. Other methods which may be used to retrieve
unknown sequences
are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids
Res. 19:3055-3060).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries
(Clontech, Palo
Alto CA) to walk genomic DNA. This procedure avoids the need to screen
libraries and is useful in
finding intron/exon junctions. For all PCR-based methods, primers may be
designed using
commercially available software, such as OLIGO 4.06 primer analysis software
(National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30
nucleotides in length,
to have a GC content of about 50% or more, and to anneal to the template at
temperatures of about
68°C to 72°C.
When screening for full length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence
into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQiJENCE NAVIGATOR, Applied Biosystems), and
the entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof which
encode TRICH may be cloned in recombinant DNA molecules that direct expression
of TRICH, 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 TRICH.
The nucleotide sequences of the present invention can be engineered using
methods generally
43
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
known in the art in order to alter TRICH-encoding sequences for a variety of
purposes including, but
not limited to, modification of the cloning, processing, and/or expression of
the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction
sites, alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
No.
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or improve
the biological properties of TRICH, such as its biological or enzymatic
activity or its ability to bind to
other molecules or compounds. DNA shuffling is a process by which a library of
gene variants is
produced using PCR-mediated recombination of gene fragments. The library is
then subjected to
selection or screening procedures that identify those gene variants with the
desired properties. These
preferred variants may then be pooled and further subjected to recursive
rounds of DNA shuffling and
selection/screening. Thus, genetic diversity is created through "artificial"
breeding and rapid
molecular evolution. For example, fragments of a single gene containing random
point mutations may
be recombined, screened, and then reshuffled until the desired properties are
optimized. Alternatively,
fragments of a given gene may be recombined with fragments of homologous genes
in the same gene
family, either from the same or different species, thereby maximizing the
genetic diversity of multiple
naturally occurring genes in a directed and controllable manner.
In another embodiment, sequences encoding TRICH 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,
TRICH itself or a fragment thereof may be synthesized using chemical methods.
For example,
peptide synthesis can be performed using various solution-phase or solid-phase
techniques. (See, e.g.,
Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH
Freeman, New York NY, pp.
55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated
synthesis may be achieved
using the ABI 431A peptide synthesizer (Applied Biosystems). Additionally, the
amino acid sequence
of TRICH, or any part thereof, may be altered during direct synthesis and/or
combined with
sequences from other proteins, or any part thereof, to produce a variant
polypeptide or a polypeptide
having a sequence of a naturally occurnng 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.)
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CA 02438206 2003-08-06
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The composition of the synthetic peptides may be confirmed by amino acid
analysis or by sequencing.
(See, e.g., Creighton, supra, pp. 28-53.)
In order to express a biologically active TRICH, the nucleotide sequences
encoding TRICH or
derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which contains
the necessary elements for transcriptional and translational control of the
inserted coding sequence in
a suitable host. These elements include regulatory sequences, such as
enhancers, constitutive and
inducible promoters, and 5' and 3' untranslated regions in the vector and in
polynucleotide sequences
encoding TRICH. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
TRICH. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding TRICH 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 TRICH 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 TRICH. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV, or
tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or
animal cell systems. (See, e.g., Sambrook, supra; Ausubel, supra; Van Heeke,
G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl.
Acad. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu,
N. (1987) EMBO
J. 6:307-311; The McGraw Hill Yearbook of Science and TechnoloQV (1992) McGraw
Hill, New
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA
81:3655-3659; and
Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors
derived from retroviruses,
adenoviruses, or herpes or vaccinia viruses, or from various bacterial
plasmids, may be used for
delivery of nucleotide sequences to the targeted organ, tissue, or cell
population. (See, e.g., Di Nicola,
M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc.
Natl. Acad. Sci. USA
90(13):6340-6344; Butler, R.M. et al. (1985) Nature 317(G040):813-815;
McGregor, D.P. et al. (1994)
Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature 389:239-
242.) The
invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding TRICH. For
example, routine cloning,
subcloning, and propagation of polynucleotide sequences encoding TRICH 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 TRICH into the
vector's multiple
cloning site disrupts the lacZ gene, allowing a colorimetric screening
procedure for identification of
transformed bacteria containing recombinant molecules. In addition, these
vectors may be useful for
in vitro transcription, dideoxy sequencing, single strand rescue with helper
phage, and creation of
nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M.
Schuster (1989) J. Biol.
Chem. 264:5503-5509.) When large quantities of TRICH are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of TRICH may be used.
For example, vectors
containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of TRICH. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomvces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable integration
of foreign sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, 1995, supra;
Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et
al. (1994)
Bio/Technology 12:181-184.) ,
Plant systems may also be used for expression of TRICH. Transcription of
sequences
encoding TRICH may be driven by viral promoters, e.g., the 355 and 195
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
46
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technolo~y
(1992) McGraw Hill,
New York NY, pp. 191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding TRICH
may be ligated into
an adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses TRICH 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-
l0 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
TRICH in cell lines is preferred. For example, sequences encoding TRICH can be
transformed into
cell lines using expression vectors which may contain viral origins of
replication and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media before
being switched to selective media. The purpose of the selectable marker is to
confer resistance to a
selective agent, and its presence allows growth and recovery of cells which
successfully express the
introduced sequences. Resistant clones of stably transformed cells may be
propagated using tissue
culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk- and apr cells, respectively.
(See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic, or
herbicide resistance can be used as the basis for selection. For example, dhfr
confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. (1981)
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hisD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan (1988) Proc.
47
CA 02438206 2003-08-06
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Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech),13 glucuronidase and its substrate f3-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest
is also present, the presence and expression of the gene may need to be
confirmed. For example, if
the sequence encoding TRICH is inserted within a marker gene sequence,
transformed cells
containing sequences encoding TRICH can be identified by the absence of marker
gene function.
Alternatively, a marker gene can be placed in tandem with a sequence encoding
TRICH 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 TRICH
and that express
TRICH may be identified by a variety of procedures known to those of skill in
the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations,
PCR
amplification, and protein bioassay or immunoassay techniques which include
membrane, solution, or
chip based technologies for the detection and/or quantification of nucleic
acid or protein sequences.
Immunological methods for detecting and measuring the expression of TRICH
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 TRICH 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
Fress, St. Paul MN,
Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in ImmunoloQV, Greene
Pub. Associates and
Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical
Protocols, 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
TRICH include
oligolabeling, nick translation, end-labeling, or PCR amplification using a
labeled nucleotide.
Alternatively, the sequences encoding TRICH, or any fragments thereof, may be
cloned into a vector
for the production of an mRNA probe. Such vectors are known in the art, are
commercially available,
and may be used to synthesize RNA probes in vitro by addition of an
appropriate RNA polymerase
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CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
such as T7, T3, or SP6 and labeled nucleotides. These procedures may be
conducted using a variety
of commercially available kits, such as those provided by Amersham Pharmacia
Biotech, Promega
(Madison WI), and US Biochemical. Suitable reporter molecules or labels which
may be used for
ease of detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic
agents, as well as substrates, cofactors, inhibitors, magnetic particles, and
the like.
Host cells transformed with nucleotide sequences encoding TRICH may be
cultured under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the sequence
and/or the vector used. As will be understood by those of skill in the art,
expression vectors containing
polynucleotides which encode TRICH may be designed to contain signal sequences
which direct
secretion of TRICH 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, phosphorylatior
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 TRICH 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 TRICH protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of TRICH
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 TRICH encoding sequence and the heterologous protein
sequence, so that
49
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
TRICH may be cleaved away from the heterologous moiety following purification.
Methods for
fusion protein expression and purification are discussed in Ausubel (1995,
supra, ch. 10). A variety of
commercially available kits may also be used to facilitate expression and
purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled TRICH may
be achieved in
vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These
systems couple transcription and translation of protein-coding sequences
operably associated with the
T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
precursor, for example, 35S-methionine.
TRICH of the present invention or fragments thereof may be used to screen for
compounds
l0 that specifically bind to TRICH. At least one and up to a plurality of test
compounds may be screened
for specific binding to TRICH. Examples of test compounds include antibodies,
oligonucleotides,
proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the
natural ligand of
TRICH, e.g., a ligand or fragment thereof, a natural substrate, a structural
or functional mimetic, or a
natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current
Protocols in Immunoloey 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which TRICH
binds, or to at least a fragment of the receptor, e.g., the ligand binding
site. In either case, the
compound can be rationally designed using known techniques. In one embodiment,
screening for
these compounds involves producing appropriate cells which express TRICH,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or E.
coli. Cells expressing TRICH or cell membrane fractions which contain TRICH
are then contacted
with a test compound and binding, stimulation, or inhibition of activity of
either TRICH or the
compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide,
wherein binding is
detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable
label. For example, the
assay may comprise the steps of combining at least one test compound with
TRICH, either in solution
or affixed to a solid support, and detecting the binding of TRICH to the
compound. Alternatively, the
assay may detect or measure binding of a test compound in the presence of a
labeled competitor.
Additionally, the assay may be carried out using cell-free preparations,
chemical libraries, or natural
product mixtures, and the test compounds) may be free in solution or affixed
to a solid support.
TRICH of the present 'invention or fragments thereof may be used to screen for
compounds
that modulate the activity of TRICH. Such compounds may include agonists,
antagonists, or partial or
inverse agonists. In one embodiment, an assay is performed under conditions
permissive for TRICH
activity, wherein TRICH is combined with at least one test compound, and the
activity of TRICH in
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the presence of a test compound is compared with the activity of TRICH in the
absence of the test
compound. A change in the activity of TRICH in the presence of the test
compound is indicative of a
compound that modulates the activity of TRICH. Alternatively, a test compound
is combined with an
in vitro or cell-free system comprising TRICH under conditions suitable for
TRICH activity, and the
assay is performed. In either of these assays, a test compound which modulates
the activity of
TRICH may do so indirectly and need not come in direct contact with the test
compound. At least
one and up to a plurality of test compounds may be screened.
In another embodiment, polynucleotides encoding TRICH or their mammalian
homologs may
be "knocked out" in an animal model system using homologous recombination in
embryonic stem (ES)
cells. Such techniques are well known in the art and are useful for the
generation of animal models of
human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No.
5,767,337.) For example,
mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the
early mouse embryo and
grown in culture. The ES cells are transformed with a vector containing the
gene of interest disrupted
by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi,
M.R. (1989) Science
244:1288-1292). The vector integrates into the corresponding region of the
host genome by
homologous recombination. Alternatively, homologous recombination takes place
using the Cre-loxP
system to knockout a gene of interest in a tissue- or developmental stage-
specific manner (Marth, J.D.
(1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids
Res. 25:4323-4330).
Transformed ES cells are identified and microinjected into mouse cell
blastocysts such as those from
the C57BL/6 mouse strain. The blastocysts are surgically transferred to
pseudopregnant dams, and
the resulting chimeric progeny are genotyped and bred to produce heterozygous
or homozygous
strains. Transgenic animals thus generated may be tested with potential
therapeutic or toxic agents.
Polynucleotides encoding TRICH may also be manipulated in vitro in ES cells
derived from
human blastocysts. Human ES cells have the potential to differentiate into at
least eight separate cell
lineages including endoderm, mesoderm, and ectodermal cell types. These cell
lineages differentiate
into, for example, neural cells, hematopoietic lineages, and cardiomyocytes
(Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding TRICH can also be used to create "knockin" humanized
animals
(pigs) or transgenic animals (mice or rats) to model human disease. With
knockin technology, a region
of a polynucleotide encoding TRICH is injected into animal ES cells, and the
injected sequence
integrates into the animal cell genome. Transformed cells are injected into
blastulae, and the blastulae
are implanted as described above. Transgenic progeny or inbred lines are
studied and treated with
potential pharmaceutical agents to obtain information on treatment of a human
disease. Alternatively,
a mammal inbred to overexpress TRICH, e.g., by secreting TRICH in its milk,
may also serve as a
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convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu.
Rev. 4:55-74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists between
regions of TRICH and transponers and ion channels. In addition, examples of
tissues expressing
TRICH are primary human breast epithelial cells and also can be found in Table
6. Therefore,
TRICH appears to play a role in transport, neurological, muscle, immunological
and cell proliferative
disorders. In the treatment of disorders associated with increased TRICH
expression or activity, it is
desirable to decrease the expression or activity of TRICH. In the treatment of
disorders associated
with decreased TRICH expression or activity, it is desirable to increase the
expression or activity of
l0 TRICH.
Therefore, in one embodiment, TRICH 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 TRICH. Examples of such disorders include, but are not limited to,
a transport disorder
such as akinesia, amyotrophic lateral sclerosis, ataxia telangiectasia, cystic
fibrosis, Becker's muscular
dystrophy, Bell's palsy, Charcot-Marie Tooth disease, diabetes mellitus,
diabetes insipidus, diabetic
neuropathy, Duchenne muscular dystrophy, hyperkalemic periodic paralysis,
normokalemic periodic
paralysis, Parkinson's disease, malignant hyperthermia, multidrug resistance,
myasthenia gravis,
myotonic dystrophy, catatonia, tardive dyskinesia, dystonias, peripheral
neuropathy, cerebral
neoplasms, prostate cancer, cardiac disorders associated with transport, e.g.,
angina, bradyarrythmia,
tachyarrythmia, hypertension, Long QT syndrome, myocarditis, cardiomyopathy,
nemaline myopathy,
centronuclear myopathy, lipid myopathy, mitochondrial myopathy, thyrotoxic
myopathy, ethanol
myopathy, dermatomyositis, inclusion body myositis, infectious myositis,
polymyositis, neurological
disorders associated with transport, e.g., Alzheimer's disease, amnesia,
bipolar disorder, dementia,
depression, epilepsy, Tourette's disorder, paranoid psychoses, and
schizophrenia, and other disorders
associated with transport, e.g., neurofibromatosis, postherpetic neuralgia,
trigeminal neuropathy,
sarcoidosis, sickle cell anemia, 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, glycogen
storage disease,
hypercholesterolemia, adrenoleukodystrophy, Zellweger syndrome, Menkes
disease, occipital horn
syndrome, von Gierke disease, pseudohypoaldosteronism type 1, Liddle's
syndrome, cystinuria,
iminoglycinuria, Hartup disease, Fanconi disease, and Banter syndrome; a
neurological disorder such
as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms,
Alzheimer's disease, Pick's
disease, Huntington's disease, dementia, Parkinson's disease and other
extrapyramidal disorders,
amyotrophic lateral sclerosis and other motor neuron disorders, progressive
neural muscular atrophy,
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retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other
demyelinating diseases, bacterial
and viral meningitis, brain abscess, subdural empyema, epidural abscess,
suppurative intracranial
thrombophlebitis, myelitis and radiculitis, viral central nervous system
disease, prion diseases including
kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome,
fatal familial
insomnia, nutritional and metabolic diseases of the nervous system,
neurofibromatosis, tuberous
sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal
syndrome, mental retardation
and other developmental disorders of the central nervous system including Down
syndrome, cerebral
palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial
nerve disorders, spinal cord
diseases, muscular dystrophy and other neuromuscular disorders, peripheral
nervous system disorders,
dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic
myopathies, myasthenia
gravis, periodic paralysis, mental disorders including mood, anxiety, and
schizophrenic disorders,
seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic
neuropathy, hemiplegic
migraine, tardive dyskinesia, dystonias, paranoid psychoses, postheipetic
neuralgia, Tourette's
disorder, progressive supranuclear palsy, corticobasal degeneration, and
familial frontotemporal
dementia; a muscle disorder such as cardiomyopathy, myocarditis, Duchenne's
muscular dystrophy,
Becker's muscular dystrophy, myotonic dystrophy, central core disease,
nemaline myopathy,
centronuclear myopathy, lipid myopathy, mitochondrial myopathy, infectious
myositis, polymyositis,
dermatomyositis, inclusion body myositis, thyrotoxic myopathy, ethanol
myopathy, angina, anaphylactic
shock, arrhythmias, asthma, cardiovascular shock, Cushing's syndrome,
hypertension, hypoglycemia,
myocardial infarction, migraine, pheochromocytoma, and myopathies including
encephalopathy,
epilepsy, Kearns-Sayre syndrome, lactic acidosis, myoclonic disorder,
ophthalmoplegia, acid maltase
deficiency (AMD, also known as Pompe's disease), generalized myotonia, and
myotonia congenita; an
immunological disorder such as acquired immunodeficiency syndrome (A)DS),
Addison's disease,
adult respiratory distress syndrome, allergies, ankylosing spondylitis,
amyloidosis, anemia, asthma,
atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis,
autoimmune
polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis,
cholecystitis, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema, episodic
lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum,
atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis,
hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's
syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial,
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fungal, parasitic, protozoal, and helminthic infections, and trauma; and a
cell proliferative disorder such
as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis,
hepatitis, mixed connective
tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria,
polycythemia vera,
psoriasis, primary thrombocythemia, and cancers including adenocarcinoma,
leukemia, lymphoma,
melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of
the adrenal gland,
bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract, heart,
kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate,
salivary glands, skin, spleen,
testis, thymus, thyroid, and uterus.
In another embodiment, a vector capable of expressing TRICH 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 TRICH including, but not limited to, those described
above.
In a further embodiment, a composition comprising a substantially purified
TRICH 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 TRICH
including, but not limited to,
those provided above.
In still another embodiment, an agonist which modulates the activity of TRICH
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of TRICH including, but not limited to, those listed above. ,
In a further embodiment, an antagonist of TRICH may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of TRICH.
Examples of such
disorders include, but are not limited to, those transport, neurological,
muscle, immunological and cell
proliferative disorders described above. In one aspect, an antibody which
specifically binds TRICH
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 TRICH.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding TRICH may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of TRICH 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.
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An antagonist of TRICH may be produced using methods which are generally known
in the
art. In particular, purified TRICH may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind TRICH.
Antibodies to TRICH 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. Single chain
antibodies (e.g., from
camels or llamas) may be potent enzyme inhibitors and may have advantages in
the design of peptide
mimetics, and in the development of immuno-adsorbents and biosensors
(Muyldermans, S. (2001) J.
Biotechno1.74:277-302).
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, camels,
dromedaries, llamas, humans, and others may be immunized by injection with
TRICH 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 Corynebacterium
parvum are especially
preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
TRICH have an amino acid sequence consisting of at least about 5 amino acids,
and generally will
consist of at least about 10 amino acids. It is also preferable that these
oligopeptides, peptides, or
fragments are identical to a portion of the amino acid sequence of the natural
protein. Short stretches
of TRICH 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 TRICH 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,
CA 02438206 2003-08-06
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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
TRICH-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 TRICH may also be
generated.
For example, such fragments include, but are not limited to, F(ab~2 fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab~2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.) ,
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
TRICH and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive
to two non-interfering TRICH 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 TRICH. Affinity is
expressed as an association
constant, K.~, which is defined as the molar concentration of TRICH-antibody
complex divided by the
molar concentrations of free antigen and free antibody under equilibrium
conditions. The Ka
determined for a preparation of polyclonal antibodies, which are heterogeneous
in their affinities for
multiple TRICH epitopes, represents the average affinity, or avidity, of the
antibodies for TRICH.
The I~ determined for a preparation of monoclonal antibodies, which are
monospecific for a particular
TRICH epitope, represents a true measure of affinity. High-affinity antibody
preparations with Ka
ranging from about 109 to 10'2 L/mole are preferred for use in immunoassays in
which the TRICH-
antibody complex must withstand rigorous manipulations. Low-affinity antibody
preparations with Ka
ranging from about 106 to 10' L/mole are preferred for use in
immunopurification and similar
procedures which ultimately require dissociation of TRICH, preferably in
active form, from the
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antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL
Press, Washington DC;
Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies,
John Wiley & Sons,
New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to determine
the quality and suitability of such preparations for certain downstream
applications. For example, a
polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml, preferably 5-10 mg
specific antibody/ml, is generally employed in procedures requiring
precipitation of TRICH-antibody
complexes. Procedures for evaluating antibody specificity, titer, and avidity,
and guidelines for
antibody quality and usage in various applications, are generally available.
(See, e.g., Catty, supra, and
Coligan et al. supra.)
In another embodiment of the invention, the polynucleotides encoding TRICH, or
any
fragment or complement thereof, may be used for therapeutic purposes. In one
aspect, modifications
of gene expression can be achieved by designing complementary sequences or
antisense molecules
(DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory
regions of the gene
encoding TRICH. Such technology is well known in the art, and antisense
oligonucleotides or larger
fragments can be designed from various locations along the coding or control
regions of sequences
encoding TRICH. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics,
Humana Press Inc.,
Totawa NJ.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. (1990) Blood
76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et
al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al. (1997)
Nucleic Acids Res.
25(14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding TRICH may be
used for
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease
characterized by X-
linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672),
severe combined
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immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-
242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites (e.g.,
against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore, D. (1988)
Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA
93:11395-11399), hepatitis
B or C virus (HBV, HCV); fungal parasites, such as Candida albicans and
Paracoccidioides
brasiliensis; and protozoan parasites such as Plasmodium falciparum and
Trv_panosoma cruzi). In the
case where a genetic deficiency in TRICH expression or regulation causes
disease, the expression of
TRICH from an appropriate population of transduced cells may alleviate the
clinical manifestations
caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by
deficiencies in
TRICH are treated by constructing mammalian expression vectors encoding TRICH
and introducing
these vectors by mechanical means into TRICH-deficient cells. Mechanical
transfer technologies for
use with cells in vivo or ex vitro include (i) direct DNA microinjection into
individual cells, (ii) ballistic
gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-
mediated gene transfer, and
(v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu.
Rev. Biochem.
62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H. Recipon
(1998) Curr. Opin.
Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of TRICH include,
but are not
limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors
(Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La
Jolla CA),
and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
TRICH
may be expressed using (i) a constitutively active promoter, (e.g., from
cytomegalovirus (CMV), Rous
sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or (3-actin genes),
(ii) an inducible promoter
(e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992)
Proc. Natl. Acad. Sci.
USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi,
F.M.V. and H.M. Blau
(1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen));
the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the
FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible
promoter (Rossi, F.M.V.
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and H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native
promoter of the endogenous
gene encoding TRICH from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPll~
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of these
standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to TRICH expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding TRICH under the control of an independent promoter or
the retrovirus long
terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and
(iii) a Rev-responsive
element (RRE) along with additional retrovirus cis-acting RNA sequences and
coding sequences
required for efficient vector propagation. Retrovirus vectors (e.g., PFB and
PFBNEO) are
commercially available (Stratagene) and are based on published data (Riviere,
I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The
vector is propagated in
an appropriate vector producing cell line (VPCL) that expresses an envelope
gene with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R. et
al. (1998) J. Virol. 72:9873-9880). U.S. Patent No. 5,910,434 to Rigg ("Method
for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant") discloses
a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by reference.
Propagation of retrovirus vectors, transduction of a population of cells
(e.g., CD4+ T-cells), and the
return of transduced cells to a patient are procedures well known to persons
skilled in the art of gene
therapy and have been well documented (Ranga, U. et al. (1997) J. Virol.
71:7020-7029; Bauer, G. et
al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-4716;
Ranga, U. et al. (1998)
Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding TRICH to cells which have one or more genetic
abnormalities with respect
to the expression of TRICH. The construction and packaging of adenovirus-based
vectors are well
known to those with ordinary skill in the art. Replication defective
adenovirus vectors have proven to
be versatile for importing genes encoding immunoregulatory proteins into
intact islets in the pancreas
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(Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful
adenoviral vectors are
described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors for
gene therapy"),
hereby incorporated by reference. For adenoviral vectors, see also Antinozzi,
P.A. et al. (1999)
Annu. Rev. Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature
18:389:239-242, both
incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding TRICH to target cells which have one or more genetic
abnormalities with
respect to the expression of TRICH. The use of herpes simplex virus (HSV)-
based vectors may be
especially valuable for introducing TRICH to cells of the central nervous
system, for which HSV has
a tropism. The construction and packaging of herpes-based vectors are well
known to those with
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
type 1-based vector has
been used to deliver a reporter gene to the eyes of primates (Liu, X. et al.
(1999) Exp. Eye Res.
169:385-395). The construction of a HSV-1 virus vector has also been disclosed
in detail in U.S.
Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is hereby
incorporated by reference. U.S. Patent No. 5,804,413 teaches the use of
recombinant HSV d92
which consists of a genome containing at least one exogenous gene to be
transferred to a cell under
the control of the appropriate promoter for purposes including human gene
therapy. Also taught by
this patent are the construction and use of recombinant HSV strains deleted
for ICP4, ICP27 and
ICP22. For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-
532 and Xu, H. et al.
(1994) Dev. Biol. 163:152-161, hereby incorporated by reference. The
manipulation of cloned
herpesvirus sequences, the generation of recombinant virus following the
transfection of multiple
plasmids containing different segments of the large herpesvirus genomes, the
growth and propagation
of herpesvirus, and the infection of cells with herpesvirus are techniques
well known to those of
ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding TRICH to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based
on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During
alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full length
genomic RNA,
resulting in the overproduction of capsid proteins relative to the viral
proteins with enzymatic activity
(e.g., protease and polymerase). Similarly, inserting the coding sequence for
TRICH into the
alphavirus genome in place of the capsid-coding region results in the
production of a large number of
TRICH-coding RNAs and the synthesis of high levels of TRICH in vector
transduced cells. While
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alphavirus infection is typically associated with cell lysis within a few
days, the ability to establish a
persistent infection in hamster normal kidney cells (BHK-21) with a variant of
Sindbis virus (SIN)
indicates that the lytic replication of alphaviruses can be altered to suit
the needs of the gene therapy
application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host
range of alphaviruses will
allow the introduction of TRICH into a variety of cell types. The specific
transduction of a subset of
cells in a population may require the sorting of cells prior to transduction.
The methods of
manipulating infectious cDNA clones of alphaviruses, performing alphavirus
cDNA and RNA
transfections, and performing alphavirus infections, are well known to those
with ordinary skill in the
art.
Oligonucleotides derived from the transcription initiation site, e.g., between
about positions -10
and +10 from the start site, may also be employed to inhibit gene expression.
Similarly, inhibition can
be achieved using triple helix base-pairing methodology. Triple helix pairing
is useful because it
causes inhibition of the ability of the double helix to open sufficiently for
the binding of polymerases,
transcription factors, or regulatory molecules. Recent therapeutic advances
using triplex DNA have
been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in
Huber, B.E. and B.I. Carr,
Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-
177.) A
complementary sequence or antisense molecule may also be designed to block
translation of mRNA
by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding TRICH.
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
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sequences encoding TRICH. Such DNA sequences may be incorporated into a wide
variety of
vectors with suitable RNA polymerise promoters such as T7 or SP6.
Alternatively, these cDNA
constructs that synthesize complementary RNA, constitutively or inducibly, can
be introduced into cell
lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half-
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3' ends
of the molecule, or the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages
within the backbone of the molecule. This concept is inherent in the
production of PNAs and can be
extended in all of these molecules by the inclusion of nontraditional bases
such as inosine, queosine,
and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified
forms of adenine, cytidine,
guanine, thymine, and uridine which are not as easily recognized by
endogenous, endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding TRICH. Compounds
which may be effective in altering expression of a specific polynucleotide may
include, but are not
limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming
oligonucleotides,
transcription factors and other polypeptide transcriptional regulators, and
non-macromolecular
chemical entities which are capable of interacting with specific
polynucleotide sequences. Effective
compounds may alter polynucleotide expression by acting as either inhibitors
or promoters of
polynucleotide expression. Thus, in the treatment of disorders associated with
increased TRICH
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding TRICH may be therapeutically useful, and in the treatment of
disorders associated with
decreased TRICH expression or activity, a compound which specifically promotes
expression of the
polynucleotide encoding TRICH may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for
effectiveness in
altering expression of a specific polynucleotide. A test compound may be
obtained by any method
commonly known in the art, including chemical modification of a compound known
to be effective in
altering polynucleotide expression; selection from an existing, commercially-
available or proprietary
library of naturally-occurring or non-natural chemical compounds; rational
design of a compound
based on chemical and/or structural properties of the target polynucleotide;
and selection from a
library of chemical compounds created combinatorially or randomly. A sample
comprising a
polynucleotide encoding T'RICH is exposed to at least one test compound thus
obtained. The sample
may comprise, for example, an intact or permeabilized cell, or an in vitro
cell-free or reconstituted
biochemical system. Alterations in the expression of a polynucleotide encoding
TRICH are assayed
by any method commonly known in the art. Typically, the expression of a
specific nucleotide is
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detected by hybridization with a probe having a nucleotide sequence
complementary to the sequence
of the polynucleotide encoding TRICH. The amount of hybridization may be
quantified, thus forming
the basis for a comparison of the expression of the polynucleotide~both with
and without exposure to
one or more test compounds. Detection of a change in the expression of a
polynucleotide exposed to
a test compound indicates that the test compound is effective in altering the
expression of the
polynucleotide. A screen for a compound effective in altering expression of a
specific polynucleotide
can be carned out, for example, using a Schizosaccharom~pombe gene expression
system (Atkins,
D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic
Acids Res. 28:E15) or a
human cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem.
Biophys. Res. Commun.
268:8-13). A particular embodiment of the present invention involves screening
a combinatorial library
of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide
nucleic acids, and modified
oligonucleotides) for antisense activity against a specific polynucleotide
sequence (Bruice, T.W. et al.
(1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No.
6,022,691).
Many methods for introducing vectors into cells or tissues are available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
B iotechnol . 15 :462-466. )
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
composition which
generally comprises an active ingredient formulated with a pharmaceutically
acceptable excipient.
Excipients may include, for example, sugars, starches, celluloses, gums, and
proteins. Various
formulations are commonly known and are thoroughly discussed in the latest
edition of Remin tg on's
Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may
consist of TRICH,
antibodies to TRICH, and mimetics, agonists, antagonists, or inhibitors of
TRICH.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, infra-
arterial, intramedullary, intrathecal,
intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal,
intranasal, enteral, topical,
sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
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case of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of fast-
acting formulations is well-known in the art. In the case of macromolecules
(e.g. larger peptides and
proteins), recent developments in the field of pulmonary delivery via the
alveolar region of the lung
have enabled the practical delivery of drugs such as insulin to blood
circulation (see, e.g., Patton, J.S.
et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage of
administration without
needle injection, and obviates the need for potentially toxic penetration
enhancers.
Compositions suitable for use in the invention include compositions wherein
the active
ingredients are contained in an effective amount to achieve the intended
purpose. The determination
of an effective dose is well within the capability of those skilled in the
art.
Specialized forms of compositions may be prepared for direct intracellular
delivery of
macromolecules comprising TRICH or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of the
macromolecule. Alternatively, TRICH or a fragment thereof may be joined to a
short cationic N-
terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated
have been found to
transduce into the cells of all tissues, including the brain, in a mouse model
system (Schwarze, S.R. et
al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs, monkeys,
or pigs. An animal model may also be used to determine the appropriate
concentration range and
route of administration. Such information can then be used to determine useful
doses and routes for
administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example
TRICH or fragments thereof, antibodies of TRICH, and agonists, antagonists or
inhibitors of TRICH,
which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity
may be determined
by standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDso (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDso/EDSO ratio. Compositions
which exhibit large
therapeutic indices are preferred. The data obtained from cell culture assays
and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDSO
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
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subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and response
to therapy. Long-acting compositions may be administered every 3 to 4 days,
every week, or
biweekly depending on the half-life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about 0.1 ~cg to 100,000 ~sg, 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 TRICH may be used
for the
diagnosis of disorders characterized by expression of TRICH, or in assays to
monitor patients being
treated with TRICH or agonists, antagonists, or inhibitors of TRICH.
Antibodies useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic
assays for TRICH include methods which utilize the antibody and a label to
detect TRICH 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 TRICH, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
TRICH expression. Normal
or standard values for TRICH expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibodies to TRICH under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of TRICH
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 TRICH 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 TRICH
may be correlated
with disease. The diagnostic assay may be used to determine absence, presence,
and excess
CA 02438206 2003-08-06
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expression of TRICH, and to monitor regulation of TRICH levels during
therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding TRICH or closely related
molecules may be used
to identify nucleic acid sequences which encode TRICH. 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 TRICH, 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 TRICH encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ 1D
N0:21-40 or from
genomic sequences including promoters, enhancers, and introns of the TRICH
gene.
Means for producing specific hybridization probes for DNAs encoding TRICH
include the
cloning of polynucleotide sequences encoding TRICH or TRICH derivatives into
vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and may
be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as 32P or 35S,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding TRICH may be used for the diagnosis of
disorders
associated with expression of TRICH. Examples of such disorders include, but
are not limited to, a
transport disorder such as akinesia, amyotrophic lateral sclerosis, ataxia
telangiectasia, cystic fibrosis,
Becker's muscular dystrophy, Bell's palsy, Charcot-Marie Tooth disease,
diabetes mellitus, diabetes
insipidus, diabetic neuropathy, Duchenne muscular dystrophy, hyperkalemic
periodic paralysis,
normokalemic periodic paralysis, Parkinson's disease, malignant hyperthermia,
multidrug resistance,
myasthenia gravis, myotonic dystrophy, catatonia, tardive dyskinesia,
dystonias, peripheral neuropathy,
cerebral neoplasms, prostate cancer, cardiac disorders associated with
transport, e.g., angina,
bradyarrythmia, tachyarrythmia, hypertension, Long QT syndrome, myocarditis,
cardiomyopathy,
nemaline myopathy, centronuclear myopathy, lipid myopathy, mitochondrial
myopathy, thyrotoxic
myopathy, ethanol myopathy, dermatomyositis, inclusion body myositis,
infectious myositis,
polymyositis, neurological disorders associated with transport, e.g.,
Alzheimer's disease, amnesia,
bipolar disorder, dementia, depression, epilepsy, Tourette's disorder,
paranoid psychoses, and
schizophrenia, and other disorders associated with transport, e.g.,
neurofibromatosis, postherpetic
neuralgia, trigeminal neuropathy, sarcoidosis, sickle cell anemia, Wilson's
disease, cataracts, infertility,
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pulmonary artery stenosis, sensorineural autosomal deafness, hyperglycemia,
hypoglycemia, Grave's
disease, goiter, Cushing's disease, Addison's disease, glucose-galactose
malabsorption syndrome,
glycogen storage disease, hypercholesterolemia, adrenoleukodystrophy,
Zellweger syndrome, Menkes
disease, occipital horn syndrome, von Gierke disease, pseudohypoaldosteronism
type 1, Liddle's
syndrome, cystinuria, iminoglycinuria, Hartup disease, Fanconi disease, and
Banter syndrome; a
neurological disorder such as epilepsy, ischemic cerebrovascular disease,
stroke, cerebral neoplasms,
Alzheimer's disease, Pick's disease, Huntington's disease, dementia,
Parkinson's disease and other
extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron
disorders, progressive
neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple
sclerosis and other
demyelinating diseases, bacterial and viral meningitis, brain abscess,
subdural empyema, epidural
abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis,
viral central nervous system
disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and
Gerstrnann-Straussler-Scheinker
syndrome, fatal familial insomnia, nutritional and metabolic diseases of the
nervous system,
neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis,
encephalotrigeminal
syndrome, mental retardation and other developmental disorders of the central
nervous system
including Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic
nervous system
disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy
and other neuromuscular
disorders, peripheral nervous system disorders, dermatomyositis and
polymyositis, inherited, metabolic,
endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental
disorders including
mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD),
akathesia, amnesia,
catatonia, diabetic neuropathy, hemiplegic migraine, tardive dyskinesia,
dystonias, paranoid psychoses,
postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy,
conicobasal degeneration,
and familial frontotemporal dementia; a muscle disorder such as
cardiomyopathy, myocarditis,
Duchenne's muscular dystrophy, Becker's muscular dystrophy, myotonic
dystrophy, central core
disease, nemaline myopathy, centronuclear myopathy, lipid myopathy,
mitochondrial myopathy,
infectious myositis, polymyositis, dermatomyositis, inclusion body myositis,
thyrotoxic myopathy,
ethanol myopathy, angina, anaphylactic shock, arrhythmias, asthma,
cardiovascular shock, Cushing's
syndrome, hypertension, hypoglycemia, myocardial infarction, migraine,
pheochromocytoma, and
myopathies including encephalopathy, epilepsy, Kearns-Sayre syndrome, lactic
acidosis, myoclonic
disorder, ophthalmoplegia, acid maltase deficiency (AMD, also known as Pompe's
disease),
generalized myotonia, and myotonia congenita; an immunological disorder such
as acquired
immunodeficiency syndrome (A>DS), Addison's disease, adult respiratory
distress syndrome, allergies,
ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis,
autoimmune hemolytic anemia,
autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal
dystrophy
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(APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease,
atopic dermatitis,
dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with
lymphocytotoxins,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's
syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,
irritable bowel syndrome,
multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation,
osteoarthritis,
osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome,
rheumatoid arthritis, scleroderma,
Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus,
systemic sclerosis,
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome,
complications of cancer,
hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,
parasitic, protozoal, and helminthic
infections, and trauma; and a cell proliferative disorder such as actinic
keratosis, arteriosclerosis,
atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue
disease (MCTD), myelofibrosis,
paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary
thrombocythemia, and
cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,
sarcoma,
teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder,
bone, bone marrow, brain,
breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney,
liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis,
thymus, thyroid, and uterus.
The polynucleotide sequences encoding TRICH may be used in Southern or
northern analysis, dot blot,
or other membrane-based technologies; in PCR technologies; in dipstick, pin,
and multiformat ELISA-
like assays; and in microarrays utilizing fluids or tissues from patients to
detect altered TRICH
expression. Such qualitative or quantitative methods are well known in the
art.
In a particular aspect, the nucleotide sequences encoding TRICH may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding TRICH 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
TRICH 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
TRICH, 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 TRICH, under conditions suitable for
hybridization or
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amplification. Standard hybridization may be quantified by comparing the
values obtained from normal
subjects with values from an experiment in which a known amount of a
substantially purified
polynucleotide is used. Standard values obtained in this manner may be
compared with values
obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard
values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the development
of the disease, or may provide a means for detecting the disease prior to the
appearance of actual
clinical symptoms. A more definitive diagnosis of this type may allow health
professionals to employ
preventative measures or aggressive treatment earlier thereby preventing the
development or further
progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding TRICH
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 TRICH, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
TRICH, and will be employed under optimized conditions for identification of a
specific gene or
condition. Oligomers may also be employed under less stringent conditions for
detection or
quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding TRICH may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are
substitutions, insertions and deletions that are a frequent cause of inherited
or acquired genetic disease
in humans. Methods of SNP detection include, but are not limited to, single-
stranded conformation
polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP,
oligonucleotide primers
derived from the polynucleotide sequences encoding TRICH are used to amplify
DNA using the
polymerase chain reaction (PCR). The DNA may be derived, for example, from
diseased or normal
tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause
differences in the
secondary and tertiary structures of PCR products in single-stranded form, and
these differences are
detectable using gel electrophoresis in non-denaturing gels. In fSCCP, the
oligonucleotide primers are
fluorescently labeled, which allows detection of the amplimers in high-
throughput equipment such as
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DNA sequencing machines. Additionally, sequence database analysis methods,
termed in silico SNP
(isSNP), are capable of identifying polymorphisms by comparing the sequence of
individual
overlapping DNA fragments which assemble into a common consensus sequence.
These computer-
based methods filter out sequence variations due to laboratory preparation of
DNA and sequencing
errors using statistical models and automated analyses of DNA sequence
chromatograms. In the
alternative, SNPs may be detected and characterized by mass spectrometry
using, for example, the
high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
SNPs may be used to study the genetic basis of human disease. For example, at
least 16
common SNPs have been associated with non-insulin-dependent diabetes mellitus.
SNPs are also
useful for examining differences in disease outcomes in monogenic disorders,
such as cystic fibrosis,
sickle cell anemia, or chronic granulomatous disease. For example, variants in
the mannose-binding
lectin, MBL2, have been shown to be correlated with deleterious pulmonary
outcomes in cystic
fibrosis. SNPs also have utility in pharmacogenomics, the identification of
genetic variants that
influence a patient's response to a drug, such as life-threatening toxicity.
For example, a variation in
N-acetyl transferase is associated with a high incidence of peripheral
neuropathy in response to the
anti-tuberculosis drug isoniazid, while a variation in the core promoter of
the ALOXS gene results in
diminished clinical response to treatment with an anti-asthma drug that
targets the S-lipoxygenase
pathway. Analysis of the distribution of SNPs in different populations is
useful for investigating
genetic drift, mutation, recombination, and selection, as well as for tracing
the origins of populations
and their migrations. (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512;
Kwok, P.-Y. and Z. Gu
(1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) Curr. Opin.
Neurobiol. 11:637-641.)
Methods which may also be used to quantify the expression of TRICH include
radiolabeling or
biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives rapid
quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described below. The microarray may also be
used to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, to monitor
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progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used
to develop a pharmacogenomic profile of a patient in order to select the most
appropriate and effective
treatment regimen for that patient. For example, therapeutic agents which are
highly effective and
display the fewest side effects may be selected for a patient based on his/her
pharmacogenomic
profile.
In another embodiment, TRICH, fragments of TRICH, or antibodies specific for
TRICH may
be used as elements on a microarray. The microarray may be used to monitor or
measure protein-
protein interactions, drug-target interactions, and gene expression profiles,
as described above.
A particular embodiment relates to the use of the polynucleotides of the
present invention to
generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern of
gene expression by a particular tissue or cell type. Global gene expression
patterns are analyzed by
quantifying the number of expressed genes and their relative abundance under
given conditions and at
a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent No.
5,840,484, expressly incorporated by reference herein.) Thus a transcript
image may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines, biopsies,
or other biological samples. The transcript image may thus reflect gene
expression in vivo, as in the
case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the
present invention
may also be used in conjunction with in vitro model systems and preclinical
evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and toxicity
(Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L.
Anderson (2000)
Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein).
If a test compound has a
signature similar to that of a compound with known toxicity, it is likely to
share those toxic properties.
These fingerprints or signatures are most useful and refined when they contain
expression information
from a large number of genes and gene families. Ideally, a genome-wide
measurement of expression
provides the highest quality signature. Even genes whose expression is not
altered by any tested
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compounds are important as well, as the levels of expression of these genes
are used to normalize the
rest of the expression data. The normalization procedure is useful for
comparison of expression data
after treatment with different compounds. While the assignment of gene
function to elements of a
toxicant signature aids in interpretation of toxicity mechanisms, knowledge of
gene function is not
necessary for the statistical matching of signatures which leads to prediction
of toxicity. (See, for
example, Press Release 00-02 from the National Institute of Environmental
Health Sciences, released
February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm.)
Therefore, it is
important and desirable in toxicological screening using toxicant signatures
to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological sample
containing nucleic acids with the test compound. Nucleic acids that are
expressed in the treated
biological sample are hybridized with one or more probes specific to the
polynucleotides of the present
invention, so that transcript levels corresponding to the polynucleotides of
the present invention may be
quantified. The transcript levels in the treated biological sample are
compared with levels in an
untreated biological sample. Differences in the transcript levels between the
two samples are
indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell .type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a proteome
can be subjected individually to further analysis. Proteome expression
patterns, or profiles, are
analyzed by quantifying the number of expressed proteins and their relative
abundance under given
conditions and at a given time. A profile of a cell's proteome may thus be
generated by separating
and analyzing the polypeptides of a particular tissue or cell type. In one
embodiment, the separation is
achieved using two-dimensional gel electrophoresis, in which proteins from a
sample are separated by
isoelectric focusing in the first dimension, and then according to molecular
weight by sodium dodecyl
sulfate slab gel electrophoresis in the second dimension (Steiner and
Anderson, supra). The proteins
are visualized in the gel as discrete and uniquely positioned spots, typically
by staining the gel with an
agent such as Coomassie Blue or silver or fluorescent stains. The optical
density of each protein spot
is generally proportional to the -level of the protein in the sample. The
optical densities of equivalently
positioned protein spots from different samples, for example, from biological
samples either treated or
untreated with a test compound or therapeutic agent, are compared to identify
any changes in protein
spot density related to the treatment. The proteins in the spots are partially
sequenced using, for
example, standard methods employing chemical or enzymatic cleavage followed by
mass
spectrometry. The identity of the protein in a spot may be determined by
comparing its partial
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sequence, preferably of at least 5 contiguous amino acid residues, to the
polypeptide sequences of the
present invention. In some cases, further sequence data may be obtained for
definitive protein
identification.
A proteomic profile may also be generated using antibodies specific for TRICH
to quantify
the levels of TRICH expression. In one embodiment, the antibodies are used as
elements on a
microarray, and protein expression levels are quantified by exposing the
microarray to the sample and
detecting the levels of protein bound to each array element (Lueking, A. et
al. (1999) Anal. Biochem.
270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection
may be performed by
a variety of methods known in the art, for example, by reacting the proteins
in the sample with a thiol-
or amino-reactive fluorescent compound and detecting the amount of
fluorescence bound at each
array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
correlation between transcript and protein abundances for some proteins in
some tissues (Anderson,
N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures may be
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to rapid
degradation of mRNA, so proteomic profiling may be more reliable and
informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
each protein is compared to the amount of the corresponding protein in an
untreated biological sample.
A difference in the amount of protein between the two samples is indicative of
a toxic response to the
test compound in the treated sample. Individual proteins are identified by
sequencing the amino acid
residues of the individual proteins and comparing these partial sequences to
the polypeptides of the
present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are incubated
with antibodies specific to the polypeptides of the present invention. The
amount of protein recognized
by the antibodies is quantified. The amount of protein in the treated
biological sample is compared
with the amount in an untreated biological sample. A difference in the amount
of protein between the
two samples is indicative of a toxic response to the test compound in the
treated sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Sehena, M. et al.
(1996) Proc. Natl. Acad.
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Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application
W095/251116; Shalom D. et
al. (1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA
94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.)
Various types of
microarrays are well known and thoroughly described in DNA Microarrays: A
Practical Approach,
M. Schena, ed. (1999) Oxford University Press, London, hereby expressly
incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding TRICH
may be
used to generate hybridization probes useful in mapping the naturally
occurring genomic sequence.
Either coding or noncoding sequences may be used, and in some instances,
noncoding sequences may
be preferable over coding sequences. For example, conservation of a coding
sequence among
members of a multi-gene family may potentially cause undesired cross
hybridization during
chromosomal mapping. The sequences may be mapped to a particular chromosome,
to a specific
region of a chromosome, or to artificial chromosome constructions, e.g., human
artificial chromosomes
(HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes
(BACs), bacterial P1
constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop
genetic linkage maps, for example, which correlate the inheritance of a
disease state with the
inheritance of a particular chromosome region or restriction fragment length
polymorphism (RFLP).
(See, for example, Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci.
USA 83:7353-7357.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic
map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-
968.) Examples of genetic
map data can be found in various scientific journals or at the Online
Mendelian Inheritance in Man
(OMIM) World Wide Web site. Correlation between the location of the gene
encoding TRICH on a
physical map and a specific disorder, or a predisposition to a specific
disorder, may help define the
region of DNA associated with that disorder and thus may further positional
cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
may reveal associated markers even if the exact chromosomal locus is not
known. This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely
localized by genetic linkage to a particular genomic region, e.g., ataxia-
telangiectasia to l 1q22-23, any
sequences mapping to that area may represent associated or regulatory genes
for further investigation.
(See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide
sequence of the instant
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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, TRICH, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used for screening libraries of compounds ,in any
of a variety of drug
screening techniques. The fragment employed in such screening may be free in
solution, affixed to a
solid support, borne on a cell surface, or located intracellularly. The
formation of binding complexes
between TRICH and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with TRICH,
or fragments thereof,
and washed. Bound TRICH is then detected by methods well known in the art.
Purified TRICH 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 TRICH specifically compete with a test compound
for binding TRICH.
In this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with TRICH.
In additional embodiments, the nucleotide sequences which encode TRICH 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 embodiments are, therefore,
to be construed as merely illustrative, and not limitative of the remainder of
the disclosure in any way
whatsoever.
The disclosures of all patents, applications and publications, mentioned above
and below, in
particular U.S. Ser. No. 60/267,892, U.S. Ser. No. 60/271,168, U.S. Ser. No.
60/272,890, U.S. Ser.
No. 60/276,860, U.S. Ser. No. 60/278,255, U.S. Ser. No. 60/280,538 and U.S.
Ser. No. [Attorney
Docket No. PF-1366, filed January 25, 2002] are expressly incorporated by
reference herein.
EXAMPLES
I. Construction of cDNA Libraries
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Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database (Incyte Genomics, Palo Alto CA). Some tissues were homogenized and
lysed in guanidinium
isothiocyanate, while others were homogenized and lysed in phenol or in a
suitable mixture of
denaturants, such as TRIZOL (Life Technologies), a monophasic solution of
phenol and guanidine
isothiocyanate. The resulting lysates were centrifuged over CsCI cushions or
extracted with
chloroform. RNA was precipitated from the lysates with either isopropanol or
sodium acetate and
ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A)+ RNA was
isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX
latex particles
(QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
Alternatively,
RNA was isolated directly from tissue lysates using other RNA isolation kits,
e.g., the
POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the
UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life
Technologies), using
the recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra,
units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
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), PSPORTI plasmid (Life Technologies),
PCDNA2.1 plasmid
(Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid
(Invitrogen),
PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE
(Incyte
Genomics), or pINCY (Incyte Genomics), or derivatives thereof. Recombinant
plasmids were
transformed into competent E. coli cells including XL1-Blue, XL1-BIueMRF, or
SOLR from
Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo
excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega); an
AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid,
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QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
R.E.A.L. PREP
96 plasmid purification kit from QIAGEN. Following precipitation, plasmids
were resuspended in 0.1
ml of distilled water and stored, with or without lyophilization, at
4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically using
PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence
scanner
(Labsystems Oy, Helsinki, Finland).
l0 III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation such
as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200
thermal cycler
(MJ Research) in conjunction with the HYDRA microdispenser (Robbins
Scientific) or the
MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as
the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied
Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carned out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics); the
ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction
with standard ABI
protocols and base calling software; or other sequence analysis systems known
in the art. Reading
frames within the cDNA sequences were identified using standard methods
(reviewed in Ausubel,
1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension
using the
techniques disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by
removing
vector, linker, and poly(A) sequences and by masking ambiguous bases, using
algorithms and
programs based on BLAST, dynamic programming, and dinucleotide nearest
neighbor analysis. The
Incyte cDNA sequences or translations thereof 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; PROTEOME databases with sequences from Homo
Sapiens, Rattus norve icus, Mus musculus, Caenorhabditis eleQans,
Saccharomyces cerevisiae,
Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto
CA); hidden Markov
model (HMM)-based protein family databases such as PFAM; and HMM-based protein
domain
databases such as SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA
95:5857-5864; Letunic,
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I. et al. (2002) Nucleic Acids Res. 30:242-244). (HMM is a probabilistic
approach which analyzes
consensus primary structures of gene families. See, for example, Eddy, S.R.
(1996) Curr. Opin.
Struct. Biol. 6:361-365.) The queries were performed using programs based on
BLAST, FASTA,
BLIMPS, and HMMER. The Incyte cDNA sequences were assembled to produce full
length
polynucleotide sequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitched
sequences,
stretched sequences, or Genscan-predicted coding sequences (see Examples IV
and V) were used to
extend Incyte cDNA assemblages to full length. Assembly was performed using
programs based on
Phred, Phrap, and Consed, and cDNA assemblages 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 polypeptide sequences.
Alternatively, a polypeptide
of the invention may begin at any of the methionine residues of the full
length translated polypeptide.
Full length polypeptide sequences were subsequently analyzed by querying
against databases such as
the GenBank protein databases (genpept), SwissProt, the PROTEOME databases,
BLOCKS,
PRINTS, DOMO, PRODOM, Prosite, hidden Markov model (HMM)-based protein family
databases
such as PFAM; and HMM-based protein domain databases such as SMART. Full
length
polynucleotide sequences are also analyzed using MACDNASIS PRO software
(Hitachi Software
Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
Polynucleotide
and polypeptide sequence alignments are generated using 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.
Table 7 summarizes the tools, programs, and algorithms used for the analysis
and assembly of
Incyte cDNA and full length sequences and provides applicable descriptions,
references, and threshold
parameters. The first column of Table 7 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 or the lower the probability
value, the greater the
identity between two sequences).
The programs described above for the assembly and analysis of full length
polynucleotide and
polypeptide sequences were also used to identify polynucleotide sequence
fragments from SEQ ID
N0:21-40. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
amplification technologies are described in Table 4, column 2.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative transporters and ion channels were initially identified by running
the Genscan gene
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identification program against public genomic sequence databases (e.g., gbpri
and gbhtg). Genscan is
a general-purpose gene identification program which analyzes genomic DNA
sequences from a
variety of organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-
94, and Burge, C. and
S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354). The program
concatenates predicted exons to
form an assembled cDNA sequence extending from a methionine to a stop codon.
The output of
Genscan is a FASTA database of polynucleotide and polypeptide sequences. The
maximum range of
sequence for Genscan to analyze at once was set to 30 kb. To determine which
of these Genscan
predicted cDNA sequences encode transporters and ion channels, the encoded
polypeptides were
analyzed by querying against PFAM models for transporters and ion channels.
Potential transporters
l0 and ion channels were also identified by homology to Incyte cDNA sequences
that had been
annotated as transporters and ion channels. These selected Genscan-predicted
sequences were then
compared by BLAST analysis to the genpept and gbpri public databases. Where
necessary, the
Genscan-predicted sequences were then edited by comparison to the top BLAST
hit from genpept to
correct errors in the sequence predicted by Genscan, such as extra or omitted
exons. BLAST
analysis was also used to find any Incyte cDNA or public cDNA coverage of the
Genscan-predicted
sequences, thus providing evidence for transcription. When Incyte cDNA
coverage was available,
this information was used to correct or confirm the Genscan predicted
sequence. Full length
polynucleotide sequences were obtained by assembling Genscan-predicted coding
sequences with
Incyte cDNA sequences and/or public cDNA sequences using the assembly process
described in
Example III. Alternatively, full length polynucleotide sequences were derived
entirely from edited or
unedited Genscan-predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
"Stitched" Sequences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification program described in Example IV. Partial cDNAs assembled as
described in Example
III were mapped to genomic DNA and parsed into clusters containing related
cDNAs and Genscan
exon predictions from one or more genomic sequences. Each cluster was analyzed
using an algorithm
based on graph theory and dynamic programming to integrate cDNA and genomic
information,
generating possible splice variants that were subsequently confirmed, edited,
or extended to create a
full length sequence. Sequence intervals in which the entire length of the
interval was present on
more than one sequence in the cluster were identified, and intervals thus
identified were considered to
be equivalent by transitivity. For example, if an interval was present on a
cDNA and two genomic
sequences, then all three intervals were considered to be equivalent. This
process allows unrelated
but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals
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thus identified were then "stitched" together by the stitching algorithm in
the order that they appear
along their parent sequences to generate the longest possible sequence, as
well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence
(cDNA to cDNA or
genomic sequence to genomic sequence) were given preference over linkages
which change parent
type (cDNA to genomic sequence). The resultant stitched sequences were
translated and compared
by BLAST analysis to the genpept and gbpri public databases. Incorrect exons
predicted by Genscan
were corrected by comparison to the top BLAST hit from genpept. Sequences were
further extended
with additional cDNA sequences, or by inspection of genomic DNA, when
necessary.
"Stretched" Sequences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example III were
queried against public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases
using the BLAST program. The nearest GenBank protein homolog was then compared
by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences
described in
Example IV. A chimeric protein was generated by using the resultant high-
scoring segment pairs
(HSPs) to map the translated sequences onto the GenBank protein homolog.
Insertions or deletions
may occur in the chimeric protein with respect to the original GenBank protein
homolog. The
GenBank protein homolog, the chimeric protein, or both were used as probes to
search for homologous
genomic sequences from the public human genome databases. Partial DNA
sequences were
therefore "stretched" or extended by the addition of homologous genomic
sequences. The resultant
stretched sequences were examined to determine whether it contained a complete
gene.
VI. Chromosomal Mapping of TRICH Encoding Polynucleotides
The sequences which were used to assemble SEQ ID N0:21-40 were compared with
sequences from the Incyte L1FESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ ID N0:21-40 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for
Genome Research (WIGR), and Genethon were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ ID NO:, to that
map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map
position of an interval, in centiMorgans, is measured relative to the terminus
of the chromosome's p-
arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
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chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb)
of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM distances
are based on genetic markers mapped by Genethon which provide boundaries for
radiation hybrid
markers whose sequences were included in each of the clusters. Human genome
maps and other
resources available to the public, such as the NCBI "GeneMap'99" World Wide
Web site
(http://www.ncbi.nlm.nih.gov/genemap/), can be employed to determine if
previously identified disease
genes map within or in proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
from a particular cell type or tissue have been bound. (See, e.g., Sambrook,
supra, ch. 7; Ausubel
(1995) supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the computer
search can be modified to determine whether any particular match is
categorized as exact or similar.
The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity
S x minimum { length(Seq. 1 ), length(Seq. 2) }
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. The product score is a normalized value between
0 and 100, and is
calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
product is divided by (S times the length of the shorter of the two
sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by
gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate
the product score. The product score represents a balance between fractional
overlap and quality in a
BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the
entire length of the shorter of the two sequences being compared. A product
score of 70 is produced
either by 100% identity and 70% overlap at one end, or by 88% identity and
100% overlap at the
other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
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Alternatively, polynucleotide sequences encoding TRICH are analyzed with
respect to the
tissue sources from which they were derived. For example, some full length
sequences are
assembled, at least in part, with overlapping Incyte cDNA sequences (see
Example III). Each cDNA
sequence is derived from a cDNA library constructed from a human tissue. Each
human tissue is
classified into one of the following organ/tissue categories: cardiovascular
system; connective tissue;
digestive system; embryonic structures; endocrine system; exocrine glands;
genitalia, female; genitalia,
male; germ cells; heroic and immune system; liver; musculoskeletal system;
nervous system;
pancreas; respiratory system; sense organs; skin; stomatognathic system;
unclassified/mixed; or
urinary tract. The number of libraries in each category is counted and divided
by the total number of
libraries across all categories. Similarly, each human tissue is classified
into one of the following
disease/condition categories: cancer, cell line, developmental, inflammation,
neurological, trauma,
cardiovascular, pooled, and other, and the number of libraries in each
category is counted and divided
by the total number of libraries across all categories. The resulting
percentages reflect the tissue- and
disease-specific expression of cDNA encoding TRICH. cDNA sequences and cDNA
library/tissue
information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto
CA).
VIII. Extension of TRICH Encoding Polynucleotides
Full length polynucleotide sequences were also 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 was
synthesized 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 Mgz*, (NH4)zSO4,
and 2-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,
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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 ~,1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 p.1 of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(I,absystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ~cl to 10 ~1 aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose gel to determine which reactions were
successful in extending the
sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to relegation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended
clones were relegated using T4 ligase (New England Biolabs, Beverly MA) into
pUC 18 vector
(Amersham Pharmacia Biotech), treated with Pfu DNA polymerise (Stratagene) to
fill-in restriction
site overhangs, and transfected into competent E. coli cells. Transformed
cells were selected on
antibiotic-containing media, and individual colonies were picked and cultured
overnight at 37°C in 384-
well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerise
(Amersham Pharmacia Biotech) and Pfu DNA polymerise (Stratagene) with the
following
parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min; Step
5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7:
storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted with
20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer
sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the
above procedure or
are used to obtain 5' regulatory sequences using the above procedure along
with oligonucleotides
designed for such extension, and an appropriate genomic library.
IX. Identification of Single Nucleotide Polymorphisms in TRICH Encoding
Polynucleotides
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Common DNA sequence variants known as single nucleotide polymorphisms (SNPs)
were
identified in SEQ m N0:21-40 using the LIFESEQ database (Incyte Genomics).
Sequences from the
same gene were clustered together and assembled as described in Example III,
allowing the
identification of all sequence variants in the gene. An algorithm consisting
of a series of filters was
used to distinguish SNPs from other sequence variants. Preliminary filters
removed the majority of
basecall errors by requiring a minimum Phred quality score of 15, and removed
sequence alignment
errors and errors resulting from improper trimming of vector sequences,
chimeras, and splice variants.
An automated procedure of advanced chromosome analysis analysed the original
chromatogram files
in the vicinity of the putative SNP. Clone error filters used statistically
generated algorithms to identify
errors introduced during laboratory processing, such as those caused by
reverse transcriptase,
polymerase, or somatic mutation. Clustering error filters used statistically
generated algorithms to
identify errors resulting from clustering of close homologs or pseudogenes, or
due to contamination by
non-human sequences. A final set of filters removed duplicates and SNPs found
in immunoglobulins
or T-cell receptors.
Certain SNPs weie selected for further characterization by mass spectrometry
using the high
throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at
the SNP sites in
four different human populations. The Caucasian population comprised 92
individuals (46 male, 46
female), including 83 from Utah, four French, three Venezualan, and two Amish
individuals. The
African population comprised 194 individuals (97 male, 97 female), all African
Americans. The
Hispanic population comprised 324 individuals (162 male, 162 female), all
Mexican Hispanic. The
Asian population comprised 126 individuals (64 male, 62 female) with a
reported parental breakdown
of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian.
Allele
frequencies were first analyzed in the Caucasian population; in some cases
those SNPs which showed
no allelic variance in this population were not further tested in the other
three populations.
X. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ 1D N0:21-40 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
~,~, 32P~ adenosine- triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-based
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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.
XI. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, supra.), mechanical
microspotting technologies, and derivatives thereof. The substrate in each of
the aforementioned
technologies should be uniform and solid with a non-porous surface (Schena
(1999), supra).
Suggested substrates include silicon, silica, glass slides, glass chips, and
silicon wafers. Alternatively, a
procedure analogous to a dot or slot blot may also be used to arrange and link
elements to the surface
of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
A typical array may
be produced using available methods and machines well known to those of
ordinary skill in the art and
may contain any appropriate number of elements. (See, e.g., Schena, M. et al.
(1995) Science
270:467-470; Shalom D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and
J. Hodgson (1998)
Nat. Biotechnol. 16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The
array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element on
the microarray may be assessed. In one embodiment, microarray preparation and
usage is described
in detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is
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reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/~,l oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 units/p,l RNase inhibitor, 500 ~.M dATP, 500 ~,M dGTP, 500
~,M dTTP, 40 ~.M
dCTP, 40 p.M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)+ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of O.SM sodium
hydroxide and
incubated for 20 minutes at 85° C to the stop the reaction and degrade
the RNA. Samples are purified
using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONT'ECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is
then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
NY) and resuspended
in 14 ~.1 SX SSC/0.2% SDS.
For SEQ ID N0:36, for example, HMECs, which are a primary human breast
epithelial cell
line isolated from a normal donor, were grown in Mammary Epithelial Cell
Growth Medium (Clonetics,
Walkersville MD) supplemented with 10 ng/ml human recombinant epidermal growth
factor, 5 mg/ml
insulin, 0.5 mg/ml hydrocortisone, 50 mg/ml gentamicin, 50 ng/ml amphotericin-
B, and 0.5 mg/ml
bovine pituitary extract. Cells were grown to 70-80% confluence prior to
harvesting. About 1 x 10'
cells were harvested at passage 8 (progenitor cells), passages 10 and 12
(progressively senescent
cells), passage 14 (presenescent cells), and passage 15 (senescent cells). In
this manner, it was
demonstrated that the expression in senescent cells of component 2812176 of
SEQ ID N0:36 is
increased by a factor of at least 2.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses
primers complementary to the vector sequences flanking the cDNA insert. Array
elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5 ~.g.
Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water, and
coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are
cured in a 110°C
oven.
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Array elements are applied to the coated glass substrate using a procedure
described in U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 ~.l of the array
element DNA, at an average
concentration of 100 ng/~,1, is loaded into the open capillary printing
element by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATAL1NKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°C
followed by washes in 0.2%
SDS and distilled water as before.
to Hybridization
Hybridization reactions contain 9 p,1 of sample mixture consisting of 0.2 ~.g
each of Cy3 and
Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65° C for 5 minutes and is aliquoted onto the
microarray surface and covered with
an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber
having a cavity just slightly
larger than a microscope slide. The chamber is kept at 100% humidity
internally by the addition of 140
~,1 of 5X SSC in a corner of the chamber. The chamber containing the arrays is
incubated for about
6.5 hours at 60° C. The arrays are washed for 10 min at 45° C in
a first wash buffer ( 1 X SSC, 0.1 %
SDS), three times for 10 minutes each at 45°C in a second wash buffer
(0.1X SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
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cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that location
to be correlated with a weight ratio of hybridizing species of 1:100,000. When
two samples from
different sources (e.g., representing test and control cells), each labeled
with a different fluorophore,
are hybridized to a single array for the purpose of identifying genes that are
differentially expressed,
the calibration is done by labeling samples of the calibrating cDNA with the
two fluorophores and
adding identical amounts of each to the hybridization mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping emission
spectra) between the fluorophores using each fluorophore's emission spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each spot
is centered in each element of the grid. The fluorescence signal within each
element is then integrated
to obtain a numerical value corresponding to the average intensity of the
signal. The software used
for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
XII. Complementary Polynucleotides
Sequences complementary to the TRICH-encoding sequences, or any parts thereof,
are used
to detect, decrease, or inhibit expression of naturally occurring TRICH.
Although use of
oligonucleotides comprising from about 15 to 30 base pairs is described,
essentially the same
procedure is used with smaller or with larger sequence fragments. Appropriate
oligonucleotides are
designed using OLIGO 4.06 software (National Biosciences) and the coding
sequence of TRICH. 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 TRICH-encoding
transcript.
XIII. Expression of TRICH
Expression and purification of TRICH is achieved using bacterial or virus-
based expression
systems. For expression of TRICH 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).
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Antibiotic resistant bacteria express TRICH upon induction with isopropyl beta-
D-
thiogalactopyranoside (IPTG). Expression of TRICH in eukaryotic cells is
achieved by infecting
insect or mammalian cell lines with recombinant Au~aphica californica nuclear
polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding TRICH by either homologous recombination or
bacterial-mediated
transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect Spodoptera frugiperda (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
l0 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, TRICH 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 Lnonicum, enables the purification of fusion proteins
on immobilized
glutathione under conditions that maintain protein activity and antigenicity
(Amersham Pharmacia
Biotech). Following purification, the GST moiety can be proteolytically
cleaved from TRICH at
specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity purification
using commercially available monoclonal and polyclonal anti-FLAG antibodies
(Eastman Kodak). 6-
His, a stretch of six consecutive histidine residues, enables purification on
metal-chelate resins
(QIAGEN). Methods for protein expression and purification are discussed in
Ausubel (1995, supra,
ch. 10 and 16). Purified TRICH obtained by these methods can be used directly
in the assays shown
in Examples XVII, XVIII, and XIX, where applicable.
XIV. Functional Assays
TRICH function is assessed by expressing the sequences encoding TRICH 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 ~cg 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;
89
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WO 02/077237 PCT/US02/03657
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate
the apoptotic state of the cells and other cellular properties. FCM detects
and quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events
include changes in nuclear DNA content as measured by staining of DNA with
propidium iodide;
changes in cell size and granularity as measured by forward light scatter and
90 degree side light
scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as
measured by reactivity with
specific antibodies; and alterations in plasma membrane composition as
measured by the binding of
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York NY.
The influence of TRICH on gene expression can be assessed using highly
purified populations
of cells transfected with sequences encoding TRICH and either CD64 or CD64-
GFP. CD64 and
CD64-GFP are expressed on the surface of transfected cells and bind to
conserved regions of human
immunoglobulin G (IgG). Transfected cells are efficiently separated from
nontransfected cells using
magnetic beads coated with either human IgG or antibody against CD64 (DYNAL,
Lake Success
NY). mRNA can be purified from the cells using methods well known by those of
skill in the art.
Expression of mRNA encoding TRICH and other genes of interest can be analyzed
by northern
analysis or microarray techniques.
XV. Production of TRICH Specific Antibodies
TRICH 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 animals (e.g., rabbits, mice, etc.) and to produce antibodies using
standard protocols.
Alternatively, the TRICH 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, supra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
IG.H (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) to
increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are
immunized with the
oligopeptide-ICL,H complex in complete Freund's adjuvant. Resulting antisera
are tested for
antipeptide and anti-TRICH activity by, for example, binding the peptide or
TRICH to a substrate,
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting
with radio-iodinated goat
anti-rabbit IgG.
XVI. Purification of Naturally Occurring TRICH Using Specific Antibodies
Naturally occurring or recombinant TRICH is substantially purified by
immunoaffinity
chromatography using antibodies specific for TRICH. An immunoaffinity column
is constructed by
covalently coupling anti-TRICH 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 TRICH are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of TRICH (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/TRICH 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 TRICH is collected.
XVII. Identification of Molecules Which Interact with TRICH
TRICH, or biologically active fragments thereof, are labeled with'ZSI Bolton-
Hunter reagent.
(See, e.g., Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.)
Candidate molecules
previously arrayed in the wells of a multi-well plate are incubated with the
labeled TRICH, washed,
and any wells with labeled TRICH complex are assayed. Data obtained using
different
concentrations of TRICH are used to calculate values for the number, affinity,
and association of
TRICH with the candidate molecules.
Alternatively, molecules interacting with TRICH are analyzed using the yeast
two-hybrid
system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or
using commercially
available kits based on the two-hybrid system, such as the MATCHMAKER system
(Clontech).
TRICH may also be used in the PATHCALLING process (CuraGen Corp., New Haven
CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S.
Patent No. 6,057,101).
XVII. Identification of Molecules Which Interact with TRICH
Molecules which interact with TRICH may include transporter substrates,
agonists or
antagonists, modulatory proteins such as G(3y proteins (Reimann, supra) or
proteins involved in TRICH
localization or clustering such as MAGUKs (Craven, supra). TRICH, or
biologically active fragments
thereof, are labeled with'ZSI Bolton-Hunter reagent. (See, e.g., Bolton A.E.
and W.M. Hunter (1973)
Biochem. J. 133:529-539.) Candidate molecules previously arrayed in the wells
of a multi-well plate
are incubated with the labeled TRICH, washed, and any wells with labeled TRICH
complex are
91
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
assayed. Data obtained using different concentrations of TRICH are used to
calculate values for the
number, affinity, and association of TRICH with the candidate molecules.
Alternatively, proteins that interact with TRICH are isolated using the yeast
2-hybrid system
(Fields, S. and O. Song (1989) Nature 340:245-246). TRICH, or fragments
thereof, are expressed as
fusion proteins with the DNA binding domain of Gal4 or IexA, and potential
interacting proteins are
expressed as fusion proteins with an activation domain. Interactions between
the TRICH fusion
protein and the TRICH interacting proteins (fusion proteins with an activation
domain) reconstitute a
transactivation function that is observed by expression of a reporter gene.
Yeast 2-hybrid systems are
commercially available, and methods for use of the yeast 2-hybrid system with
ion channel proteins
to are discussed in Niethammer, M. and M. Sheng (1998, Meth. Enzymol. 293:104-
122).
TRICH may also be used in the PATHCALLING process (CuraGen Corp., New Haven
CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S.
Patent No. 6,057,101).
Potential TRICH agonists or antagonists may be tested for activation or
inhibition of TRICH
ion channel activity using the assays described in section XVIII.
XVIII. Demonstration of TRICH Activity
Ion channel activity of TRICH is demonstrated using an electrophysiological
assay for ion
conductance. TRICH can be expressed by transforming a mammalian cell line such
as COS7, HeLa
or CHO with a eukaryotic expression vector encoding TRICH. Eukaryotic
expression vectors are
commercially available, and the techniques to introduce them into cells are
well known to those skilled
in the art. A second plasmid which expresses any one of a number of marker
genes, such as 13-
galactosidase, is co-transformed into the cells to allow rapid identification
of those cells which have
taken up and expressed the foreign DNA. The cells are incubated for 48-72
hours after
transformation under conditions appropriate for the cell line to allow
expression and accumulation of
TRICH and 13-galactosidase.
Transformed cells expressing Q-galactosidase are stained blue when a suitable
colorimetric
substrate is added to the culture media under conditions that are well known
in the art. Stained cells
are tested for differences in membrane conductance by electrophysiological
techniques that are well
known in the art. Untransformed cells, and/or cells transformed with either
vector sequences alone or
Li-galactosidase sequences alone, are used as controls and tested in parallel.
Cells expressing TRICH
will have higher anion or cation conductance relative to control cells. The
contribution of TRICH to
conductance can be confirmed by incubating the cells using antibodies specific
for TRICH. The
antibodies will bind to the extracellular side of TRICH, thereby blocking the
pore in the ion channel,
92
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
and the associated conductance.
Alternatively, ion channel activity of TRICH is measured as current flow
across a TRICH-
containing Xenopus laevis oocyte membrane using the two-electrode voltage-
clamp technique (Ishi et
al., suyra; Jegla, T. and L. Salkoff (1997) J. Neurosci. 17:32-44). TRICH is
subcloned into an
appropriate Xenopus oocyte expression vector, such as pBF, and 0.5-5 ng of
mRNA is injected into
mature stage IV oocytes. Injected oocytes are incubated at 18°C for 1-5
days. Inside-out
macropatches are excised into an intracellular solution containing 116 mM K-
gluconate, 4 mM KCI,
and 10 mM Hepes (pH 7.2). The intracellular solution is supplemented with
varying concentrations of
the TRICH mediator, such as cAMP, cGMP, or Ca+z (in the form of CaClz), where
appropriate.
Electrode resistance is set at 2-5 MS2 and electrodes are filled with the
intracellular solution lacking
mediator. Experiments are performed at room temperature from a holding
potential of 0 mV. Voltage
ramps (2.5 s) from -100 to 100 mV are acquired at a sampling frequency of 500
Hz. Current
measured is proportional to the activity of TRICH in the assay.
Transport activity of TRICH is assayed by measuring uptake of labeled
substrates into
Xenopus laevis oocytes. Oocytes at stages V and VI are injected with TRICH
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 z,
1mM MgCI z, 1mM NazHP04, 5 mM Hepes, 3.8 mM NaOH , SO~,g/ml gentamycin, pH
7.8) to allow
expression of TRICH. Oocytes are then transferred to standard uptake medium
(100mM NaCI, 2
mM KCI, 1mM CaClz, 1mM MgClz, 10 mM Hepes/Tris pH 7.5). Uptake of various
substrates (e.g.,
amino acids, sugars, drugs, ions, and neurotransmitters) is initiated by
adding labeled substrate (e.g.
radiolabeled with 3H, fluorescently labeled with rhodamine, etc.) to the
oocytes. After incubating for
minutes, uptake is terminated by washing the oocytes three times in Na+-free
medium, measuring
the incorporated label, and comparing with controls. TRICH activity is
proportional to the level of
internalized labeled substrate. In particular, test substrates include glucose
and other sugars for
25 TRICH-1, aminophospholipids for TRICH-2, HC03- for TRICH-3, sulfate and
other anions for
TRICH-4, nucleotides for TRICH-5, Na+ and bile acids for TRICH-6, TRICH-8,
cationic amino acids
for TRICH-11, amino acids for TRICH-7, protons for TRICH-9, drugs for TRICH-
12, bile acids for
TRICH-13 and TRICH-17, nucleosides for TRICH-15, drugs and other xenobiotics
for TRICH-16,
and neurotransmitters or organic osmolytes for TRICH-18.
30 ATPase activity associated with TRICH can be measured by hydrolysis of
radiolabeled ATP-
['y-3zp], separation of the hydrolysis products by chromatographic methods,
and quantitation of the
recovered 3zP using a scintillation counter. The reaction mixture contains ATP-
['y-3zP] and varying
amounts of TRICH 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
93
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
aliquot of the reaction mixture is subjected to membrane or filter paper-based
chromatography to
separate the reaction products. The amount of 32P liberated is counted in a
scintillation counter. The
amount of radioactivity recovered is proportional to the ATPase activity of
TRICH in the assay.
Lipocalin activity of TRICH is measured by ligand fluorescence enhancement
spectrofluorometry (Lin et al. (1997) Molecular Vision 3:17). Examples of
ligands include retinol
(Sigma, St. Louis MO) and 16-anthryloxy-palmitic acid (16-AP) (Molecular
Probes Inc., Eugene OR).
Ligand is dissolved in 100% ethanol and its concentration is estimated using
known extinction
coefficents (retinol: 46,000 A/M/cm at 325 nm; 16-AP: 8,200 A/M/cm at 361 nm).
A 700 ~.1 aliquot of
1 ~,M TRICH in 10 mM Tris (pH 7.5), 2 mM EDTA, and 500 mM NaCI is placed in a
1 cm path
length quartz cuvette and 1 ~,1 aliquots of ligand solution are added.
Fluorescence is measured 100
seconds after each addition until readings are stable. Change in fluorescence
per unit change in ligand
concentration is proportional to TRICH activity.
In particular, the activity of TRICH-10 is measured as Ca2+ conductance, the
activity of
TRICH-14 is measured as K+ conductance and the activity of TRICH-19 is
measured as calcium-
activated K+ conductance.
XIX. Identification of TRICH Agonists and Antagonists
TRICH is expressed in a eukaryotic cell line such as CHO (Chinese Hamster
Ovary) or HEK
(Human Embryonic Kidney) 293. Ion channel activity of the transformed cells is
measured in the
presence and absence of candidate agonists or antagonists. Ion channel
activity is assayed using
patch clamp methods well known in the art or as described in Example XVIII.
Alternatively, ion
channel activity is assayed using fluorescent techniques that measure ion flux
across the cell
membrane (Velicelebi, G. et al. (1999) Meth. Enzymol. 294:20-47; West, M.R.
and C.R. Molloy
(1996) Anal. Biochem. 241:51-58). These assays may be adapted for high-
throughput screening using
microplates. Changes in internal ion concentration are measured using
fluorescent dyes such as the
Caz+ indicator Fluo-4 AM, sodium-sensitive dyes such as SBFI and sodium green,
or the CY indicator
MQAE (all available from Molecular Probes) in combination with the FLIPR
fluorimetric plate reading
system (Molecular Devices). In a more generic version of this assay, changes
in membrane potential
caused by ionic flux across the plasma membrane are measured using oxonyl dyes
such as DiBAC4
(Molecular Probes). DiBAC4 equilibrates between the extracellular solution and
cellular sites
according to the cellular membrane potential. The dye's fluorescence intensity
is 20-fold greater
when bound to hydrophobic intracellular sites, allowing detection of DiBAC4
entry into the cell
(Gonzalez, J.E. and P.A. Negulescu (1998) Curr. Opin. Biotechnol. 9:624-631).
Candidate agonists or
antagonists may be selected from known ion channel agonists or antagonists,
peptide libraries, or
combinatorial chemical libraries.
94
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
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.
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
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CA 02438206 2003-08-06
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Table 5
PolynucleotideIncyte ProjectRepresentative Library
SEQ ID:
ID NO:
_21 6911460CB1 BRAXTDR15
22 55138203CB THYMNOR02
1
23 7478871CB KIDNNOT32 _ _ _
1
_25 7487851CB1 LUNGNOT37
26 7472881CB1 LIVRTUE01
27 _ 7612560CB1 K>DCTME01 _ ___ _
28 2880370CB ISLTNOTO1
1
29 6267489CB KIDETXS02
1
_30 7484777CB1 BRADDIROi
31 2493969CB BRAINOY02
1
32 3244593CB BRAENOT02
1
33 4921451CB PANCTUTO1
1
34 5547443CB1 TESTNOT11
35 56008413CB LIVRTUE01
1
36 6127911CB1 BRSTNOTO1
37 6427133CB TLYMNOT08
1
39 8463147CB BRAIFET02
1
40 7506408CB BONSTUTO1
1
129
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CA 02438206 2003-08-06
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< 110 > INCYTE GENOMICS , ?~cj~7 ..
LEE, Ernestine A.
DING, Li
BAUGHN, Mariah R.
TRIBOULEY, Catherine M.
BRUNS, Christopher M.
ELLIOTT, Vicki S.
WALIA, Narinder K.
FORSYTHE, Ian
RAUMANN, Brigitte E.
BURFORD, Neil
LAL, Preeti G.
THORNTON, Michael
GANDHI, Ameena R.
ARIVZU, Chandra
YAO, Monique G.
YUE, Henry
XU, Yuming
HAFALIA, April J.A.
ISON, Craig H.
CHEN, Huei-Mei
<120> TRANSPORTERS AND ION CHANNELS
<130> PI-0356 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/267,892; 60/271,168; 60/272,890; 60/276,860; 60/278,255;
60/280,538; 60/351,359
<151> 2001-02-09; 2001-02-23; 2001-03-02; 2001-03-16; 2001-03-23;
2001-03-30; 2002-O1-25
<160> 40
<170> PERL Program
<210> 1
<211> 617
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte ID No: 6911460CD1
<400> 1
Met Val Pro Val Glu Asn Thr Glu Gly Pro Ser Leu Leu Asn Gln
1 5 10 15
Lys Gly Thr Ala Val Glu Thr,Glu Gly Ser Gly Ser Arg His Pro
20 25 30
Pro Trp Ala Arg Gly Cys Gly Met Phe Thr Phe Leu Ser Ser Val
35 40 45
Thr Ala Ala Val Ser Gly Leu Leu Val Gly Tyr Glu Leu Gly Ile
50 55 60
Ile Ser Gly Ala Leu Leu Gln Ile Lys Thr Leu Leu Ala Leu Ser
1 ..
CA 02438206 2003-08-06
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65 70 75
Cys His Glu Gln Glu Met Val Val Ser Ser Leu Val Ile Gly Ala
80 85 90
Leu Leu Ala Ser Leu.Thr Gly Gly Val Leu Ile Asp Arg Tyr Gly
95 100 105
Arg Arg Thr Ala Ile Ile Leu Ser Ser Cys Leu Leu Gly Leu Gly
110 115 120
Ser Leu Val Leu Ile Leu Ser Leu Ser Tyr Thr Val Leu Ile Val
125 130 135
Gly Arg Ile Ala Ile Gly Val Ser Ile Ser Leu Ser Ser Ile Ala
140 145 150
Thr Cys Val Tyr Ile Ala Glu Ile Ala Pro Gln His Arg Arg Gly
155 160 165
Leu Leu Val Ser Leu Asn Glu Leu Met Ile Val Ile Gly Ile Leu
170 175 180
Ser Ala Tyr Ile Ser Asn Tyr Ala Phe Ala Asn Val Phe His Gly
185 190 195
Trp Lys Tyr Met Phe Gly Leu Val Ile Pro Leu Gly Val Leu Gln
200 205 210
Ala Ile Ala Met Tyr Phe Leu Pro Pro Ser Pro Arg Phe Leu Val
215 220 225
Met Lys Gly Gln Glu Gly Ala Ala Ser Lys Val Leu Gly Arg Leu
230 235 240
Arg Ala Leu Ser Asp Thr Thr Glu Glu Leu Thr Val Ile Lys Ser
245 250 255
Ser Leu Lys Asp Glu Tyr Gln Tyr Ser Phe Trp Asp Leu Phe Arg
260 265 270
Ser Lys Asp Asn Met Arg Thr Arg Ile Met Ile Gly Leu Thr Leu
275 280 285
Val Phe Phe Val Gln Ile Thr Gly Gln Pro Asn Ile Leu Phe Tyr
290 295 300
Ala Ser Thr Val Leu Lys Ser Val Gly Phe Gln Ser Asn Glu Ala
305 310 315
Ala Ser Leu Ala Ser Thr Gly Val Gly Val Val Lys Val Ile Ser
320 325 330
Thr Ile Pro Ala Thr Leu Leu Val Asp His Val Gly Ser Lys Thr
335 340 345
Phe Leu Cys Ile Gly Ser Ser Val Met Ala Ala Ser Leu Val Thr
350 355 360
Met Gly Ile Val Asn Leu Asn Ile His Met Asn Phe Thr His Ile
365 370 375
Cys Arg Ser His Asn Ser Ile Asn Gln Ser Leu Asp Glu Ser Val
380 385 390
Ile Tyr Gly Pro Gly Asn Leu Ser Thr Asn Asn Asn Thr Leu Arg
395 400 405
Asp His Phe Lys Gly Ile Ser Ser His Ser Arg Ser Ser Leu Met
410 415 420
Pro Leu Arg Asn Asp Val Asp Lys Arg Gly Glu Thr Thr Ser Ala
425 430 435
Ser Leu Leu Asn Ala Gly Leu Ser His Thr Glu Tyr Gln Ile Val
440 445 450
Thr Asp Pro Gly Asp Val Pro Ala Phe Leu Lys Trp Leu Ser Leu
455 460 465
Ala Ser Leu Leu Val~Tyr Val Ala Ala Phe Ser Ile Gly Leu Gly
470 475 480
Pro Met Pro Trp Leu Val Leu Ser Glu Ile Phe Pro Gly Gly Ile
2
CA 02438206 2003-08-06
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485 490 495
Arg Gly Arg Ala Met Ala Leu Thr Ser Ser Met Asn Trp Gly Ile
500 505 510
Asn Leu Leu Ile Ser Leu Thr Phe Leu Thr Val Thr Asp Leu Ile
515 520 525
Gly Leu Pro Trp Val Cys Phe Ile Tyr Thr Ile Met Ser Leu Ala
530 535 540
Ser Leu Leu Phe Val Val Met Phe Ile Pro Glu Thr Lys Gly Cys
545 550 555
Ser Leu Glu Gln Ile Ser Met Glu Leu Ala Lys Val Asn Tyr Val
560 565 570
Lys Asn Asn Ile Cys Phe Met Ser His His Gln Glu Glu Leu Val
575 580 585
Pro Lys Gln Pro Gln Lys Arg Lys Pro Gln Glu Gln Leu Leu Glu
590 595 600
Cys Asn Lys Leu Cys Gly Arg Gly Gln Ser Arg Gln Leu Ser Pro
605 610 615
Glu Thr
<210> 2
<211> 1193
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte ID No: 55138203CD1
<400> 2
Met Tyr Ser Ala Asn Ile Gly Tyr Leu Leu Phe Val Gly Thr Gly
1 5 10 15
Val Glu Lys Met Asn Asn Thr Pro Ser Met Ala Leu Gly Ser Ser
20 25 30
His Ser Gly Arg Gly Asn Leu Thr Gln Ala Ala Thr Lys Pro Ser
35 40 45
Gly Tyr Glu Lys Thr Asp Asp Val Ser Glu Lys Thr Ser Leu Ala
50 55 60
Asp Gln Glu Glu Val Arg Thr Ile Phe Ile Asn Gln Pro Gln Leu
65 70 75
Thr Lys Phe Cys Asn Asn His Val Ser Thr Ala Lys Tyr Asn Ile
80 85 90
Ile Thr Phe Leu Pro Arg Phe Leu Tyr Ser Gln Phe Arg Arg Ala
95 100 105
Ala Asn Ser Phe Phe Leu Phe Ile Ala Leu Leu Gln Gln Ile Pro
110 115 120
Asp Val Ser Pro Thr Gly Arg Tyr Thr Thr Leu Val Pro Leu Leu
125 130 135
Phe Ile Leu Ala Val Ala Ala Ile Lys Glu Ile Ile Glu Asp Ile
140 145 150
Lys Arg His Lys Ala Asp Asn Ala Val Asn Lys Lys Gln Thr Gln
155' 160 165
Val Leu Arg Asn Gly Ala Trp Glu Ile Val His Trp Glu Lys Val
170 175 180
Asn Val Gly Asp Ile Val Ile Ile Lys Gly Lys Glu Tyr Ile Pro
185 190 195
3
CA 02438206 2003-08-06
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Ala Asp Thr Val Leu Leu Ser Ser Ser Glu Pro Gln Ala Met Cys
200 205 210
Tyr Ile Glu Thr Ser Asn Leu Asp Gly Glu Thr Asn Leu Lys Ile
215 220 225
Arg Gln Gly Leu Pro Ala Thr Ser Asp Ile Lys Asp Val Asp Ser
230 235 240
Leu Met Arg Ile Ser Gly Arg Ile Glu Cys Glu Ser Pro Asn Arg
245 250 255
His Leu Tyr Asp Phe Val Gly Asn Ile Arg Leu Asp Gly His Gly
260 265 270
Thr Val Pro Leu Gly Ala Asp Gln Ile Leu Leu Arg Gly Ala Gln
275 280 285
Leu Arg Asn Thr Gln Trp Val His Gly Ile Val Val Tyr Thr Gly
290 295 300
His Asp Thr Lys Leu Met Gln Asn Ser Thr Ser Pro Pro Leu Lys
305 310 315
Leu Ser Asn Val Glu Arg Ile Thr Asn Val Gln Ile Leu Ile Leu
320 325 330
Phe Cys Ile Leu Ile Ala Met Ser Leu Val Cys Ser Val Gly Ser
335 340 345
Ala Ile Trp Asn Arg Arg His Ser Gly Lys Asp Trp Tyr Leu Asn
350 355 360
Leu Asn Tyr Gly Gly Ala Ser Asn Phe Gly Leu Asn Phe Leu Thr
365 370 375
Phe Ile Ile Leu Phe Asn Asn Leu Ile Pro Ile Ser Leu Leu Val
380 385 390
Thr Leu Glu Val Val Lys Phe Thr Gln Ala Tyr Phe Ile Asn Trp
395 400 405
Asp Leu Asp Met His Tyr Glu Pro Thr Asp Thr Ala Ala Met Ala
410 415 420
Arg Thr Ser Asn Leu Asn Glu Glu Leu Gly Gln Val Lys Tyr Ile
425 430 435
Phe Ser Asp Lys Thr Gly Thr Leu Thr Cys Asn Val Met Gln Phe
440 445 450
Lys Lys Cys Thr Ile Ala Gly Val Ala Tyr Gly His Val Pro Glu
455 460 465
Pro Glu Asp Tyr Gly Cys Ser Pro Asp Glu Trp Gln Asn Ser Gln
470 475 480
Phe Gly Asp Glu Lys Thr Phe Ser Asp Ser Ser Leu Leu Glu Asn
485 490 495
Leu Gln Asn Asn His Pro Thr Ala Pro Ile Ile Cys Glu Phe Leu
500 505 510
Thr Met Met Ala Val Cys His Thr Ala Val Pro~Glu Arg Glu Gly
515 520 525
Asp Lys Ile Ile Tyr Gln Ala Ala Ser Pro Asp Glu Gly Ala Leu
530 535 540
Val Arg Ala Ala Lys Gln Leu Asn Phe Val Phe Thr Gly Arg Thr
545 550 555
Pro Asp Ser Val Ile Ile Asp Ser Leu Gly Gln Glu Glu Arg Tyr
560 565 570
Glu Leu Leu Asn Val Leu Glu Phe Thr Ser Ala Arg Lys Arg Met
575 580 585
Ser Val Ile Val Arg Thr Pro Ser Gly Lys Leu Arg Leu Tyr Cys
590 595 600
Lys Gly Ala Asp Thr Val Ile Tyr Asp Arg Leu Ala Glu Thr Ser
605 610 615
4
CA 02438206 2003-08-06
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Lys Tyr Lys Glu Ile Thr Leu Lys His Leu Glu Gln Phe Ala Thr
620 625 630
Glu Gly Leu Arg Thr Leu Cys Phe Ala Val Ala Glu Ile Ser Glu
635 640 645
Ser Asp Phe Gln Glu Trp Arg Ala Val Tyr Gln Arg Ala Ser Thr
650 655 660
Ser Val Gln Asn Arg Leu Leu Lys Leu Glu Glu Ser Tyr Glu Leu
665 670 675
Ile Glu Lys Asn Leu Gln Leu Leu Gly Ala Thr Ala Ile Glu Asp
680 685 690
Lys Leu Gln Asp Gln Val Pro Glu Thr Ile Glu Thr Leu Met Lys
695 700 705
Ala Asp Ile Lys Ile Trp Ile Leu Thr Gly Asp Lys Gln Glu Thr
710 715 720
Ala Ile Asn Ile Gly His Ser Cys Lys Leu Leu Lys Lys Asn Met
725 730 735
Gly Met Ile Val Ile Asn Glu Gly Ser Leu Asp Gly Thr Arg Glu
740 745 750
Thr Leu Ser Arg His Cys Thr Thr Leu Gly Asp Ala Leu Arg Lys
755 760 - 765
Glu Asn Asp Phe Ala Leu Ile Ile Asp Gly Lys Thr Leu Lys Tyr
770 775 780
Ala Leu Thr Phe Gly Val Arg Gln Tyr Phe Leu Asp Leu Ala Leu
785 790 795
Ser Cys Lys Ala Val Ile Cys Cys Arg Val Ser Pro Leu Gln Lys
800 805 810
Ser Glu Val Val Glu Met Val Lys Lys Gln Val Lys Val Val Thr
815 820 825
Leu Ala Ile Gly Asp Gly Ala Asn Asp Val Ser Met Ile Gln Thr
830 835 840
Ala His Val Gly Val Gly Ile Ser Gly Asn Glu Gly Leu Gln Ala
845 850 855
Ala Asn Ser Ser Asp Tyr Ser Ile Ala Gln Phe Lys Tyr Leu Lys
860 865 870
Asn Leu Leu Met Ile His Gly Ala Trp Asn Tyr Asn Arg Val Ser
875 880 885
Lys Cys Ile Leu Tyr Cys Phe Tyr Lys Asn Ile Val Leu Tyr Ile
890 895 900
Ile Glu Ile Trp Phe Ala Phe Val Asn Gly Phe Ser Gly Gln Ile
905 910 915
Leu Phe Glu Arg Trp Cys Ile Gly Leu Tyr Asn Val Met Phe Thr
920 925 930
Ala Met Pro Pro Leu Thr Leu Gly Ile Phe Glu Arg Ser Cys Arg
935 940 945
Lys Glu Asn Met Leu Lys Tyr Pro Glu Leu Tyr Lys Thr Ser Gln
950 955 960
Asn Ala Leu Asp Phe Asn Thr Lys Val Phe Trp Val His Cys Leu
965 970 975
Asn Gly Leu Phe His Ser Val Ile Leu Phe Trp Phe Pro Leu Lys
980 985 990
Ala Leu Gln Tyr Gly Thr Ala Phe Gly Asn Gly Lys Thr Ser Asp
995 1000 1005
Tyr Leu Leu Leu Gly Asn Phe Val Tyr Thr Phe Val Val Ile Thr
1010 1015 1020
Val Cys Leu Lys Ala Gly Leu Glu Thr Ser Tyr Trp Thr Trp Phe
1025 1030 1035
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
Ser His Ile Ala Ile Trp Gly Ser Ile Ala Leu Trp Val Val Phe
1040 1045 1050
Leu Gly Ile Tyr Ser Ser Leu Trp Pro Ala Ile Pro Met Ala Pro
1055 1060 1065
Asp Met Ser Gly Glu Ala Ala Met Leu Phe Ser Ser Gly Val Phe
1070 1075 1080
Trp Met Gly Leu Leu Phe Ile Pro Val Ala Ser Leu Leu Leu Asp
1085 1090 1095
Val Val Tyr Lys Val Ile Lys Arg Thr Ala Phe Lys Thr Leu Val
1100 1105 1110
Asp Glu Val Gln Glu Leu Glu Ala Lys Ser Gln Asp Pro Gly Ala
1115 1120 1125
Val Val Leu Gly Lys Ser Leu Thr Glu Arg Ala Gln Leu Leu Lys
1130 1135 1140
Asn Val Phe Lys Lys Asn His Val Asn Leu Tyr Arg Ser Glu Ser
1145 1150 1155
Leu Gln Gln Asn Leu Leu His Gly Tyr Ala Phe Ser Gln Asp Glu
1160 1165 1170
Asn Gly Ile Val Ser Gln Ser Glu Val Ile Arg Ala Tyr Asp Thr
1175 1180 1185
Thr Lys Gln Arg Pro Asp Glu Trp
1190
<210> 3
<211> 989
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No:.7478871CD1
<400> 3
Met Gln Pro Ala Arg Gly Pro Leu Ala Ser Glu Pro Arg Thr Val
1 5 10 15
Leu Val Leu Arg Phe Cys Ala Ser Leu Met Glu Met Lys Leu Pro
20 25 30
Gly Gln Glu Gly Phe Glu Ala Ser Ser Ala Pro Arg Asn Ile Pro
35 40 45
Ser Gly Glu Leu Asp Ser Asn Pro Asp Pro Gly Thr Gly Pro Ser
50 55 60
Pro Asp Gly Pro Ser Asp Thr Glu Ser Lys Glu Leu Gly Val Pro
65 70 75
Lys Asp Pro Leu Leu Phe Ile Gln Leu Asn Glu Leu Leu Gly Trp
80 85 90
Pro Gln Ala Leu Glu Trp Arg Glu Thr Gly Thr Trp Val Leu Phe
95 100 105
Glu Glu Lys Leu Glu Val Ala Ala Gly Arg Trp Ser Ala Pro His
110 115 120
Val Pro Thr Leu Ala Leu Pro Ser Leu Gln Lys Leu Arg Ser Leu
125 130 135
Leu Ala Glu Gly Leu Val Leu Leu Asp Cys Pro Ala Gln Ser Leu
140 145 150
Leu Glu Leu Val Glu Gln Val Thr Arg Val Glu Ser Leu Ser Pro
155 160 165
Glu Leu Arg Gly Gln Leu Gln Ala Leu Leu Leu Gln Arg Pro Gln
6
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
170 175 180
His Tyr Asn Gln Thr Thr Gly Thr Arg Pro Cys Trp Gly Glu Ser
185 190 195
Pro Ser Leu Gly Pro Gly Pro Arg Pro Cys Thr Thr Arg Pro Gln
200 205 210
Ala Pro Gly Pro Ala Gly Gln Cys Gln Asn Pro Leu Arg Gln Lys
215 220 225
Leu Pro Pro Gly Ala Glu Ala Gly Thr Val Leu Ala Gly Glu Leu
230 235 240
Gly Phe Leu Ala Gln Pro Leu Gly Ala Phe Val Arg Leu Arg Asn
245 250 255
Pro Val Val Leu Gly Ser Leu Thr Glu Val Ser Leu Pro Ser Arg
260 265 270
Phe Phe Cys Leu Leu Leu Gly Pro Cys Met Leu Gly Lys Gly Tyr
275 280 285
His Glu Met Gly Arg Ala Ala Ala Val Leu Leu Ser Asp Pro Gln
290 295 300
Phe Gln Trp Ser Val Arg Arg Ala Ser Asn Leu His Asp Leu Leu
305 310 315
Ala Ala Leu Asp Ala Phe Leu Glu Glu Val Thr Val Leu Pro Pro
320 325 330
Gly Arg Trp Asp Pro Thr Ala Arg Ile Pro Pro Pro Lys Cys Leu
335 340 345
Pro Ser Gln His Lys Arg Leu Pro Ser Gln Gln Arg Glu Ile Arg
350 355 360
Gly Pro Ala Val Pro Arg Leu Thr Ser Ala Glu Asp Arg His Arg
365 370 375
His Gly Pro His Ala His Ser Pro Glu Leu Gln Arg Thr Gly Arg
380 385 390
Leu Phe Gly Gly Leu Ile Gln Asp Val Arg Arg Lys Val Pro Trp
395 400 405
Tyr Pro Ser Asp Phe Leu Asp Ala Leu His Leu Gln Cys Phe Ser
410 415 420
Ala Val Leu Tyr Ile Tyr Leu Ala Thr Val Thr Asn Ala Ile Thr
425 430 435
Phe Gly Gly Leu Leu Gly Asp Ala Thr Asp Gly Ala Gln Gly Val
440 445 450
Leu Glu Ser Phe Leu Gly Thr Ala Val Ala Gly Ala Ala Phe Cys
455 460 465
Leu Met Ala Gly Gln Pro Leu Thr Ile Leu Ser Ser Thr Gly Pro
470 475 480
Val Leu Val Phe Glu Arg Leu Leu Phe Ser Phe Ser Arg Asp Tyr
485 490 495
Ser Leu Asp Tyr Leu Pro Phe Arg Leu Trp Val Gly Ile Trp Val
500 505 510
Ala Thr Phe Cys Leu Val Leu Val Ala Thr Glu Ala Ser Val Leu
515 520 525
Val Arg Tyr Phe Thr Arg Phe Thr Glu Glu Gly Phe Cys Ala Leu
530 535 540
Ile Ser Leu Ile Phe Ile Tyr Asp Ala Val Gly Lys Met Leu Asn
545 550 555
Leu Thr His Thr Tyr Pro Ile Gln Lys Pro Gly Ser Ser Ala Tyr
560 565 570
Gly Cys Leu Cys Gln Tyr Pro Gly Pro Gly Gly Asn Glu Ser Gln
575 580 585
Trp Ile Arg Thr Arg Pro Lys Asp Arg Asp Asp Ile Val Ser Met
7
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
590 595 600
Asp Leu Gly Leu Ile Asn Ala Ser Leu Leu Pro Pro Pro Glu Cys
605 610 615
Thr Arg Gln Gly Gly His Pro Arg Gly Pro Gly Cys His Thr Val
620 625 630
Pro Asp Ile Ala Phe Phe Ser Leu Leu Leu Phe Leu Thr Ser Phe
635 640 645
Phe Phe Ala Met Ala Leu Lys Cys Val Lys Thr Ser Arg Phe Phe
650 655 660
Pro Ser Val Val Arg Lys Gly Leu Ser Asp Phe Ser Ser Val Leu
665 670 675
Ala Ile Leu Leu Gly Cys Gly Leu Asp Ala Phe Leu Gly Leu Ala
680 685 690
Thr Pro Lys Leu Met Val Pro Arg Glu Phe Lys Pro Thr Leu Pro
695 700 705
Gly Arg Gly Trp Leu Val Ser Pro Phe Gly Ala Asn Pro Trp Trp
710 715 720
Trp Ser Val Ala Ala Ala Leu Pro Ala Leu Leu Leu Ser Ile Leu
725 730 735
Ile Phe Met Asp Gln Gln Ile Thr Ala Val Ile Leu Asn Arg Met
740 745 750
Glu Tyr Arg Leu Gln Lys Gly Ala Gly Phe His Leu Asp Leu Phe
755 760 765
Cys Val Ala Val Leu Met Leu Leu Thr Ser Ala Leu Gly Leu Pro
770 775 780
Trp Tyr Val Ser Ala Thr Val Ile Ser Leu Ala His Met Asp Ser
785 790 795
Leu Arg Arg Glu Ser Arg Ala Cys Ala Pro Gly Glu Arg Pro Asn
800 805 810
Phe Leu Gly Ile Arg Glu Gln Arg Leu Thr Gly Leu Val Val Phe
815 820 825
Ile Leu Thr Gly Ala Ser Ile Phe Leu Ala Pro Val Leu Lys Phe
830 835 840
Ile Pro Met Pro Val Leu Tyr Gly Ile Phe Leu Tyr Met Gly Val
845 850 855
Ala Ala Leu Ser Ser Ile Gln Phe Thr Asn Arg Val Lys Leu Leu
860 865 870
Leu Met Pro Ala Lys His Gln Pro Asp Leu Leu Leu Leu Arg His
875 880 885
Val Pro Leu Thr Arg Val His Leu Phe Thr Ala Ile Gln Leu Ala
890 895 900
Cys Leu Gly Leu Leu Trp Ile Ile Lys Ser Thr Pro Ala Ala Ile
905 910 915
Ile Phe Pro Leu Met Leu Leu Gly Leu Val Gly Val Arg Lys Ala
920 925 930
Leu Glu Arg Val Phe Ser Pro Gln Glu Leu Leu Trp Leu Asp Glu
935 940 945
Leu Met Pro Glu Glu Glu Arg Ser Ile Pro Glu Lys Gly Leu Glu
950 955 960
Pro Glu His Ser Phe Ser Gly Ser Asp Ser Glu Asp Ser Glu Leu
965 970 975
Met Tyr Gln Pro Lys Ala Pro Glu Ile Asn Ile Ser Val Asn
980 985
<210> 4
<211> 505
8
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7483601CD1
<400> 4
Met Asp His Ala Glu Glu Asn Glu Ile Leu Ala Ala Thr Gln Arg
1 5 10 15
Tyr Tyr Val Glu Arg Pro Ile Phe Ser His Pro Val Leu Gln Glu
20 25 30
Arg Leu His Thr Lys Asp Lys Val Pro Asp Ser Ile Ala Asp Lys
35 40 45
Leu Lys Gln Ala Phe Thr Cys Thr Pro Lys Lys Ile Arg Asn Ile
50 55 60
Ile Tyr Met Phe Leu Pro Ile Thr Lys Trp Leu Pro Ala Tyr Lys
65 70 75
Phe Lys Glu Tyr Val Leu Gly Asp Leu Val Ser Gly Ile Ser Thr
80 85 90
Gly Val Leu Gln Leu Pro Gln Gly Leu Ala Phe Ala Met Leu Ala
95 100 105
Ala Val Pro Pro Ile Phe Gly Leu Tyr Pro Ser Phe Tyr Pro Val
110 115 120
Ile Met Tyr Cys Phe Leu Gly Thr Ser Arg His Ile Ser Ile Gly
125 130 135
Pro Phe Ala Val Ile Ser Leu Met Ile Gly Gly Val Ala Val Arg
140 145 150
Leu Val Pro Asp Asp Ile Val Ile Pro Gly Gly Val Asn Ala Thr
155 160 165
Asn Gly Thr Glu Ala Arg Asp Ala Leu Arg Val Lys Val Ala Met
170 175 180
Ser Val Thr Leu Leu Ser Gly Ile Ile Gln Phe Cys Leu Gly Val
185 190 195
Cys Arg Phe Gly Phe Val Ala Ile Tyr Leu Thr Glu Pro Leu Val
200 205 210
Arg Gly Phe Thr Thr Ala Ala Ala Val His Val Phe Thr Ser Met
215 220 225
Leu Lys Tyr Leu Phe Gly Val Lys Thr Lys Arg Tyr Ser Gly Ile
230 235 240
Phe Ser Val Val Tyr Ser Thr Val Ala Val Leu Gln Asn Val Lys
245 250 255
Asn Leu Asn Val Cys Ser Leu Gly Val Gly Leu Met Val Phe Gly
260 265 270
Leu Leu Leu Gly Gly Lys Glu Phe Asn Glu Arg Phe Lys Glu Lys
275 280 285
Leu Pro Ala Pro Ile Pro Leu Glu Phe Phe Ala Val Val Met Gly
290 295 300
Thr Gly Ile Ser Ala Gly Phe Asn Leu Lys Glu Ser Tyr Asn Val
305 310 315
Asp Val Val Gly Thr Leu Pro Leu Gly Leu Leu Pro Pro Ala Asn
320 325 330
Pro Asp Thr Ser Leu Phe His Leu Val Tyr Val Asp Ala Ile Ala
335 340 345
Ile Ala Ile Val Gly Phe Ser Val Thr Ile Ser Met Ala Lys Thr
350 355 360
9
CA 02438206 2003-08-06
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Leu Ala Asn Lys His Gly Tyr Gln Val Asp Gly Asn Gln Glu Leu
365 370 375
Ile Ala Leu Gly Leu Cys Asn Ser Ile Gly Ser Leu Phe Gln Thr
380 385 390
Phe Ser Ile Ser Cys Ser Leu Ser Arg Ser Leu Val Gln Glu Gly
395 400 405
Thr Gly Gly Lys Thr Gln Leu Ala Gly Cys Leu Ala Ser Leu Met
410 415 420
Ile Leu Leu Val Ile Leu Ala Thr Gly Phe Leu Phe Glu Ser Leu
425 430 435
Pro Gln Ala Val Leu Ser Ala Ile Val Ile Val Asn Leu Lys Gly
440 445 450
Met Phe Met Gln Phe Ser Asp Leu Pro Phe Phe Trp Arg Thr Ser
455 460 465
Lys Ile Glu Leu Thr Ile Trp Leu Thr Thr Phe Val Ser Ser Leu
470 475 480
Phe Leu Gly Leu Asp Tyr Gly Leu Ile Thr Ala Val Ile Ile Ala
485 490 495
Leu Leu Thr Val Ile Tyr Arg Thr Gln Arg
500 505
<210> 5
<211> 618
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7487851CD1
<400> 5
Met Ser Arg Ser Pro Leu Asn Pro Ser Gln Leu Arg Ser Val Gly
1 5 10 15
Ser Gln Asp Ala Leu Ala Pro Leu Pro Pro Pro Ala Pro Gln Asn
20 25 30
Pro Ser Thr His Ser Trp Asp Pro Leu Cys Gly Ser Leu Pro Trp
35 40 45
Gly Leu Ser Cys Leu Leu Ala Leu Gln His Val Leu Val Met Ala
50 55 60
Ser Leu Leu Cys Val Ser His Leu Leu Leu Leu Cys Ser Leu Ser
65 70 75
Pro Gly Gly Leu Ser Tyr Ser Pro Ser Gln Leu Leu Ala Ser Ser
80 85 90
Phe Phe Ser Arg Gly Met Ser Thr Ile Leu Gln Thr Trp Met Gly
95 100 105
Ser Arg Leu Pro Leu Val Gln Ala Pro Ser Leu Glu Phe Leu Ile
110 115 120
Pro Ala Leu Val Leu Thr Ser Gln Lys Leu Pro Arg Ala Ile Gln
125 130 135
Thr Pro Gly Asn Cys Glu His Arg Ala Arg Ala Arg Ala Ser Leu
140 145 150
Met Leu His Leu Cys Arg Gly Pro Ser Cys His Gly Leu Gly His
155 160 165
Trp Asn Thr Ser Leu Gln Glu Val Ser Gly Ala Val Val Val Ser
170 175 180
Gly Leu Leu Gln Gly Met Met Gly Leu Leu Gly Ser Pro Gly His
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
185 190 195
Val Phe Pro His Cys Gly Pro Leu Val Leu Ala Pro Ser Leu Val
200 205 210
Val Ala Gly Leu Ser Ala His Arg Glu Val Ala Gln Phe Cys Phe
215 220 225
Thr His Trp Gly Leu Ala Leu Leu Val Ile Leu Leu Met Val Val
230 235 240
Cys Ser Gln His Leu Gly Ser Cys Gln Phe His Val Cys Pro Trp
245 250 255
Arg Arg Ala Ser Thr Ser Ser Thr His Thr Pro Leu Pro Val Phe
260 265 270
Arg Leu Leu Ser Val Leu Ile Pro Val Ala Cys Val Trp Ile Val
275 280 285
Ser Ala Phe Val Gly Phe Ser Val Ile Pro Gln Glu Leu Ser Ala
290 295 300
Pro Thr Lys Ala Pro Trp Ile Trp Leu Pro His Pro Gly Glu Trp
305 310 315
Asn Trp Pro Leu Leu Thr Pro Arg Ala Leu Ala Ala Gly Ile Ser
320 325 330
Met Ala Leu Ala Ala Ser Thr Ser Ser Leu Gly Cys Tyr Ala Leu
335 340 345
Cys Gly Arg Leu Leu His Leu Pro Pro Pro Pro Pro His Ala Cys
350 355 360
Ser Arg Gly Leu Ser Leu Glu Gly Leu Gly Ser Val Leu Ala Gly
365 370 375
Leu Leu Gly Ser Pro Met Gly Thr Ala Ser Ser Phe Pro Asn Val
380 385 390
Gly Lys Val Gly Leu Ile Gln Ala Gly Ser Gln Gln Val Ala His
395 400 405
Leu Val Gly Leu Leu Cys Val Gly Leu Gly Leu Ser Pro Arg Leu
410 415 420
Ala Gln Leu Leu Thr Thr Ile Pro Leu Pro Val Val Gly Gly Val
425 430 435
Leu Gly Val Thr Gln Ala Val Val Leu Ser Ala Gly Phe Ser Ser
440 445 450
Phe Tyr Leu Ala Asp Ile Asp Ser Gly Arg Asn Ile Phe Ile Val
455 460 465
Gly Phe Ser Ile Phe Met Ala Leu Leu Leu Pro Arg Trp Phe Arg
470 475 480
Glu Ala Pro Val Leu Phe Ser Thr Gly Trp Ser Pro Leu Asp Val
485 490 495
Leu Leu His Ser Leu Leu Thr Gln Pro Ile Phe Leu Ala Gly Leu
500 505 510
Ser Gly Phe Leu Leu Glu Asn Thr Ile Pro Gly Thr Gln Leu Glu
515 520 525
Arg Gly Leu Gly Gln Gly Leu Pro Ser Pro Phe Thr Ala Gln Glu
530 535 540
Ala Arg Met Pro Gln Lys Pro Arg Glu Lys Ala Ala Gln Val Tyr
545 550 555
Arg Leu Pro Phe Pro Ile Gln Asn Leu Cys Pro Cys Ile Pro Gln
560 565 570
Pro Leu His Cys Leu Cys Pro Leu Pro Glu Asp Pro Gly Asp Glu
575 580 585
Glu Gly Gly Ser Ser Glu Pro Glu Glu Met Ala Asp Leu Leu Pro
590 595 600
Gly Ser Gly Glu Pro Cys Pro Glu Ser Ser Arg Glu Gly Phe Arg
11
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Ser Gln Lys
605 610 615
<210> 6
<211> 377
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7472881CD1
<400> 6
Met Arg Ala Asn Cys Ser Ser Ser Ser Ala Cys Pro Ala Asn Ser
1 5 10 15
Ser Glu Glu Glu Leu Pro Val Gly Leu Glu Ala His Gly Asn Leu
20 25 30
Glu Leu Val Phe Thr Val Val Pro Thr Val Met Met Gly Leu Leu
35 40 45
Met Phe Ser Leu Gly Cys Ser Val Glu Ile Arg Lys Leu Trp Ser
50 55 60
His Ile Arg Arg Pro Trp Gly Ile Ala Val Gly Leu Leu Cys Gln
65 70 75
Phe Gly Leu Met Pro Phe Thr Ala Tyr Leu Leu Ala Ile Ser Phe
80 85 90
Ser Leu Lys Pro Val Gln Ala Ile Ala Val Leu Ile Met Gly Cys
95 100 105
Cys Pro Gly Gly Thr Ile Ser Asn Ile Phe Thr Phe Trp Val Asp
110 115 120
Gly Asp Met Asp Leu Ser Ile Ser Met Thr Thr Cys Ser Thr Val
125 130 135
Ala Ala Leu Gly Met Met Pro Leu Cys Ile Tyr Leu Tyr Thr Trp
140 145 150
Ser Trp Ser Leu Gln Gln Asn Leu Thr Ile Pro Tyr Gln Asn Ile
155 160 165
Gly Ile Thr Leu Val Cys Leu Thr Ile Pro Val Ala Phe Gly Val
170 175 180
Tyr Val Asn Tyr Arg Trp Pro Lys Gln Ser Lys Ile Ile Leu Lys
185 190 195
Ile Gly Ala Val Val Gly Gly Val Leu Leu Leu Val Val Ala Val
200 205 210
Ala Gly Val Val Leu Ala Lys Gly Ser Trp Asn Ser Asp Ile Thr
215 220 225
Leu Leu Thr Ile Ser Phe Ile Phe Pro Leu Ile Gly His Val Thr
230 235 240
Gly Phe Leu Leu Ala Leu Phe Thr His Gln Ser Trp Gln Arg Cys
245 250 255
Arg Thr Ile Ser Leu Glu Thr Gly Ala Gln Asn Ile Gln Met Cys
260 265 270
Ile Thr Met Leu Gln Leu Ser Phe Thr Ala Glu His Leu Val Gln
275 280 285
Met Leu Ser Phe Pro Leu Ala Tyr Gly Leu Phe Gln Leu Ile Asp
290 295 300
Gly Phe Leu Ile Val Ala Ala Tyr Gln Thr Tyr Lys Arg Arg Leu
305 310 315
12
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Lys Asn Lys His Gly Lys Lys Asn Ser Gly Cys Thr Glu Val Cys
320 325 330
His Thr Arg Lys Ser Thr Ser Ser Arg Glu Thr Asn Ala Phe Leu
335 340 345
Glu Val Asn Glu Glu Gly Ala Ile Thr Pro Gly Pro Pro Gly Pro
350 355 360
Met Asp Cys His Arg Ala Leu Glu Pro Val Gly His Ile Thr Ser
365 370 375
Cys Glu
<210> 7
<211> 507
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7612560CD1
<400> 7
Met Ser Val Thr Lys Ser Thr Glu Gly Pro Gln Gly Ala Val Ala
1 5 10 15
Ile Lys Leu Asp Leu Met Ser Pro Pro Glu Ser Ala Lys Lys Leu
20 25 30
Glu Asn Lys Asp Ser Thr Phe Leu Asp Glu Ser Pro Ser Glu Ser
35 40 45
Ala Gly Leu Lys Lys Thr Lys Gly Ile Thr Val Phe Gln Ala Leu
50 55 60
Ile His Leu Val Lys Gly Asn Met Gly Thr Gly Ile Leu Gly Leu
65 70 75
Pro Leu Ala Val Lys Asn Ala Gly Ile Leu Met Gly Pro Leu Ser
80 85 90
Leu Leu Val Met Gly Phe Ile Ala Cys His Cys Met His Ile Leu
95 100 105
Val Lys Cys Ala Gln Arg Phe Cys Lys Arg Leu Asn Lys Pro Phe
110 115 120
Met Asp Tyr Gly Asp Thr Val Met His Gly Leu Glu Ala Asn Pro
125 130 135
Asn Ala Trp Leu Gln Asn His Ala His Trp Gly Arg His Ile Val
140 145 150
Ser Phe Phe Leu Ile Ile Thr Gln Leu Gly Phe Cys Cys Val Tyr
155 160 165
Ile Val Phe Leu Ala Asp Asn Leu Lys Gln Val Val Glu Ala Val
170 175 180
Asn Ser Thr Thr Asn Asn Cys Tyr Ser Asn Glu Thr Val Ile Leu
185 190 195
Thr Pro Thr Met Asp Ser Arg Leu Tyr Met Leu Ser Phe Leu Pro
200 205 210
Phe Leu Val Leu Leu Val Leu Ile Arg Asn Leu Arg Ile Leu Thr
215 220 225
Ile Phe Ser Met Leu Ala Asn Ile Ser Met Leu Val Ser Leu Val
230 235 240
Ile Ile Ile Gln Tyr Ile Thr Gln Glu Ile Pro Asp Pro Ser Arg
245 250 255
Leu Pro Leu Val Ala Ser Trp Lys Thr Tyr Pro Leu Phe Phe Gly
13
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260 265 270
Thr Ala Ile Phe Ser Phe Glu Ser Ile Gly Val Val Leu Pro Leu
275 280 285
Glu Asn Lys Met Lys Asn Ala Arg His Phe Pro Ala Ile Leu Ser
290 295 300
Leu Gly Met Ser Ile Val Thr Ser Leu Tyr Ile Gly Met Ala Ala
305 310 315
Leu Gly Tyr Leu Arg Phe Gly Asp Asp Ile Lys Ala Ser Ile Ser
320 325 330
Leu Asn Leu Pro Asn Cys Trp Leu Tyr Gln Ser Val Lys Leu Leu
335 340 345
Tyr Ile Ala Gly Ile Leu Cys Thr Tyr Ala Leu Gln Phe Tyr Val
350 355 360
Pro Ala Glu Ile Ile Ile Pro Phe Ala Ile Ser Arg Val Ser Thr
365 370 375
Arg Trp Ala Leu Pro Leu Asp Leu Ser Ile Arg Leu Val Met Val
380 385 390
Cys Leu Thr Cys Leu Leu Ala Ile Leu Ile Pro Arg Leu Asp Leu
395 400 405
Val Ile Ser Leu Val Gly Ser Val Ser Gly Thr Ala Leu Ala Leu
410 415 420
Ile Ile Pro Pro Leu Leu Glu Val Thr Thr Phe Tyr Ser Glu Gly
425 430 435
Met Ser Pro Leu Thr Ile Phe Lys Asp Val Leu Ile Ser Ile Leu
440 445 450
Gly Phe Val Gly Phe Val Val Gly Thr Tyr Gln Ala Leu Asp Glu
455 460 465
Leu Leu Lys Ser Glu Asp Ser His Pro Phe Ser Asn Ser Thr Thr
470 475 480
Phe Val Arg Val Glu Leu Cys Lys Lys Gln Pro Pro Glu Gly Pro
485 490 495
Lys Trp Gln Gln Leu Ala Lys Gly Asp Ala Ala Ser
500 505
<210> 8
<211> 438
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2880370CD1
<400> 8
Met Ile Arg Lys Leu Phe Ile Val Leu Leu Leu Leu Leu Val Thr
1 5 10 15
Ile Glu Glu Ala Arg Met Ser Ser Leu Ser Phe Leu Asn Ile Glu
20 25 30
Lys Thr Glu Ile Leu Phe Phe Thr Lys Thr Glu Glu Thr Ile Leu
35 40 45
Val Ser Ser Ser Tyr Glu Asn Lys Arg Pro Asn Ser Ser His Leu
50 55 60
Phe Val Lys Ile Glu Asp Pro Lys Ile Leu Gln Met Val Asn Val
65 70 75
Ala Lys Lys Ile Ser Ser Asp Ala Thr Asn Phe Thr Ile Asn Leu
80 85 90
14
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Val Thr Asp Glu Glu Gly Glu Thr Asn Val Thr Ile Gln Leu Trp
95 100 105
Asp Ser Glu Gly Arg Gln Glu Arg Leu Ile Glu Glu Ile Lys Asn
110 115 120
Val Lys Val Lys Val Leu Lys Gln Lys Asp Ser Leu Leu Gln Ala
125 130 135
Pro Met His Ile Asp Arg Asn Ile Leu Met Leu Ile Leu Pro Leu
140 145 150
Ile Leu Leu Asn Lys Cys Ala Phe Gly Cys Lys Ile Glu Leu Gln
155 160 165
Leu Phe Gln Thr Val Trp Lys Arg Pro Leu Pro Val Ile Leu Gly
170 175 180
Ala Val Thr Gln Phe Phe Leu Met Pro Phe Cys Gly Phe Leu Leu
185 190 195
Ser Gln Ile Val Ala Leu Pro Glu Ala Gln Ala Phe Gly Val Val
200 205 210
Met Thr Cys Thr Cys Pro Gly Gly Gly Gly Gly Tyr Leu Phe Ala
215 220 225
Leu Leu Leu Asp Gly Asp Phe Thr Leu Ala Ile Leu Met Thr Cys
230 235 240
Thr Ser Thr Leu Leu Ala Leu Ile Met Met Pro Val Asn Ser Tyr
245 250 255
Ile Tyr Ser Arg Ile Leu Gly Leu Ser Gly Thr Phe His Ile Pro
260 265 270
Val Ser Lys Ile Val Ser Thr Leu Leu Phe Ile Leu Val Pro Val
275 280 285
Ser Ile Gly Ile Val Ile Lys His Arg Ile Pro Glu Lys Ala Ser
290 295 300
Phe Leu Glu Arg Ile Ile Arg Pro Leu Ser Phe Ile Leu Met Phe
305 310 315
Val Gly Ile Tyr Leu Thr Phe Thr Val Gly Leu Val Phe Leu Lys
320 325 330
Thr Asp Asn Leu Glu Val Ile Leu Leu Gly Leu Leu Val Pro Ala
335 340 345
Leu Gly Leu Leu Phe Gly Tyr Ser Phe Ala Lys Val Cys Thr Leu
350 355 360
Pro Leu Pro Val Cys Lys Thr Val Ala Ile Glu Ser Gly Met Leu
365 370 375
Asn Ser Phe Leu Ala Leu Ala Val Ile Gln Leu Ser Phe Pro Gln
380 385 390
Ser Lys Ala Asn Leu Ala Ser Val Ala Pro Phe Thr Val Ala Met
395 400 405
Cys Ser Gly Cys Glu Met Leu Leu Ile Ile Leu Val Tyr Lys Ala
410 415 420
Lys Lys Arg Cys Ile Phe Phe Leu Gln Asp Lys Arg Lys Arg Asn
425 430 435
Phe Leu Ile
<210> 9
<211> 350
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
<223> Incyte ID No: 6267489CD1
<400> 9
Met Leu Glu Gly Ala Glu Leu Tyr Phe Asn Val Asp His Gly Tyr
1 5 10 15
Leu Glu Gly Leu Val Arg Gly Cys Lys Ala Ser Leu Leu Thr Gln
20 25 30
Gln Asp Tyr Ile Asn Leu Val Gln Cys Glu Thr Leu Glu Asp Leu
35 40 45
Lys Ile His Leu Gln Thr Thr Asp Tyr Gly Asn Phe Leu Ala Asn
50 55 60
His Thr Asn Pro Leu Thr Val Ser Lys Ile Asp Thr Glu Met Arg
65 70 75
Lys Arg Leu Cys Gly Glu Phe Glu Tyr Phe Arg Asn His Ser Leu
80 85 90
Glu Pro Leu Ser Thr Phe Leu Thr Tyr Met Thr Cys Ser Tyr Met
95 100 105
Ile Asp Asn Val Ile Leu Leu Met Asn Gly Ala Leu Gln Lys Lys
110 115 120
Ser Val Lys Glu Ile Leu Gly Lys Cys His Pro Leu Gly Arg Phe
125 130 135
Thr Glu Met Glu Ala Val Asn Ile Ala Glu Thr Pro Ser Asp Leu
140 145 150
Phe Asn Ala Ile Leu Ile Glu Thr Pro Leu Ala Pro Phe Phe Gln
155 160 165
Asp Cys Met Ser Glu Asn Ala Leu Asp Glu Leu Asn Ile Glu Leu
170 175 180
Leu Arg Asn Lys Leu Tyr Lys Ser Tyr Leu Glu Ala Phe Tyr Lys
185 190 195
Phe Cys Lys Asn His Gly Asp Val Thr Ala Glu Val Met Cys Pro
200 205 210
Ile Leu Glu Phe Glu Ala Asp Arg Arg Ala Phe Ile Ile Thr Leu
215 220 225
Asn Ser Phe Gly Thr Glu Leu Ser Lys Glu Asp Arg Glu Thr Leu
230 235 240
Tyr Pro Thr Phe Gly Lys Leu Tyr Pro~Glu Gly Leu Arg Leu Leu
245 250 255
Ala Gln Ala Glu Asp Phe Asp Gln Met Lys Asn Val Ala Asp His
260 265 270
Tyr Gly Val Tyr Lys Pro Leu Phe Glu Ala Val Gly Gly Ser Gly
275 280 285
Gly Lys Thr Leu Glu Asp Val Phe Tyr Glu Arg Glu Val Gln Met
290 295 300
Asn Val Leu Ala Phe Asn Arg Gln Phe His Tyr Gly Val Phe Tyr
305 310 315
Ala Tyr Val Lys Leu Lys Glu Gln Glu Ile Arg Asn Ile Val Trp
320 325 330
Ile Ala Glu Cys Ile Ser Gln Arg His Arg Thr Lys Ile Asn Ser
335 340 345
Tyr Ile Pro Ile Leu
350
<210> 10
<211> 1707
<212> PRT
<213> Homo sapiens
16
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<220>
<221> misc feature
<223> Incyte ID No: 7484777CD1
<400> 10
Met Pro Glu Pro Trp Gly Thr Val Tyr Phe Leu Gly Ile Ala Gln
1 5 10 15
Val Phe Ser Phe Leu Phe Ser Trp Trp Asn Leu Glu Gly Val Met
20 25 30
Asn Gln Ala Asp Ala Pro Arg Pro Leu Asn Trp Thr Ile Arg Lys
35 40 45
Leu Cys His Ala Ala Phe Leu Pro Ser Val Arg Leu Leu Lys Ala
50 55 60
Gln Lys Ser Trp Ile Glu Arg Ala Phe Tyr Lys Arg Glu Cys Val
65 70 75
His Ile Ile Pro Ser Thr Lys Asp Pro His Arg Cys Cys Cys Gly
80 85 90
Arg Leu Ile Gly Gln His Val Gly Leu Thr Pro Ser Ile Ser Val
95 100 105
Leu Gln Asn Glu Lys Asn Glu Ser Arg Leu Ser Arg Asn Asp Ile
110 115 120
Gln Ser Glu Lys Trp Ser Ile Ser Lys His Thr Gln Leu Ser Pro
125 130 135
Thr Asp Ala Phe Gly Thr Ile Glu Phe Gln Gly Gly Gly His Ser
140 145 150
Asn Lys Ala Met Tyr Val Arg Val Ser Phe Asp Thr Lys Pro Asp
155 160 165
Leu Leu Leu His Leu Met Thr Lys Glu Trp Gln Leu Glu Leu Pro
170 175 180
Lys Leu Leu Ile Ser Val His Gly Gly Leu Gln Asn Phe Glu Leu
185 190 195
Gln Pro Lys Leu Lys Gln Val Phe Gly Lys Gly Leu Ile Lys Ala
200 205 210
Ala Met Thr Thr Gly Ala Trp Ile Phe Thr Gly Gly Val Asn Thr
215 220 225
Gly Val Ile Arg His Val Gly Asp Ala Leu Lys Asp His Ala Ser
230 235 240
Lys Ser Arg Gly Lys Ile Cys Thr Ile Gly Ile Ala Pro Trp Gly
245 250 255
Ile Val Glu Asn Gln Glu Asp Leu Ile Gly Arg Asp Val Val Arg
260 265 270
Pro Tyr Gln Thr Met Ser Asn Pro Met Ser Lys Leu Thr Val Leu
275 280 285
Asn Ser Met His Ser His Phe Ile Leu Ala Asp Asn Gly Thr Thr
290 295 300
Gly Lys Tyr Gly Ala Glu Val Lys Leu Arg Arg Gln Leu Glu Lys
305 310 315
His Ile Ser Leu Gln Lys Ile Asn Thr Arg Ile Gly Gln Gly Val
320 325 330
Pro Val Val Ala Leu Ile Val Glu Gly Gly Pro Asn Val Ile Ser
335 340 345
Ile Val Leu Glu Tyr Leu Arg Asp Thr Pro Pro Val Pro Val Val
350 355 360
Val Cys Asp Gly Ser Gly Arg Ala Ser Asp Ile Leu Ala Phe Gly
365 370 375
His Lys Tyr Ser Glu Glu Gly Gly Leu Ile Asn Glu Ser Leu Arg
17
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380 385 390
Asp Gln Leu Leu Val Thr Ile Gln Lys Thr Phe Thr Tyr Thr Arg
395 400 405
Thr Gln Ala Gln His Leu Phe Ile Ile Leu Met Glu Cys Met Lys
410 415 420
Lys Lys Glu Leu Ile Thr Val Phe Arg Met Gly Ser Glu Gly His
425 430 435
Gln Asp Ile Asp Leu Ala Ile Leu Thr Ala Leu Leu Lys Gly Ala
440 445 450
Asn Ala Ser Ala Pro Asp Gln Leu Ser Leu Ala Leu Ala Trp Asn
455 460 465
Arg Val Asp Ile Ala Arg Ser Gln Ile Phe Ile Tyr Gly Gln Gln
470 475 480
Trp Pro Val Gly Ser Leu Glu Gln Ala Met Leu Asp Ala Leu Val
485 490 495
Leu Asp Arg Val Asp Phe Val Lys Leu Leu Ile Glu Asn Gly Val
500 505 510
Ser Met His Arg Phe Leu Thr Ile Ser Arg Leu Glu Glu Leu Tyr
515 520 525
Asn Thr Arg His Gly Pro Ser Asn Thr Leu Tyr His Leu Val Arg
530 535 540
Asp Val Lys Lys Gly Asn Leu Pro Pro Asp Tyr Arg Ile Ser Leu
545 550 555
Ile Asp Ile Gly Leu Val Ile Glu Tyr Leu Met Gly Gly Ala Tyr
560 565 570
Arg Cys Asn Tyr Thr Arg Lys Arg Phe Arg Thr Leu Tyr His Asn
575 580 585
Leu Phe Gly Pro Lys Arg Pro Lys Ala Leu Lys Leu Leu Gly Met
590 595 600
Glu Asp Asp Ile Pro Leu Arg Arg Gly Arg Lys Thr Thr Lys Lys
605 610 615
Arg Glu Glu Glu Val Asp Ile Asp Leu Asp Asp Pro Glu Ile Asn
620 625 630
His Phe Pro Phe Pro Phe His Glu Leu Met Val Trp Ala Val Leu
635 640 645
Met Lys Arg Gln Lys Met Ala Leu Phe Phe Trp Gln His Gly Glu
650 655 660
Glu Ala Met Ala Lys Ala Leu Val Ala Cys Lys Leu Cys Lys Ala
665 670 675
Met Ala His Glu Ala Ser Glu Asn Asp Met Val Asp Asp Ile Ser
680 685 690
Gln Glu Leu Asn His Asn Ser Arg Asp Phe Gly Gln Leu Ala Val
695 700 705
Glu Leu Leu Asp Gln Ser Tyr Lys G1n Asp Glu Gln Leu Ala Met
710 715 720
Lys Leu Leu Thr Tyr Glu Leu Lys Asn Trp Ser Asn Ala Thr Cys
725 730 735
Leu Gln Leu Ala Val Ala Ala Lys His Arg Asp Phe Ile Ala His
740 745 750
Thr Cys Ser Gln Met Leu Leu Thr Asp Met Trp Met Gly Arg Leu
755 760 765
Arg Met Arg Lys Asn Ser Gly Leu Lys Val Ile Leu Gly Ile Leu
770 775 780
Leu Pro Pro Ser Ile Leu Ser Leu Glu Phe Lys Asn Lys Asp Asp
785 790 795
Met Pro Tyr Met Ser Gln Ala Gln Glu Ile His Leu Gln Glu Lys
18
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800 805 810
Glu Ala Glu Glu Pro Glu Lys Pro Thr Lys Glu Lys Glu Glu Glu
815 820 825
Asp Met Glu Leu Thr Ala Met Leu Gly Arg Asn Asn Gly Glu Ser
830 835 840
Ser Arg Lys Lys Asp Glu Glu Glu Val Gln Ser Lys His Arg Leu
845 850 855
Ile Pro Leu Gly Arg Lys Ile Tyr Glu Phe Tyr Asn Ala Pro Ile
860 865 870
Val Lys Phe Trp Phe Tyr Thr Leu Ala Tyr Ile Gly Tyr Leu Met
875 880 885
Leu Phe Asn Tyr Ile Val Leu Val Lys Met Glu Arg Trp Pro Ser
890 895 900
Thr Gln Glu Trp Ile Val Ile Ser Tyr Ile Phe Thr Leu Gly Ile
905 910 915
Glu Lys Met Arg. Glu Ile Leu Met Ser Glu Pro Gly Lys Leu Leu
920 925 930
Gln Lys Val Lys Val Trp Leu Gln Glu Tyr Trp Asn Val Thr Asp
935 940 945
Leu Ile Ala Ile Leu Leu Phe Ser Val Gly Met Ile Leu Arg Leu
950 955 960
Gln Asp Gln Pro Phe Arg Ser Asp Gly Arg Val Ile Tyr Cys Val
965 970 975
Asn Ile Ile Tyr Trp Tyr Ile Arg Leu Leu Asp Ile Phe Gly Val
980 985 990
Asn Lys Tyr Leu Gly Pro Tyr Val Met Met Ile Gly Lys Met Met
995 1000 1005
Ile Asp Met Met Tyr Phe Val Ile Ile Met Leu Val Val Leu Met
1010 1015 1020
Ser Phe Gly Val Ala Arg Gln Ala Ile Leu Phe Pro Asn Glu Glu
1025 1030 1035
Pro Ser Trp Lys Leu Ala Lys Asn Ile Phe Tyr Met Pro Tyr Trp
1040 1045 1050
Met Ile Tyr Gly Glu Val Phe Ala Asp Gln Ile Asp Pro Pro Cys
1055 1060 1065
Gly Gln Asn Glu Thr Arg Glu Asp Gly Lys Ile Ile Gln Leu Pro
1070 1075 1080
Pro Cys Lys Thr Gly Ala Trp Ile Val Pro Ala Ile Met Ala Cys
1085 1090 1095
Tyr Leu Leu Val Ala Asn Ile Leu Leu Val Asn Leu Leu Ile Ala
1100 1105 1110
Val Phe Asn Asn Thr Phe Phe Glu Val Lys Ser Ile Ser Asn Gln
1115 1120 1125
Val Trp Lys Phe Gln Arg Tyr Gln Leu Ile Met Thr Phe His Glu
1130 1135 1140
Arg Pro Val Leu Pro Pro Pro Leu Ile Ile Phe Ser His Met Thr
1145 1150 1155
Met Ile Phe Gln His Leu Cys Cys Arg Trp Arg Lys His Glu Ser
1160 1165 1170
Asp Pro Asp Glu Arg Asp Tyr Gly Leu Lys Leu Phe Ile Thr Asp
1175 1180 1185
Asp Glu Leu Lys Lys Val His Asp Phe Glu Glu Gln Cys Ile Glu
1190 1195 1200
Glu Tyr Phe Arg Glu Lys Asp Asp Arg Phe Asn Ser Ser Asn Asp
1205 1210 1215
Glu Arg Ile Arg Val Thr Ser Glu Arg Val Glu Asn Met Ser Met
19
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1220 1225 1230
Arg Leu Glu Glu Val Asn Glu Arg Glu His Ser Met Lys Ala Ser
1235 1240 1245
Leu Gln Thr Val Asp Ile Arg Leu Ala Gln Leu Glu Asp Leu Ile
1250 1255 1260
Gly Arg Met Ala Thr Ala Leu Glu Arg Leu Thr Gly Leu Glu Arg
1265 1270 1275
Ala Glu Ser Asn Lys Ile Arg Ser Arg Thr Ser Ser Asp Cys Thr
1280 1285 1290
Asp Ala Ala Tyr Ile Val Arg Gln Ser Ser Phe Asn Ser Gln Glu
1295 1300 1305
Gly Asn Thr Phe Lys Leu Gln Glu Ser Ile Asp Pro Ala Gly Glu
1310 1315 1320
Glu Thr Met Ser Pro Thr Ser Pro Thr Leu Met Pro Arg Met Arg
1325 1330 1335
Ser His Ser Phe Tyr Ser Val Asn Met Lys Asp Lys Gly Gly Ile
1340 1345 1350
Glu Lys Leu Glu Ser Ile Phe Lys Glu Arg Ser Leu Ser Leu His
1355 1360 1365
Arg Ala Thr Ser Ser His Ser Val Ala Lys Glu Pro Lys Ala Pro
1370 1375 1380
Ala Ala Pro Ala Asn Thr Leu Ala Ile Val Pro Asp Ser Arg Arg
1385 1390 1395
Pro Ser Ser Cys Ile Asp Ile Tyr Val Ser Ala Met Asp Glu Leu
1400 1405 1410
His Cys Asp Ile Asp Pro Leu Asp Asn Ser Val Asn Ile Leu Gly
1415 1420 1425
Leu Gly Glu Pro Ser Phe Ser Thr Pro Val Pro Ser Thr Ala Pro
1430 1435 1440
Ser Ser Ser Ala Tyr Ala Thr Leu Ala Pro Thr Asp Arg Pro Pro
1445 1450 1455
Ser Arg Ser Ile Asp Phe Glu Asp Ile Thr Ser Met Asp Thr Arg
1460 1465 1470
Ser Phe Ser Ser Asp Tyr Thr His Leu Pro Glu Cys Gln Asn Pro
1475 1480 1485
Trp Asp Ser Glu Pro Pro Met Tyr His Thr Ile Glu Arg Ser Lys
1490 1495 1500
Ser Ser Arg Tyr Leu Ala Thr Thr Pro Phe Leu Leu Glu Glu Ala
1505 1510 1515
Pro Ile Val Lys Ser His Ser Phe Met Phe Ser Pro Ser Arg Ser
1520 1525 1530
Tyr Tyr Ala Asn Phe Gly Val Pro Val Lys Thr Ala Glu Tyr Thr
1535 1540 1545
Ser Ile Thr Asp Cys Ile Asp Thr Arg Cys Val Asn Ala Pro Gln
1550 1555 1560
Ala Ile Ala Asp Arg Ala Ala Phe Pro Gly Gly Leu Gly Asp Lys
1565 1570 1575
Val Glu Asp Leu Thr Cys Cys His Pro Glu Arg Glu Ala Glu Leu
1580 1585 1590
Ser His Pro Ser Ser Asp Ser Glu Glu Asn Glu Ala Lys Gly Arg
1595 1600 1605
Arg Ala Thr Ile Ala Ile Ser Ser Gln Glu Gly Asp Asn Ser Glu
1610 1615 1620
Arg Thr Leu Ser Asn Asn Ile Thr Val Pro Lys Ile Glu Arg Ala
1625 1630 1635
Asn Ser Tyr Ser Ala Glu Glu Pro Ser Ala Pro Tyr Ala His Thr
2C
CA 02438206 2003-08-06
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1640 1645 1650
Arg Lys Ser Phe Ser Ile Ser Asp Lys Leu Asp Arg Gln Arg Asn
1655 1660 1665
Thr Ala Ser Leu Arg Asn Pro Phe Gln Arg Ser Lys Ser Ser Lys
1670 1675 1680
Pro Glu Gly Arg Gly Asp Ser Leu Ser Met Arg Lys Leu Ser Arg
1685 1690 1695
Thr Ser Ala Phe Gln Ser Phe Glu Ser Lys His Thr
1700 1705
<210> 11
<211> 771
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2493969CD1
<400> 11
Met Ser Gly Phe Phe Thr Ser Leu Asp Pro Arg Arg Val Gln Trp
1 5 10 15
Gly Ala Ala Trp Tyr Ala Met His Ser Arg Ile Leu Arg Thr Lys
20 25 30
Pro Val Glu Ser Met Leu Glu Gly Thr Gly Thr Thr Thr Ala His
35 40 45
Gly Thr Lys Leu Ala Gln Val Leu Thr Thr Val Asp Leu Ile Ser
50 55 60
Leu Gly Val Gly Ser Cys Val Gly Thr Gly Met Tyr Val Val Ser
65 70 75
Gly Leu Val Ala Lys Glu Met Ala Gly Pro Gly Val Ile Val Ser
80 85 90
Phe Ile Ile Ala Ala Val Ala Ser Ile Leu Ser Gly Val Cys Tyr
95 100 105
Ala Glu Phe Gly Val Arg Val Pro Lys Thr Thr Gly Ser Ala Tyr
110 115 120
Thr Tyr Ser Tyr Val Thr Val Gly Glu Phe Val Ala Phe Phe Ile
125 130 135
Gly Trp Asn Leu Ile Leu Glu Tyr Leu Ile Gly Thr Ala Ala Gly
140 145 150
Ala Ser Ala Leu Ser Ser Met Phe Asp Ser Leu Ala Asn His Thr
155 160 165
Ile Ser Arg Trp Met Ala Asp Ser Val Gly Thr Leu Asn Gly Leu
170 175 180
Gly Lys Gly Glu Glu Ser Tyr Pro Asp Leu Leu Ala Leu Leu Ile
185 190 195
Ala Val Ile Val Thr Ile Ile Val Ala Leu Gly Val Lys Asn Ser
200 205 210
Ile Gly Phe Asn Asn Val Leu Asn Val Leu Asn Leu Ala Val Trp
215 220 225
Val Phe Ile Met Ile Ala Gly Leu Phe Phe Ile Asn Gly Lys Tyr
230 235 240
Trp Ala Glu Gly Gln Phe Leu Pro His Gly Trp Ser Gly Val Leu
245 250 255
Gln Gly Ala Ala Thr Cys Phe Tyr Ala Phe Ile Gly Phe Asp Ile
260 265 270
21
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Ile Ala Thr Thr Gly Glu Glu Ala Lys Asn Pro Asn Thr Ser Ile
275 280 285
Pro Tyr Ala Ile Thr Ala Ser Leu Val Ile Cys Leu Thr Ala Tyr
290 295 300
Val Ser Val Ser Val Ile Leu Thr Leu Met Val Pro Tyr Tyr Thr
305 310 315
Ile Asp Thr Glu Ser Pro Leu Met Glu Met Phe Val Ala His Gly
320 325 330
Phe Tyr Ala Ala Lys Phe Val Val Ala Ile Gly Ser Val Ala Gly
335 340 345
Leu Thr Val Ser Leu Leu Gly Ser Leu Phe Pro Met Pro Arg Val
350 355 360
Ile Tyr Ala Met Ala Gly Asp Gly Leu Leu Phe Arg Phe Leu Ala
365 370 375
His Val Ser Ser Tyr Thr Glu Thr Pro Val Val Ala Cys Ile Val
380 385 390
Ser Gly Phe Leu Ala Ala Leu Leu Ala Leu Leu Val Ser Leu Arg
395 400 405
Asp Leu Ile Glu Met Met Ser Ile Gly Thr Leu Leu Ala Tyr Thr
410 415 420
Leu Val Ser Val Cys Val Leu Leu Leu Arg Tyr Gln Pro Glu Ser
425 430 435
Asp Ile Asp Gly Phe Val Lys Phe Leu Ser Glu Glu His Thr Lys
440 445 450
Lys Lys Glu Gly Ile Leu Ala Asp Cys Glu Lys Glu Ala Cys Ser
455 460 465
Pro Val Ser Glu Gly Asp Glu Phe Ser Gly Pro Ala Thr Asn Thr
470 475 480
Cys Gly Ala Lys Asn Leu Pro Ser Leu Gly Asp Asn Glu Met Leu
485 490 495
Ile Gly Lys Ser Asp Lys Ser Thr Tyr Asn Val Asn His Pro Asn
500 505 510
Tyr Gly Thr Val Asp Met Thr Thr Gly Ile Glu Ala Asp Glu Ser
515 520 525
Glu Asn Ile Tyr Leu Ile Lys Leu Lys Lys Leu Ile Gly Pro His
530 535 540
Tyr Tyr Thr Met Arg Ile Arg Leu Gly Leu Pro Gly Lys Met Asp
545 550 555
Arg Pro Thr Ala Ala Thr Gly His Thr Val Thr Ile Cys Val Leu
560 565 570
Leu Leu Phe Ile Leu Met Phe Ile Phe Cys Ser Phe Ile Ile Phe
575 580 585
Gly Ser Asp Tyr Ile Ser Glu Gln Ser Trp Trp Ala Ile Leu Leu
590 595 600
Val Val Leu Met Val Leu Leu Ile Ser Thr Leu Val Phe Val Ile
605 610 615
Leu Gln Gln Pro Glu Asn Pro Lys Lys Leu Pro Tyr Met Ala Pro
620 625 630
Cys Leu Pro Phe Val Pro Ala Phe Ala Met Leu Val Asn Ile Tyr
635 640 645
Leu Met Leu Lys Leu Ser Thr Ile Thr Trp Ile Arg Phe Ala Val
650 655 660
Trp Cys Phe Val Gly Leu Leu Ile Tyr Phe Gly Tyr Gly Ile Trp
665 670 675
Asn Ser Thr Leu Glu Ile Ser Ala Arg Glu Glu Ala Leu His Gln
680 685 690
22
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Ser Thr Tyr Gln Arg Tyr Asp Val Asp Asp Pro Phe Ser Val Glu
695 700 705
Glu Gly Phe Ser Tyr Ala Thr Glu Gly Glu Ser Gln Glu Asp Trp
710 715 720
Gly Gly Pro Thr Glu Asp Lys Gly Phe Tyr Tyr Gln Gln Met Ser
725 730 735
Asp Ala Lys Ala Asn Gly Arg Thr Ser Ser Lys Ala Lys Ser Lys
740 745 750
Ser Lys His Lys Gln Asn Ser Glu Ala Leu Ile Ala Asn Asp Glu
755 760 765
Leu Asp Tyr Ser Pro Glu
770
<210> 12
<211> 1329
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3244593CD1
<400> 12
Met Val Gly Glu Gly Pro Tyr Leu Ile Ser Asp Leu Asp Gln Arg
1 5 10 15
Gly Arg Arg Arg Ser Phe Ala Glu Arg Tyr Asp Pro Ser Leu Lys
20 25 30
Thr Met Ile Pro Val Arg Pro Cys Ala Arg Leu Ala Pro Asn Pro
35 40 45
Val Asp Asp Ala Gly Leu Leu Ser Phe Ala Thr Phe Ser Trp Leu
50 55 60
Thr Pro Val Met Val Lys Gly Tyr Arg Gln Arg Leu Thr Val Asp
65 70 75
Thr Leu Pro Pro Leu Ser Thr Tyr Asp Ser Ser Asp Thr Asn Ala
80 85 90
Lys Arg Phe Arg Val Leu Trp Asp Glu Glu Val Ala Arg Val Gly
95 100 105
Pro Glu Lys Ala Ser Leu Ser His Val Val Trp Lys Phe Gln Arg
110 115 120
Thr Arg Val Leu Met Asp Ile Val Ala Asn Ile Leu Cys Ile Ile
125 130 135
Met Ala Ala Ile Gly Pro Thr Val Leu Ile His Gln Ile Leu Gln
140 145 150
Gln Thr Glu Arg Thr Ser Gly Lys Val Trp Val Gly Ile Gly Leu
155 160 165
Cys Ile Ala Leu Phe Ala Thr Glu Phe Thr Lys Val Phe Phe Trp
170 175 180
Ala Leu Ala Trp Ala Ile Asn Tyr Arg Thr Ala Ile Arg Leu Lys
185 190 195
Val Ala Leu Ser Thr Leu Val Phe Glu Asn Leu Val Ser Phe Lys
200 205 210
Thr Leu Thr His Ile Ser Val Gly Glu Val Leu Asn Ile Leu Ser
215 220 225
Ser Asp Ser Tyr Ser Leu Phe Glu Ala Ala Leu Phe Cys Pro Leu
230 235 240
Pro Ala Thr Ile Pro Ile Leu Met Val Phe Cys Ala Ala Tyr Ala
23
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245 250 255
Phe Phe Ile Leu Gly Pro Thr Ala Leu Ile Gly Ile Ser Val Tyr
260 265 270
Val Ile Phe Ile Pro Val Gln Met Phe Met Ala Lys Leu Asn Ser
275 280 285
Ala Phe Arg Arg Ser Ala Ile Leu Val Thr Asp Lys Arg Val Gln
290 295 300
Thr Met Asn Glu Phe Leu Thr Cys Ile Arg Leu Ile Lys Met Tyr
305 310 315
Ala Trp Glu Lys Ser Phe Thr Asn Thr Ile Gln Asp Ile Arg Arg
320 325 330
Arg Glu Arg Lys Leu Leu Glu Lys Ala Gly Phe Val Gln Ser Gly
335 340 345
Asn Ser Ala Leu Ala Pro Ile Val Ser Thr Ile Ala Ile Val Leu
350 355 360
Thr Leu Ser Cys His Ile Leu Leu Arg Arg Lys Leu Thr Ala Pro
365 370 375
Val Ala Phe Ser Val Ile Ala Met Phe Asn Val Met Lys Phe Ser
380 385 390
Ile Ala Ile Leu Pro Phe Ser Ile Lys Ala Met Ala Glu Ala Asn
395 400 405
Val Ser Leu Arg Arg Met Lys Lys Ile Leu Ile Asp Lys Ser Pro
410 415 420
Pro Ser Tyr Ile Thr Gln Pro Glu Asp Pro Asp Thr Val Leu Leu
425 430 435
Leu Ala Asn Ala Thr Leu Thr Trp Glu His Glu Ala Ser Arg Lys
440 445 450
Ser Thr Pro Lys Lys Leu Gln Asn Gln Lys Arg His Leu Cys Lys
455 460 465
Lys Gln Arg Ser Glu Ala Tyr Ser Glu Arg Ser Pro Pro Ala Lys
470 475 480
Gly Ala Thr Gly Pro Glu Glu Gln Ser Asp Ser Leu Lys Ser Val
485 490 495
Leu His Ser Ile Ser Phe Val Val Arg Lys Gly Lys Ile Leu Gly
500 505 510
Ile Cys Gly Asn Val Gly Ser Gly Lys Ser Ser Leu Leu Ala Ala
515 520 525
Leu Leu Gly Gln Met Gln Leu Gln Lys Gly Val Val Ala Val Asn
530 535 540
Gly Thr Leu Ala Tyr Val Ser Gln Gln Ala Trp Ile Phe His Gly
545 550 555
Asn Val Arg Glu Asn Ile Leu Phe Gly Glu Lys Tyr Asp His Gln
560 565 570
Arg Tyr Gln His Thr Val Arg Val Cys Gly Leu Gln Lys Asp Leu
575 580 585
Ser Asn Leu Pro Tyr Gly Asp Leu Thr Glu Ile Gly Glu Arg Gly
590 595 600
Leu Asn Leu Ser Gly Gly Gln Arg Gln Arg Ile Ser Leu Ala Arg
605 610 615
Ala Val Tyr Ser Asp Arg Gln Leu Tyr Leu Leu Asp Asp Pro Leu
620 625 630
Ser Ala Val Asp Ala His Val Gly Lys His Val Phe Glu Glu Cys
635 640 645
Ile Lys Lys Thr Leu Arg Gly Lys Thr Val Val Leu Val Thr His
650 655 660
Gln Leu Gln Phe Leu Glu Ser Cys Asp Glu Val Ile Leu Leu Glu
24
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665 670 675
Asp Gly Glu Ile Cys Glu Lys Gly Thr His Lys Glu Leu Met Glu
680 685 690
Glu Arg Gly Arg Tyr Ala Lys Leu Ile His Asn Leu Arg Gly Leu
695 700 705
Gln Phe Lys Asp Pro Glu His Leu Tyr Asn Ala Ala Met Val Glu
710 715 720
Ala Phe Lys Glu Ser Pro Ala Glu Arg Glu Glu Asp Ala Gly Ile
725 730 735
Ile Val Leu Ala Pro Gly Asn Glu Lys Asp Glu Gly Lys Glu Ser
740 745 750
Glu Thr Gly Ser Glu Phe Val Asp Thr Lys Gly Tyr Leu Leu Ser
755 760 765
Leu Phe Thr Val Phe Leu Phe Leu Leu Met Ile Gly Ser Ala Ala
770 775 780
Phe Ser Asn Trp Trp Leu Gly Leu Trp Leu Asp Lys Gly Ser Arg
785 790 795
Met Thr Cys Gly Pro Gln Gly Asn Arg Thr Met Cys Glu Val Gly
800 805 810
Ala Val Leu Ala Asp Ile Gly Gln His Val Tyr Gln Trp Val Tyr
815 820 825
Thr Ala Ser Met Val Phe Met Leu Val Phe Gly Val Thr Lys Gly
830 835 840
Phe Val Phe Thr Lys Thr Thr Leu Met Ala Ser Ser Ser Leu His
845 850 855
Asp Thr Val Phe Asp Lys Ile Leu Lys Ser Pro Met Ser Phe Phe
860 865 870
Asp Thr Thr Pro Thr Gly Arg Leu Met Asn Arg Phe Ser Lys Asp
875 880 885
Met Asp Glu Leu Asp Val Arg Leu Pro Phe His Ala Glu Asn Phe
890 895 900
Leu Gln Gln Phe Phe Met Val Val Phe Ile Leu Val Ile Leu Ala
905 910 915
Ala Val Phe Pro Ala Val Leu Leu Val Val Ala Ser Leu Ala Val
920 925 930
Gly Phe Phe Ile Leu Leu Arg Ile Phe His Arg Gly Val Gln Glu
935 940 945
Leu Lys Lys Val Glu Asn Val Ser Arg Ser Pro Trp Phe Thr His
950 955 960
Ile Thr Ser Ser Met Gln Gly Leu Gly Ile Ile His Ala Tyr Gly
965 970 975
Lys Lys Glu Ser Cys Ile Thr Tyr His Leu Leu Tyr Phe Asn Cys
980 985 990
Ala Leu Arg Trp Phe Ala Leu Arg Met Asp Val Leu Met Asn Ile
995 1000 1005
Leu Thr Phe Thr Val Ala Leu Leu Val Thr Leu Ser Phe Ser Ser
1010 1015 1020
Ile Ser.Thr Ser Ser Lys Gly Leu Ser Leu Ser Tyr Ile Ile Gln
1025 1030 1035
Leu Ser Gly Leu Leu Gln Val Cys Val Arg Thr Gly Thr Glu Thr
1040 1045 1050
Gln Ala Lys Phe Thr Ser Val Glu Leu Leu Arg Glu Tyr Ile Ser
1055 1060 1065
Thr Cys Val Pro Glu Cys Thr His Pro Leu Lys Val Gly Thr Cys
1070 1075 1080
Pro Lys Asp Trp Pro Ser Cys Gly Glu Ile Thr Phe Arg Asp Tyr
CA 02438206 2003-08-06
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1085 1090 1095
Gln Met Arg Tyr Arg Asp Asn Thr Pro Leu Val Leu Asp Ser Leu
1100 1105 1110
Asn Leu Asn Ile Gln Ser Gly Gln Thr Val Gly Ile Val Gly Arg
1115 1120 1125
Thr Gly Ser Gly Lys Ser Ser Leu Gly Met Ala Leu Phe Arg Leu
1130 1135 1140
Val Glu Pro Ala Ser Gly Thr Ile Phe Ile Asp Glu Val Asp Ile
1145 1150 1155
Cys Ile Leu Ser Leu Glu Asp Leu Arg Thr Lys Leu Thr Val Ile
1160 1165 1170
Pro Gln Asp Pro Val Leu Phe Val Gly Thr Val Arg Tyr Asn Leu
1175 1180 1185
Asp Pro Phe Glu Ser His Thr Asp Glu Met Leu Trp Gln Val Leu
1190 1195 1200
Glu Arg Thr Phe Met Arg Asp Thr Ile Met Lys Leu Pro Glu Lys
1205 1210 1215
Leu Gln Ala Glu Val Thr Glu Asn Gly Glu Asn Phe Ser Val Gly
1220 1225 1230
Glu Arg Gln Leu Leu Cys Val Ala Arg Ala Leu Leu Arg Asn Ser
1235 1240 , 1245
Lys Ile Ile Leu Leu Asp Glu Ala Thr Ala Ser Met Asp Ser Lys
1250 1255 1260
Thr Asp Thr Leu Val Gln Asn Thr Ile Lys Asp Ala Phe Lys Gly
1265 1270 1275
Cys Thr Val Leu Thr Ile Ala His Arg Leu Asn Thr Val Leu Asn
1280 1285 1290
Cys Asp His Val Leu Val Met Glu Asn Gly Lys Val Ile Glu Phe
1295 1300 1305
Asp Lys Pro Glu Val Leu Ala Glu Lys Pro Asp Ser Ala Phe Ala
1310 1315 1320
Met Leu Leu Ala Ala Glu Val Arg Leu
1325
<210> 13
<211> 1353
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4921451CD1
<400> 13
Met Gly Thr Gly Pro Ala Gln Thr Pro Arg Ser Thr Arg Ala Gly
1 5 10 15
Pro Glu Pro Ser Pro Ala Pro Pro Gly Pro Gly Asp Thr Gly Asp
20 25 30
Ser Asp Val Thr Gln Glu Gly Ser Gly Pro Ala Gly Ile Arg Gly
35 40 45
Ala Pro Pro Ala Trp Ala Ala Ser Ala Arg Glu Lys Ile Ser Glu
50 55 60
Met Arg Thr Gly Thr Gln Val Leu Ile Leu Gly Gly Gly Gly Gly
65 70 75
Ala Ala Phe Thr Trp Lys Val Gln Ala Asn Asn Arg Ala Tyr Asn
' 80 85 90
26
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Gly Gln Phe Lys Glu Lys Val Ile Leu Cys Trp Gln Arg Lys Lys
95 100 105
Tyr Lys Thr Asn Val Ile Arg Thr Ala Lys Tyr Asn Phe Tyr Ser
110 115 120
Phe Leu Pro Leu Asn Leu Tyr Glu Gln Phe His Arg Val Ser Asn
125 130 135
Leu Phe Phe Leu Ile Ile Ile Ile Leu Gln Ser Ile Pro Asp Ile
140 145 150
Ser Thr Leu Pro Trp Phe Ser Leu Ser Thr Pro Met Val Cys Leu
155 160 165
Leu Phe Ile Arg Ala Thr Arg Asp Leu Val Asp Asp Met Gly Arg
170 175 180
His Lys Ser Asp Arg Ala Ile Asn Asn Arg Pro Cys Gln Ile Leu
185 190 195
Met Gly Lys Ser Phe Lys Gln Lys Lys Trp Gln Asp Leu Cys Val
200 205 210
Gly Asp Val Val Cys Leu Arg Lys Asp Asn Ile Val Pro Val Ser
215 220 225
Trp Gly Gly Pro Arg Gly Pro Arg Thr Thr Arg Pro Leu Thr Glu
230 235 240
Ser Thr Pro Pro Arg Val Gly Arg Ala Ala Ala Pro Pro Ile Cys
245 250 255
Leu Ala Ser Pro Leu Ala Thr Leu Pro Pro Thr Pro His Gln Ala
260 265 270
Asp Met Leu Leu Leu Ala Ser Thr Glu Pro Ser Ser Leu Cys Tyr
275 280 285
Val Glu Thr Val Asp Ile Asp Gly Glu Thr Asn Leu Lys Phe Arg
290 295 300
Gln Ala Leu Met Val Thr His Lys Glu Leu Ala Thr Ile Lys Lys
305 310 315
Met Ala Ser Phe Gln Gly Thr Val Thr Cys Glu Ala Pro Asn Ser
320 325 330
Arg Met His His Phe Val Gly Cys Leu Glu Trp Asn Asp Lys Lys
335 340 345
Tyr Ser Leu Asp Ile Gly Asn Leu Leu Leu Arg Gly Cys Arg Ile
350 355 360
Arg Asn Thr Asp Thr Cys Tyr Gly Leu Val Ile Tyr Ala Gly Phe
365 370 375
Asp Thr Lys Ile Met Lys Asn Cys Gly Lys Ile His Leu Lys Arg
380 385 390
Thr Lys Leu Asp Leu Leu Met Asn Lys Leu Val Val Val Ile Phe
395 400 405
Ile Ser Val Val Leu Val Cys Leu Val Leu Ala Phe Gly Phe Gly
410 415 420
Phe Ser Val Lys Glu Phe Lys Asp His His Tyr Tyr Leu Ser Gly
425 430 435
Val His Gly Ser Ser Val Ala Ala Glu Ser Phe Phe Val Phe Trp
440 445 450
Ser Phe Leu Ile Leu Leu Ser Val Thr Ile Pro Met Ser Met Phe
455 460 465
Ile Leu Ser Glu Phe Ile Tyr Leu Gly Asn Ser Val Phe Ile Asp
470 475 480
Trp Asp Val Gln Met Tyr Tyr Lys Pro Gln Asp Val Pro Ala Lys
485 490 495
Ala Arg Ser Thr Ser Leu Asn Asp His Leu Gly Gln Val Glu Tyr
500 505 510
27
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Ile Phe Ser Asp Lys Thr Gly Thr Leu Thr Gln Asn Ile Leu Thr
515 520 525
Phe Asn Lys Cys Cys Ile Ser Gly Arg Val Tyr Gly Glu Pro Leu
530 535 540
Pro Leu Glu Gln Val Arg Arg Arg Glu Ala Ala Leu Pro Gln Cys
545 550 555
Gly Pro Ala Ala Pro Arg Ala Asp Gln Arg Gly Arg Gly Arg Ala
560 565 570
Gly Val Leu Ala Pro Ala Gly His Leu Pro His Gly Asp Asp Gln
575 580 585
Leu Leu Tyr Gln Ala Ala Ser Pro Asp Glu Gly Ala Leu Val Thr
590 595 600
Ala Ala Arg Asn Phe Gly Tyr Val Phe Leu Ser Arg Thr Gln Asp
605 610 615
Thr Val Thr Ile Met Glu Leu Gly Glu Glu Arg Val Tyr Gln Val
620 625 630
Leu Ala Ile Met Asp Phe Asn Ser Thr Arg Lys Arg Met Ser Val
635 640 645
Leu Val Arg Lys Pro Glu Gly Ala Ile Cys Leu Tyr Thr Lys Gly
650 655 660
Ala Asp Thr Val Ile Phe Glu Arg Leu His Arg Arg Gly Ala Met
665 670 675
Glu Phe Ala Thr Glu Glu Ala Leu Ala Ala Phe Ala Gln Glu Thr
680 685 690
Leu Arg Thr Leu Cys Leu Ala Tyr Arg Glu Val Ala Glu Asp Ile
695 700 705
Tyr Glu Asp Trp Gln Gln Arg His Gln Glu Ala Ser Leu Leu Leu
710 715 720
Gln Asn Arg Ala Gln Ala Leu Gln Gln Val Tyr Asn Glu Met Glu
725 730 735
Gln Asp Leu Arg Leu Leu Gly Ala Thr Ala Ile Glu Asp Arg Leu
740 745 750
Gln Asp Gly Val Pro Glu Thr Ile Lys Cys Leu Lys Lys Ser Asn
755 760 7~5
Ile Lys Ile Trp Val Leu Thr Gly Asp Lys Gln Glu Thr Ala Val
770 775 780
Asn Ile Gly Phe Ala Cys Glu Leu Leu Ser Glu Asn Met Leu Ile
785 790 795
Leu Glu Glu Lys Glu Ile Ser Arg Ile Leu Glu Thr Tyr Trp Glu
800 805 810
Asn Ser Asn Asn Leu Leu Thr Arg Glu Ser Leu Ser Gln Val Lys
815 820 825
Leu Ala Leu Val Ile Asn Gly Asp Phe Leu Asp Lys Leu Leu Val
830 835 840
Ser Leu Arg Lys Glu Pro Arg Ala Leu Ala Gln Asn Val Asn Met
845 850 855
Asp Glu Ala Trp Gln Glu Leu Gly Gln Ser Arg Arg Asp Phe Leu
860 865 870
Tyr Ala Arg Arg Leu Ser Leu Leu Cys Arg Arg Phe Gly Leu Pro
875 880 885
Leu Ala Ala Pro Pro Ala Gln Asp Ser Arg Ala Arg Arg Ser Ser
890 895 900
Glu Val Leu Gln Glu Arg Ala Phe Val Asp Leu Ala Ser Lys Cys
905 910 915
Gln Ala Val Ile Cys Cys Arg Val Thr Pro Lys Gln Lys Ala Leu
920 925 930
28
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Ile Val Ala Leu Val Lys Lys Tyr His Gln Val Val Thr Leu Ala
935 940 945
Ile Gly Asp Gly Ala Asn Asp Ile Asn Met Ile Lys Thr Ala Asp
950 955 960
Val Gly Val Gly Leu Ala Gly Gln Glu Gly Met Gln Ala Val Gln
965 970 975
Asn Ser Asp Phe Val Leu Gly Gln Phe Cys Phe Leu Gln Arg Leu
980 985 990
Leu Leu Val His Gly Arg Trp Ser Tyr Val Arg Ile Cys Lys Phe
995 1000 1005
Leu Arg Tyr Phe Phe Tyr Lys Ser Met Ala Ser Met Met Val Gln
1010 1015 1020
Val Trp Phe Ala Cys Tyr Asn Gly Phe Thr Gly Gln Asp Val Ser
1025 1030 1035
Ala Glu Gln Ser Leu Glu Lys Pro Glu Leu Tyr Val Val Gly Gln
1040 1045 1050
Lys Asp Glu Leu Phe Asn Tyr Trp Val Phe Val Gln Ala Ile Ala
1055 1060 1065
His Gly Val Thr Thr Ser Leu Val Asn Phe Phe Met Thr Leu Trp
1070 1075 1080
Ile Ser Arg Asp Thr Ala Gly Pro Ala Ser Phe Ser Asp His Gln
1085 1090 1095
Ser Phe Ala Val Val Val Ala Leu Ser Cys Leu Leu Ser Ile Thr
1100 1105 1110
Met Glu Val Ile Leu Ile Ile Lys Tyr Trp Thr Ala Leu Cys Val
1115 1120 1125
Ala Thr Ile Leu Leu Ser Leu Gly Phe Tyr Ala Ile Met Thr Thr
1130 1135 1140
Thr Thr Gln Ser Phe Trp Leu Phe Arg Val Ser Pro Thr Thr Phe
1145 1150 1155
Pro Phe Leu Tyr Ala Asp Leu Ser Val Met Ser Ser Pro Ser Ile
1160 1165 1170
Leu Leu Val Val Leu Leu Ser Val Ser Ile Asn Thr Phe Pro Val
1175 1180 1185
Leu Ala Leu Arg Val Ile Phe Pro Ala Leu Lys Glu Leu Arg Ala
1190 1195 1200
Lys Glu Glu Lys Val Glu Glu Gly Pro Ser Glu Glu Ile Phe Thr
1205 1210 1215
Met Glu Pro Leu Pro His Val His Arg Glu Ser Arg Ala Arg Arg
1220 1225 1230
Ser Ser Tyr Ala Phe Ser His Arg Gln Leu Thr Leu Glu Ser Gln
1235 1240 1245
Pro Asp Ser Ser Glu Glu Lys Ser Ala Phe Leu Lys Pro Ser Thr
1250 1255 1260
Pro Phe Arg Lys Ser Trp Gln Lys Glu Pro His Thr Pro Lys Glu
1265 1270 1275
Gly Thr Val Pro Leu Pro Asp Lys Thr His Lys Ser Gln Val Glu
1280 1285 1290
Thr Leu Pro Pro Ser Leu Glu Glu Ser Ser Thr Ser Thr Ser Glu
1295 1300 1305
Gln Pro Met Glu Val Glu Leu Trp Pro Ala Glu Lys Gln Ser Ser
1310 1315 1320
Ser Ser Met Glu Trp Leu Leu Val Pro Gly Glu Glu Gln Leu Ser
1325 1330 1335
Leu Pro Pro Glu Glu Gln Ser Leu Pro Ser Ala Glu Gly Thr Arg
1340 1345 1350
29
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Val Gln Gln
<210> 14
<211> 921
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5547443CD1
<400> 14
Met Ala His Glu Ser Ala Glu Asp Leu Phe His Phe Asn Val Gly
1 5 10 15
Gly Trp His Phe Ser Val Pro Arg Ser Lys Leu Ser Gln Phe Pro
20 25 30
Asp Ser Leu Leu Trp Lys Glu Ala Ser Ala Leu Thr Ser Ser Glu
35 40 45
Ser Gln Arg Leu Phe Ile Asp Arg Asp Gly Ser Thr Phe Arg His
50 55 60
Val His Tyr Tyr Leu Tyr Thr Ser Lys Leu Ser Phe Ser Ser Cys
65 70 75
Ala Glu Leu Asn Leu Leu Tyr Glu Gln Ala Leu Gly Leu Gln Leu
80 85 90
Met Pro Leu Leu Gln Thr Leu Asp Asn Leu Lys Glu Gly Lys His
95 100 105
His Leu Arg Val Arg Pro Ala Asp Leu Pro Val Ala Glu Arg Ala
110 115 120
Ser Leu Asn Tyr Trp Arg Thr Trp Lys Cys Ile Ser Lys Pro Ser
125 130 135
Glu Phe Pro Ile Lys Ser Pro Ala Phe Thr Gly Leu His Asp Lys
140 145 150
Ala Pro Leu Gly Leu Met Asp Thr Pro Leu Leu Asp Thr Glu Glu
155 160 165
Glu Val His Tyr Cys Phe Leu Pro Leu Asp Leu Val Ala Lys Tyr
170 175 180
Pro Ser Leu Val Thr Glu Asp Asn Leu Leu Trp Leu Ala Glu Thr
185 190 195
Val Ala Leu Ile Glu Cys Glu Cys Ser Glu Phe Arg Phe Ile Val
200 205 210
Asn Phe Leu Arg Ser Gln Lys Ile Leu Leu Pro Asp Asn Phe Ser
215 220 225
Asn Ile Asp Val Leu Glu Ala Glu Val Glu Ile Leu Glu Ile Pro
230 235 240
Ala Leu Thr Glu Ala Val Arg Trp Tyr Arg Met Asn Met Gly Gly
245 250 255
Cys Ser Pro Thr Thr Cys Ser Pro Leu Ser Pro Gly Lys Gly Ala
260 265 270
Arg Thr Ala Ser Leu Glu Ser Val Lys Pro Leu Tyr Thr Met Ala
275 280 285
Leu Gly Leu Leu Val Lys Tyr Pro Asp Ser Ala Leu Gly Gln Leu
290 295 300
Arg Ile Glu Ser Thr Leu Asp Gly Ser Arg Leu Tyr Ile Thr Gly
305 310 315
Asn Gly Val Leu Phe Gln His Val Lys Asn Trp Leu Gly Thr Cys
30,
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320 325 330
Arg Leu Pro Leu Thr Glu Thr Ile Ser Glu Val Tyr Glu Leu Cys
335 340 345
Ala Phe Leu Asp Lys Arg Asp Ile Thr Tyr Glu Pro Ile Lys Val
350 355 360
Ala Leu Lys Thr His Leu Glu Pro Arg Thr Leu Ala Pro Met Asp
365 370 375
Val Leu Asn Glu Trp Thr Ala Glu Ile Thr Val Tyr Ser Pro Gln
380 385 390
Gln Ile Ile Lys Val Tyr Val Gly Ser His Trp Tyr Ala Thr Thr
395 400 405
Leu Gln Thr Leu Leu Lys Tyr Pro Glu Leu Leu Ser Asn Pro Gln
410 415 420
Arg Val Tyr Trp Ile Thr Tyr Gly Gln Thr Leu Leu Ile His Gly
425 430 435
Asp Gly Gln Met Phe Arg His Ile Leu Asn Phe Leu Arg Leu Gly
440 445 450
Lys Leu Phe Leu Pro Ser Glu Phe Lys Glu Trp Pro Leu Phe Cys
455 460 465
Gln Glu Val Glu Glu Tyr His Ile Pro Ser Leu Ser Glu Ala Leu
470 475 480
Ala Gln Cys Glu Ala Tyr Lys Ser Trp Thr Gln Glu Lys Glu Ser
485 490 495
Glu Asn Glu Glu Ala Phe Ser Ile Arg Arg Leu His Val Val Thr
500 505 510
Glu Gly Pro Gly Ser Leu Val Glu Phe Ser Arg Asp Thr Lys Glu
515 520 525
Thr Thr Ala Tyr Met Pro Val Asp Phe Glu Asp Cys Ser Asp Arg
530 535 540
Thr Pro Trp Asn Lys Ala Lys Gly Asn Leu Val Arg Ser Asn Gln
545 550 555
Met Asp Glu Ala Glu Gln Tyr Thr Arg Pro Ile Gln Val Ser Leu
560 565 570
Cys Arg Asn Ala Lys Arg Ala Gly Asn Pro Ser Thr Tyr Ser His
575 580 585
Cys Arg Gly Leu Cys Thr Asn Pro Gly His Trp Gly Ser His Pro
590 595 600
Glu Ser Pro Pro Lys Lys Lys Cys Thr Thr Ile Asn Leu Thr Gln
605 610 615
Lys Ser Glu Thr Lys Asp Pro Pro Ala Thr Pro Met Gln Lys Leu
620 625 630
Ile Ser Leu Val Arg Glu Trp Asp Met Val Asn Cys Lys Gln Trp
635 640 645
Glu Phe Gln Pro Leu Thr Ala Thr Arg Ser Ser Pro Leu Glu Glu
650 655 660
Ala Thr Leu Gln Leu Pro Leu Gly Ser Glu Ala Ala Ser Gln Pro
665 670 675
Ser Thr Ser Ala Ala Trp Lys Ala His Ser Thr Ala Ser Glu Lys
680 685 690
Asp Pro Gly Pro Gln Ala Gly Ala Gly Ala Gly Ala Lys Asp Lys
695 700 705
Gly Pro Glu Pro Thr Phe Lys Pro Tyr Leu Pro Pro Lys Arg Ala
710 715 720
Gly Thr Leu Lys Asp Trp Ser Lys Gln Arg Thr Lys Glu Arg Glu
725 730 735
Ser Pro Ala Pro Glu Gln Pro Leu Pro Glu Ala Ser Glu Val Asp
31
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740 745 750
Ser Leu Gly Val Ile Leu Lys Val Thr His Pro Pro Val Val Gly
755 760 765
Ser Asp Gly Phe Cys Met Phe Phe Glu Asp Ser Ile Ile Tyr Thr
770 775 780
Thr Glu Met Asp Asn Leu Arg His Thr Thr Pro Thr Ala Ser Pro
785 790 795
Gln Pro Gln Glu Val Thr Phe Leu Ser Phe Ser Leu Ser Trp Glu
800 805 810
Glu Met Phe Tyr Ala Gln Lys Cys His Cys Phe Leu Ala Asp Ile
815 820 825
Ile Met Asp Ser Ile Arg Gln Lys Asp Pro Lys Ala Ile Thr Ala
830 835 840
Lys Val Val Ser Leu Ala Asn Arg Leu Trp Thr Leu His Ile Ser
845 850 855
Pro Lys Gln Phe Val Val Asp Leu Leu Ala Ile Thr Gly Phe Lys
860 865 870
Asp Asp Arg His Thr Gln Glu Arg Leu Tyr Ser Trp Val Glu Leu
875 880 885
Thr Leu Pro Phe Ala Arg Lys Tyr Gly Arg Cys Met Asp Leu Leu
890 895 900
Ile Gln Arg Gly Leu Ser Arg Ser Val Ser Tyr Ser Ile Leu Gly
905 910 915
Lys Tyr Leu Gln Glu Asp
920
<210> 15
<211> 530
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 56008413CD1
<400> 15
Met Gly Ser Val Gly Ser Gln Arg Leu Glu Glu Pro Ser Val Ala
1 5 10 15
Gly Thr Pro Asp Pro Gly Val Val Met Ser Phe Thr Phe Asp Ser
20 25 30
His Gln Leu Glu Glu Ala Ala Glu Ala Ala Gln Gly Gln Gly Leu
35 40 45
Arg Ala Arg Gly Val Pro Ala Phe Thr Asp Thr Thr Leu Asp Glu
50 55 60
Pro Val Pro Asp Asp Arg Tyr His Ala Ile Tyr Phe Ala Met Leu
65 70 75
Leu Ala Gly Val Gly Phe Leu Leu Pro Tyr Asn Ser Phe Ile Thr
80 85 90
Asp Val Asp Tyr Leu His His Lys Tyr Pro Gly Thr Ser Ile Val
95 100 105
Phe Asp Met Ser Leu Thr Tyr Ile Leu Val Ala Leu Ala Ala Val
110 115 120
Leu Leu Asn Asn Val Leu Val Glu Arg Leu Thr Leu His Thr Arg
125 130 135
Ile Thr Ala Gly Tyr Leu Leu Ala Leu Gly Pro Leu Leu Phe Ile
140 145 150
32
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Ser Ile Cys Asp Val Trp Leu Gln Leu Phe Ser Arg Asp Gln Ala
155 160 165
Tyr Ala Ile Asn Leu Ala Ala Val Gly Thr Val Ala Phe Gly Cys
170 175 180
Thr Val Gln Gln Ser Ser Phe Tyr Gly Tyr Thr Gly Met Leu Pro
185 190 195
Lys Arg Tyr Thr Gln Gly Val Met Thr Gly Glu Ser Thr Ala Gly
200 205 210
Val Met Ile Ser Leu Ser Arg Ile Leu Thr Lys Leu Leu Leu Pro
215 220 225
Asp Glu Arg Ala Ser Thr Leu Ile Phe Phe Leu Val Ser Val Ala
230 235 240
Leu Glu Leu Leu Cys Phe Leu Leu His Leu Leu Val Arg Arg Ser
245 250 255
Arg Phe Val Leu Phe Tyr Thr Thr Arg Pro Arg Asp Ser His Arg
260 265 270
Gly Arg Pro Gly Leu Gly Arg Gly Tyr Gly Tyr Arg Val His His
275 280 285
Asp Val Val Ala Gly Asp Val His Phe Glu His Pro Ala Pro Ala
290 295 300
Leu Ala Pro Asn Glu Ser Pro Lys Asp Ser Pro Ala His Glu Val
305 310 315
Thr Gly Ser Gly Gly Ala Tyr Met Arg Phe~Asp Val Pro Arg Pro
320 325 330
Arg Val Gln Arg Ser Trp Pro Thr Phe Arg Ala Leu Leu Leu His
335 340 345
Arg Tyr Val Val Ala Arg Val Ile Trp Ala Asp Met Leu Ser Ile
350 355 360
Ala Val Thr Tyr Phe Ile Thr Leu Cys Leu Phe Pro Gly Leu Glu
365 370 375
Ser Glu Ile Arg His Cys Ile Leu Gly Glu Trp Leu Pro Ile Leu
380 385 390
Ile Met Ala Val Phe Asn Leu Ser Asp Phe Val Gly Lys Ile Leu
395 400 405
Ala Ala Leu Pro Val Asp Trp Arg Gly Thr His Leu Leu Ala Cys
410 415 420
Ser Cys Leu Arg Val Val Phe Ile Pro Leu Phe Ile Leu Cys Val
425 430 435
Tyr Pro Ser Gly Met Pro Ala Leu Arg His Pro Ala Trp Pro Cys
440 445 450
Ile Phe Ser Leu Leu Met Gly Ile Ser Asn Gly Tyr Phe Gly Ser
455 460 465
Val Pro Met Ile Leu Ala Ala Gly Lys Val Ser Pro Lys Gln Arg
470 475 480
Glu Leu Ala Gly Asn Thr Met Thr Val Ser Tyr Met Ser Gly Leu
485 490 495
Thr Leu Gly Ser Ala Val Ala Tyr Cys Thr Tyr Ser Leu Thr Arg
500 505 510
Asp Ala His Gly Ser Cys Leu His Ala Ser Thr Ala Asn Gly Ser
515 520 525
Ile Leu Ala Gly Leu
530
<210> 16
<211> 1617
<212> PRT
33
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<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6127911CD1
<400> 16
Met Asn Met Lys Gln Lys Ser Val Tyr Gln Gln Thr Lys Ala Leu
1 5 10 15
Leu Cys Lys Asn Phe Leu Lys Lys Trp Arg Met Lys Arg Glu Ser
20 25 30
Leu Leu Glu Trp Gly Leu Ser Ile Leu Leu Gly Leu Cys Ile Ala
35 40 45
Leu Phe Ser Ser Ser Met Arg Asn Val Gln Phe Pro Gly Met Ala
50 - 55 60
Pro Gln Asn Leu Gly Arg Val Asp Lys Phe Asn Ser Ser Ser Leu
65 70 75
Met Val Val Tyr Thr Pro Ile Ser Asn Leu Thr Gln Gln Ile Met
80 85 90
Asn Lys Thr Ala Leu Ala Pro Leu Leu Lys Gly Thr Ser Val Ile
95 100 105
Gly Ala Pro Asn Lys Thr His Met Asp Glu Ile Leu Leu Glu Asn
110 115 120
Leu Pro Tyr Ala Met Gly Ile Ile Phe Asn Glu Thr Phe Ser Tyr
125 130 135
Lys Leu Ile Phe Phe Gln Gly Tyr Asn Ser Pro Leu Trp Lys Glu
140 145 150
Asp Phe Ser Ala His Cys Trp Asp Gly Tyr Gly Glu Phe Ser Cys
155 160 165
Thr Leu Thr Lys Tyr Trp Asn Arg Gly Phe Val Ala Leu Gln Thr
170 175 180
Ala Ile Asn Thr Ala Ile Ile Glu Ile Thr Thr Asn His Pro Val
185 190 195
Met Glu Glu Leu Met Ser Val Thr Ala Ile Thr Met Lys Thr Leu
200 205 210
Pro Phe Ile Thr Lys Asn Leu Leu His Asn Glu Met Phe Ile Leu
215 220 225
Phe Phe Leu Leu His Phe Ser Pro Leu Val Tyr Phe Ile Ser Leu
230 235 240
Asn Val Thr Lys Glu Arg Lys Lys Ser Lys Asn Leu Met Lys Met
245 250 255
Met Gly Leu Gln Asp Ser Ala Phe Trp Leu Ser Trp Gly Leu Ile
260 265 270
Tyr Ala Gly Phe Ile Phe Ile Ile Ser Ile Phe Ile Thr Ile Ile
275 280 285
Ile Thr Phe Thr Gln Ile Ile Val Met Thr Gly Phe Met Val Ile
290 295 300
Phe Ile Leu Phe Phe Leu Tyr Gly Leu Ser Leu Val Ala Leu Val
305 310 315
Phe Leu Met Ser Val Leu Leu Lys Lys Ala Val Leu Thr Asn Leu
320 325 330
Val Val Phe Leu Leu Thr Leu Phe Trp Gly Cys Leu Gly Phe Thr
335 340 345
Val Phe Tyr Glu Gln Leu Pro Ser Ser Leu Glu Trp Ile Leu Asn
350 355 360
Ile Cys Ser Pro Phe Ala Phe Thr Thr Gly Met Ile Gln Ile Ile
34
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365 370 375
Lys Leu Asp Tyr Asn Leu Asn Gly Val Ile Phe Pro Asp Pro Ser
380 385 390
Gly Asp Ser Tyr Thr Met Ile Ala Thr Phe Ser Met Leu Leu Leu
395 400 405
Asp Gly Leu Ile Tyr Leu Leu Leu Ala Leu Tyr Phe Asp Lys Ile
410 415 420
Leu Pro Tyr Gly Asp Glu Arg His Tyr Ser Pro Leu Phe Phe Leu
425 430 435
Asn Ser Ser Ser Cys Phe Gln His Gln Arg Thr Asn Ala Lys Val
440 445 450
Ile Glu Lys Glu Ile Asp Ala Glu His Pro Ser Asp Asp Tyr Phe
455 460 465
Glu Pro Val Ala Pro Glu Phe Gln Gly Lys Glu Ala Ile Arg Ile
470 475 480
Arg Asn Val Lys Lys Glu Tyr Lys Gly Lys Ser Gly Lys Val Glu
485 490 495
Ala Leu Lys Gly Leu Leu Phe Asp Ile Tyr Glu Gly Gln Ile Thr
500 505 510
Ala Ile Leu Gly His Ser Gly Ala Gly Lys Ser Ser Leu Leu Asn
515 520 525
Ile Leu Asn Gly Leu Ser Val Pro Thr Glu Gly Ser Val Thr Ile
530 535 540
Tyr Asn Lys Asn Leu Ser Glu Met Gln Asp Leu Glu Glu Ile Arg
545 550 555
Lys Ile Thr Gly Val Cys Pro Gln Phe Asn Val Gln Phe Asp Ile
560 565 570
Leu Thr Val Lys Glu Asn Leu Ser Leu Phe Ala Lys Ile Lys Gly
575 580 585
Ile His Leu Lys Glu Val Glu Gln Glu Val Gln Arg Ile Leu Leu
590 595 600
Glu Leu Asp Met Gln Asn Ile Gln Asp Asn Leu Ala Lys His Leu
605 610 615
Ser Glu Gly Gln Lys Arg Lys Leu Thr Phe Gly Ile Thr Ile Leu
620 625 630
Gly Asp Pro Gln Ile Leu Leu Leu Asp Glu Pro Thr Thr Gly Leu
635 640 645
Asp Pro Phe Ser Arg Asp Gln Val Trp Ser Leu Leu Arg Glu Arg
650 655 660
Arg Ala Asp His Val Ile Leu Phe Ser Thr Gln Ser Met Asp Glu
665 670 675
Ala Asp Ile Leu Ala Asp Arg Lys Val Ile Met Ser Asn Gly Arg
680 685 690
Leu Lys Cys Ala Gly~Ser Ser Met Phe Leu Lys Arg Arg Trp Gly
695 700 705
Leu Gly Tyr His Leu Ser Leu His Arg Asn Glu Ile Cys Asn Pro
710 715 720
Glu Gln Ile Thr Ser Phe Ile Thr His His Ile Pro Asp Ala Lys
725 730 735
Leu Lys Thr Glu Asn Lys Glu Lys Leu Val Tyr Thr Leu Pro Leu
740 745 750
Glu Arg Thr Asn Thr Phe Pro Asp Leu Phe Ser Asp Leu Asp Lys
755 760 765
Cys Ser Asp Gln Gly Val Thr Gly Tyr Asp Ile Ser Met Ser Thr
770 775 780
Leu Asn Glu Val Phe Met Lys Leu Glu Gly Gln Ser Thr Ile Glu
CA 02438206 2003-08-06
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785 790 795
Gln Asp Phe Glu Gln Val Glu Met Ile Arg Asp Ser Glu Ser Leu
800 805 810
Asn Glu Met Glu Leu Ala His Ser Ser Phe Ser Glu Met Gln Thr
815 820 825
Ala Val Ser Asp Met Gly Leu Trp Arg Met Gln Val Phe Ala Met
830 835 840
Ala Arg Leu Arg Phe Leu Lys Leu Lys Arg Gln Thr Lys Val Leu
845 850 855
Leu Thr Leu Leu Leu Val Phe Gly Ile Ala Ile Phe Pro Leu Ile
860 865 870
Val Glu Asn Ile Ile Tyr Ala Met Leu Asn Glu Lys Ile Asp Trp
875 880 885
Glu Phe Lys Asn Glu Leu Tyr Phe Leu Ser Pro Gly Gln Leu Pro
890 895 900
Gln Glu Pro Arg Thr Ser Leu Leu Ile Ile Asn Asn Thr Glu Ser
905 910 915
Asn Ile Glu Asp Phe Ile Lys Ser Leu Lys His Gln Asn Ile Leu
920 925 930
Leu Glu Val Asp Asp Phe Glu Asn Arg Asn Gly Thr Asp Gly Leu
935 940 945
Ser Tyr Asn Gly Ala Ile Ile Val Ser Gly Lys Gln Lys Asp Tyr
950 955 960
Arg Phe Ser Val Val Cys Asn Thr Lys Arg Leu His Cys Phe Pro
965 970 975
Ile Leu Met Asn Ile Ile Ser Asn Gly Leu Leu Gln Met Phe Asn
980 985 990
His Thr Gln His Ile Arg Ile Glu Ser Ser Pro Phe Pro Leu Ser
995 1000 1005
His Ile Gly Leu Trp Thr Gly Leu Pro Asp Gly Ser Phe Phe Leu
1010 1015 1020
Phe Leu Val Leu Cys Ser Ile Ser Pro Tyr Ile Thr Met Gly Ser
1025 1030 1035
Ile Ser Asp Tyr Lys Lys Asn Ala Lys Ser Gln Leu Trp Ile Ser
1040 1045 1050
Gly Leu Tyr Thr Ser Ala Tyr Trp Cys Gly Gln Ala Leu Val Asp
1055 1060 1065
Val Ser Phe Phe Ile Leu Ile Leu Leu Leu Met Tyr Leu Ile Phe
1070 1075 1080
Tyr Ile Glu Asn Met Gln Tyr Leu Leu Ile Thr Ser Gln Ile Val
1085 1090 1095
Phe Ala Leu Val Ile Val Thr Pro Gly Tyr Ala Ala Ser Leu Val
1100 1105 1110
Phe Phe Ile Tyr Met Ile Ser Phe Ile Phe Arg Lys Arg Arg Lys
1115 1120 1125
Asn Ser Gly Leu Trp Ser Phe Tyr Phe Phe Phe Ala Ser Thr Ile
1130 1135 1140
Met Phe Ser Ile Thr Leu Ile Asn His Phe Asp Leu Ser Ile Leu
1145 1150 1155
Ile Thr Thr Met Val Leu Val Pro Ser Tyr Thr Leu Leu Gly Phe
1160 1165 1170
Lys Thr Phe Leu Glu Val Arg Asp Gln Glu His Tyr Arg Glu Phe
1175 1180 1185
Pro Glu Ala Asn Phe Glu Leu Ser Ala Thr Asp Phe Leu Val Cys
1190 1195 1200
Phe Ile Pro Tyr Phe Gln Thr Leu Leu Phe Val Phe Val Leu Arg
36
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1205 1210 1215
Cys Met Glu Leu Lys Cys Gly Lys Lys Arg Met Arg Lys Asp Pro
1220 1225 1230
Val Phe Arg Ile Ser Pro Gln Ser Arg Asp Ala Lys Pro Asn Pro
1235 1240 1245
Glu Glu Pro Ile Asp Glu Asp Glu Asp Ile Gln Thr Glu Arg Ile
1250 1255 1260
Arg Thr Ala Thr Ala Leu Thr Thr Ser Ile Leu~Asp Glu Lys Pro
1265 1270 1275
Val Ile Ile Ala Ser Cys Leu His Lys Glu Tyr Ala Gly Gln Lys
1280 1285 1290
Lys Ser Cys Phe Ser Lys Arg Lys Lys Lys Ile Ala Ala Arg Asn
1295 1300 1305
Ile Ser Phe Cys Val Gln Glu Gly Glu Ile Leu Gly Leu Leu Gly
1310 1315 1320
Pro Ser Gly Ala Gly Lys Ser Ser Ser Ile Arg Met Ile Ser Gly
1325 1330 1335
Ile Thr Lys Pro Thr Ala Gly Glu Val Glu Leu Lys Gly Cys Ser
1340 1345 1350
Ser Val Leu Gly His Leu Gly Tyr Cys Pro Gln Glu Asn Val Leu
1355 1360 1365
Trp Pro Met Leu Thr Leu Arg Glu His Leu Glu Val Tyr Ala Ala
1370 1375 1380
Val Lys Gly Leu Arg Lys Ala Asp Ala Arg Leu Ala Ile Ala Arg
1385 1390 1395
Leu Val Ser Ala Phe Lys Leu His Glu Gln Leu Asn Val Pro Val
1400 1405 1410
Gln Lys Leu Thr Ala Gly Ile Thr Arg Lys Leu Cys Phe Val Leu
1415 1420 1425
Ser Leu Leu Gly Asn Ser Pro Val Leu Leu Leu Asp Glu Pro Ser
1430 1435 1440
Thr Gly Ile Asp Pro Thr Gly Gln Gln Gln Met Trp Gln Ala Ile
1445 1450 1455
Gln Ala Val Val Lys Asn Thr Glu Arg Gly Val Leu Leu Thr Thr
1460 1465 1470
His Asn Leu Ala Glu Ala Glu Ala Leu Cys Asp Arg Val Ala Ile
1475 1480 1485
Met Val Ser Gly Arg Leu Arg Cys Ile Gly Ser Ile Gln His Leu
1490 1495 1500
Lys Asn Lys Leu Gly Lys Asp Tyr Ile Leu Glu Leu Lys Val Lys
1505 1510 1515
Glu Thr Ser Gln Val Thr Leu Val His Thr Glu Ile Leu Lys Leu
1520 1525 1530
Phe Pro Gln Ala Ala Gly Gln Glu Arg Tyr Ser Ser Leu Leu Thr
1535 1540 1545
Tyr Lys Leu Pro Val Ala Asp Val Tyr Pro Leu Ser Gln Thr Phe
1550 1555 1560
His Lys Leu Glu Ala Val Lys His Asn Phe Asn Leu Glu Glu Tyr
1565 1570 1575
Ser Leu Ser Gln Cys Thr Leu Glu Lys Val Phe Leu Glu Leu Ser
1580 1585 1590
Lys Glu Gln Glu Val Gly Asn Phe Asp Glu Glu Ile Asp Thr Thr
1595 1600 1605
Met Arg Trp Lys Leu Leu Pro His Ser Asp Glu Pro
1610 1615
37
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<210> 17
<211> 1192
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6427133CD1
<400> 17
Met Phe Cys Ser Glu Lys Lys Leu Arg Glu Val Glu Arg Ile Val
1 5 10 15
Lys Ala Asn Asp Arg Glu Tyr Asn Glu Lys Phe Gln Tyr Ala Asp
20 25 30
Asn Arg Ile His Thr Ser Lys Tyr Asn Ile Leu Thr Phe Leu Pro
35 40 45
Ile Asn Leu Phe Glu Gln Phe Gln Arg Val Ala Asn Ala Tyr Phe
50 55 60
Leu Cys Leu Leu Ile Leu Gln Leu Ile Pro Glu Ile Ser Ser Leu
65 70 75
Thr Trp Phe Thr Thr Ile Val Pro Leu Val Leu Val Ile Thr Met
80 85 90
Thr Ala Val Lys Asp Ala Thr Asp Asp Tyr Phe Arg His Lys Ser
95 100 105
Asp Asn Gln Val Asn Asn Arg Gln Ser Glu Val Leu Ile Asn Ser
110 115 120
Lys Leu Gln Asn Glu Lys Trp Met Asn Val Lys Val Gly Asp Ile
125 130 135
Ile Lys Leu Glu Asn Asn Gln Phe Val Ala Ala Asp Leu Leu Leu
140 145 150
Leu Ser Ser Ser Glu Pro His Gly Leu Cys Tyr Val Glu Thr Ala
155 160 165
Glu Leu Asp Gly Glu Thr Asn Leu Lys Val Arg His Ala Leu Ser
170 175 180
Val Thr Ser Glu Leu Gly Ala Asp Ile Ser Arg Leu Ala Gly Phe
185 190 195
Asp Gly Ile Val Val Cys Glu Val Pro Asn Asn Lys Leu Asp Lys
200 205 210
Phe Met Gly Ile Leu Ser Trp Lys Asp Ser Lys His Ser Leu Asn
215 220 225
Asn Glu Lys Ile Ile Pro Arg Gly Cys Ile Leu Arg Asn Thr Ser
230 235 240
Trp Cys Phe Gly Met Val Ile Phe Ala Gly Pro Asp Thr Lys Leu
245 250 255
Met Gln Asn Ser Gly Lys Thr Lys Phe Lys Arg Thr Ser Ile Asp
260 265 270
Arg Leu Met Asn Thr Leu Val Leu Trp Ile Phe Gly Phe Leu Ile
275 280 285
Cys Leu Gly Ile Ile Leu Ala Ile Gly Asn Ser Ile Trp Glu Ser
290 295 300
Gln Thr Gly Asp Gln Phe Arg Thr Phe Leu Phe Trp Asn Glu Gly
305 310 315
Glu Lys Ser Ser Val Phe Ser Gly Phe Leu Thr Phe Trp Ser Tyr
320 325 330
Ile Ile Ile Leu Asn Thr Val Val Pro Ile Ser Leu Tyr Val Ser
335 340 345
38
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Val Glu Val Ile Arg Leu Gly His Ser Tyr Phe Ile Asn Trp Asp
350 355 360
Arg Lys Met Tyr Tyr Ser Arg Lys Ala Ile Pro Ala Val Ala Arg
365 370 375
Thr Thr Thr Leu Asn Glu Glu Leu Gly Gln Ile Glu Tyr Ile Phe
380 385 390
Ser Asp Lys Thr Gly Thr Leu Thr Gln Asn Ile Met Thr Phe Lys
395 400 405
Arg Cys Ser Ile Asn Gly Arg Ile Tyr Gly Glu Val His Asp Asp
410 415 420
Leu Asp Gln Lys Thr Glu Ile Thr Gln Glu Lys Glu Pro Val Asp
425 430 435
Phe Ser Val Lys Ser Gln Ala Asp Arg Glu Phe Gln Phe Phe Asp
440 445 450
His Asn Leu Met Glu Ser Ile Lys Met Gly Asp Pro Lys Val His
455 460 465
Glu Phe Leu Arg Leu Leu Ala Leu Cys His Thr Val Met Ser Glu
470 475 480
Glu Asn Ser Ala Gly Glu Leu Ile Tyr Gln Val Gln Ser Pro Asp
485 490 495
Glu Gly Ala Leu Val Thr Ala Ala Arg Asn Phe Gly Phe Ile Phe
500 505 510
Lys Ser Arg Thr Pro Glu Thr Ile Thr Ile Glu Glu Leu Gly Thr
515 520 525
Leu Val Thr Tyr Gln Leu Leu Ala Phe Leu Asp Phe Asn Asn Thr
530 535 540
Arg Lys Arg Met Ser Val Ile Val Arg Asn Pro Glu Gly Gln Ile
545 550 555
Lys Leu Tyr Ser Lys Gly Ala Asp Thr Ile Leu Phe Glu Lys Leu
560 565 570
His Pro Ser Asn Glu Val Leu Leu Ser Leu Thr Ser Asp His Leu
575 580 585
Ser Glu Phe Ala Gly Glu Gly Leu Arg Thr Leu Ala Ile Ala Tyr
590 595 600
Arg Asp Leu Asp Asp Lys Tyr Phe Lys Glu Trp His Lys Met Leu
605 610 615
Glu Asp Ala Asn Ala Ala Thr Glu Glu Arg Asp Glu Arg Ile Ala
620 625 630
Gly Leu Tyr Glu Glu Ile Glu Arg Asp Leu Met Leu Leu Gly Ala
635 640 645
Thr Ala Val Glu Asp Lys Leu Gln Glu Gly Val Ile Glu Thr Val
650 655 660
Thr Ser Leu Ser Leu Ala Asn Ile Lys Ile Trp Val Leu Thr Gly
665 670 675
Asp Lys Gln Glu Thr Ala Ile Asn Ile Gly Tyr Ala Cys Asn Met
680 685 690
Leu Thr Asp Asp Met Asn Asp Val Phe Val Ile Ala Gly Asn Asn
695 700 705
Ala Val Glu Val Arg Glu Glu Leu Arg Lys Ala Lys Gln Asn Leu
710 715 720
Phe Gly Gln Asn Arg Asn Phe Ser Asn Gly His Val Val Cys Glu
725 730 735
Lys Lys Gln Gln Leu Glu Leu Asp Ser Ile Val Glu Glu Thr Ile
740 745 750
Thr Gly Asp Tyr Ala Leu Ile Ile Asn Gly His Ser Leu Ala His
755 760 765
39
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Ala Leu Glu Ser Asp Val Lys Asn Asp Leu Leu Glu Leu Ala Cys
770 775 780
Met Cys Lys Thr Val Ile Cys Cys Arg Val Thr Pro Leu Gln Lys
785 790 795
Ala Gln Val Val Glu Leu Val Lys Lys Tyr Arg Asn Ala Val Thr
800 805 810
Leu Ala Ile Gly Asp Gly Ala Asn Asp Val Ser Met Ile Lys Ser
815 820 825
Ala His Ile Gly Val Gly Ile Ser Gly Gln Glu Gly Leu Gln Ala
830 835 840
Val Leu Ala Ser Asp Tyr Ser Phe Ala Gln Phe Arg Tyr Leu Gln
845 850 855
Arg Leu Leu Leu Val His Gly Arg Trp Ser Tyr Phe Arg Met Cys
860 865 870
Lys Phe Leu Cys Tyr Phe Phe Tyr Lys Asn Phe Ala Phe Thr Leu
875 880 885
Val His Phe Trp Phe Gly Phe Phe Cys Gly Phe Ser Ala Gln Thr
890 895 900
Val Tyr Asp Gln Trp Phe Ile Thr Leu Phe Asn Ile Val Tyr Thr
905 910 915
Ser Leu Pro Val Leu Ala Met Gly Ile Phe Asp Gln Asp Val Ser
920 925 930
Asp Gln Asn Ser Val Asp Cys Pro Gln Leu Tyr Lys Pro Gly Gln
935 940 945
Leu Asn Leu Leu Phe Asn Lys Arg Lys Phe Phe Ile Cys Val Leu
950 955 960
His Gly Ile Tyr Thr Ser Leu Val Leu Phe Phe Ile Pro Tyr Gly
965 970 975
Ala Phe Tyr Asn Val Ala Gly Glu Asp Gly Gln His Ile Ala Asp
980 985 990
Tyr Gln Ser Phe Ala Val Thr Met Ala Thr Ser Leu Val Ile Val
995 1000 1005
Val Ser Val Gln Ile Ala Leu Asp Thr Ser Tyr Trp Thr Phe Ile
1010 1015 1020
Asn His Val Phe Ile Trp Gly Ser Ile Ala Ile Tyr Phe Ser Ile
1025 1030 1035
Leu Phe Thr Met His Ser Asn Gly Ile Phe Gly Ile Phe Pro Asn
1040 1045 1050
Gln Phe Pro Phe Val Gly Asn Ala Arg His Ser Leu Thr Gln Lys
1055 1060 1065
Cys Ile Trp Leu Val Ile Leu Leu Thr Thr Val Ala Ser Val Met
1070 1075 1080
Pro Val Val Ala Phe Arg Phe Leu Lys Val Asp Leu Tyr Pro Thr
1085 1090 1095
Leu Ser Asp Gln Ile Arg Arg Trp Gln Lys Ala Gln Lys Lys Ala
1100 1105 1110
Arg Pro Pro Ser Ser Arg Arg Pro Arg Thr Arg Arg Ser Ser Ser
1115 1120 1125
Arg Arg Ser Gly Tyr Ala Phe Ala His Gln Glu Gly Tyr Gly Glu
1130 1135 1140
Leu Ile Thr Ser Gly Lys Asn Met Arg Ala Lys Asn Pro Pro Pro
1145 1150 1155
Thr Ser Gly Leu Glu Lys Thr His Tyr Asn Ser Thr Ser Trp Ile
1160 1165 1170
Glu Asn Leu Cys Lys Lys Thr Thr Asp Thr Val Ser Ser Phe Ser
1175 1180 1185
CA 02438206 2003-08-06
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Gln Asp Lys Thr Val Lys Leu
1190
<210> 18
<211> 625
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472932CD1
<400> 18
Met Ala His Ala Pro Glu Pro Asp Pro Ala Ala Ser Asp Leu Gly
1 5 10 15
Asp Glu Arg Pro Lys Trp Asp Asn Lys Ala Gln Tyr Leu Leu Ser
20 25 30
Cys Ile Gly Phe Ala Val Gly Leu Gly Asn Ile Trp Arg Phe Pro
35 40 45
Tyr Leu Cys Gln Thr Tyr Gly Gly Gly Ala Phe Leu Ile Pro Tyr
50 55 60
Val Ile Ala Leu Val Phe Glu Gly Ile Pro Ile Phe His Val Glu
65 70 75
Leu Ala Ile Gly Gln Arg Leu Arg Lys Gly Ser Val Gly Val Trp
80 85 90
Thr Ala Ile Ser Pro Tyr Leu Ser Gly Val Gly Leu Gly Cys Val
95 100 105
Thr Leu Ser Phe Leu Ile Ser Leu Tyr Tyr Asn Thr Ile Val Ala
110 115 120
Trp Val Leu Trp Tyr Leu Leu Asn Ser Phe Gln His Pro Leu Pro
125 130 135
Trp Ser Ser Cys Pro Pro Asp Leu Asn Arg Thr Gly Phe Val Glu
140 145 150
Glu Cys Gln Gly Ser Ser Ala Val Ser Tyr Phe Trp Tyr Arg Gln
155 160 165
Thr Leu Asn Ile Thr Ala Asp Ile Asn Asp Ser Gly Ser Ile Gln
170 175 180
Trp Trp Leu Leu Ile Cys Leu Ala Ala Ser Trp Ala Val Val Tyr
185 190 195
Met Cys Val Ile Arg Gly Ile Glu Thr Thr Gly Lys Val Ile Tyr
200 205 210
Phe Thr Ala Leu Phe Pro Tyr Leu Val Leu Thr Ile Phe Leu Ile
215 220 225
Arg Gly Leu Thr Leu Pro Gly Ala Thr Lys Gly Leu Ile Tyr Leu
230 235 240
Phe Thr Pro Asn Met His Ile Leu Gln Asn Pro Arg Val Trp Leu
245 250 255
Asp Ala Ala Thr Gln Ile Phe Phe Ser Leu Ser Leu Ala Phe Gly
260 265 270
Gly His Ile Ala Phe Ala Ser Tyr Asn Ser Pro Arg Asn Asp Cys
275 280 285
Gln Lys Asp Ala Val Val Ile Ala Leu Val Asn Arg Met Thr Ser
290 295 300
Leu Tyr Ala Ser Ile Ala Val Phe Ser Val Leu Gly Phe Lys Ala
305 310 315
Thr Asn Asp Cys Pro Arg Arg Asn Ile Leu Ser Leu Ile Asn Asp
41
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320 325 330
Phe Asp Phe Pro Glu Gln Ser Ile Ser Arg Asp Asp Tyr Pro Ala
335 340 345
Val Leu Met His Leu Asn Ala Thr Trp Pro Lys Arg Val Ala Gln
350 355 360
Leu Pro Leu Lys Ala Cys Leu Leu Glu Asp Phe Leu Asp Lys Ser
365 370 375
Ala Ser Gly Pro Gly Leu Ala Phe Val Val Phe Thr Glu Thr Asp
380 385 390
Leu His Met Pro Gly Ala Pro Val Trp Ala Met Leu Phe Phe Gly
395 400 405
Met Leu Phe Thr Leu Gly Leu Ser Thr Met Phe Gly Thr Val Glu
410 415 420
Ala Val Ile Thr Pro Leu Leu Asp Val Gly Val Leu Pro Arg Trp
425 430 435
Val Pro Lys Glu Ala Leu Thr Gly Leu Val Cys Leu Val Cys Phe
440 445 450
Leu Ser Ala Thr Cys Phe Thr Leu Gln Ser Gly Asn Tyr Trp Leu
455 460 465
Glu Ile Phe Asp Asn Phe Ala Ala Ser Leu Asn Leu Leu Met Leu
470 475 480
Ala Phe Leu Glu Val Val Gly Val Val Tyr Val Tyr Gly Met Lys
485 490 495
Arg Phe Cys Asp Asp Ile Ala Trp Met Thr Gly Arg Arg Pro Ser
500 505 510
Pro Tyr Trp Arg Leu Thr Trp Arg Val Val Ser Pro Leu Leu Leu
515 520 525
Thr Ile Phe Val Ala Tyr Ile Ile Leu Leu Phe Trp Lys Pro Leu
530 535 540
Arg Tyr Lys Ala Trp Asn Pro Lys Tyr Glu Leu Phe Pro Ser Arg
545 550 555
Gln Glu Lys Leu Tyr Pro Gly Trp Ala Arg Ala Ala Cys Val Leu
560 565 570
Leu Ser Leu Leu Pro Val Leu Trp Val Pro Val Ala Ala Leu Ala
575 580 585
Gln Leu Leu Thr Arg Arg Arg Arg Thr Trp Arg Asp Arg Asp Ala
590 595 600
Arg Pro Asp Thr Asp Met Arg Pro Asp Thr Asp Thr Arg Pro Asp
605 610 615
Thr Asp Met Arg Pro Asp Thr Asp Met Arg
620 625
<210> 19
<211> 1181
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 8463147CD1
<400> 19
Met Thr Gln Tyr Gln Lys Tyr Leu Glu Leu Pro
Ala Ile Lys Lys
1 5 10 15
Ser Pro Gly Lys Gly Arg Ala Pro Gly Thr Pro
Asp Trp Ser Ser
20 25 30
42
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Gly Asn Leu Leu Ser Pro Phe Met Ala Ala Ser Asn Ser Phe Pro
35 40 45
Glu Leu Cys Ser Gln Val Ser Arg Arg Glu Tyr Trp Asp Leu His
50 55 60
Gly Ile Pro Ser Asp His Phe Ser Val Arg Val Gln Val Glu Phe
65 70 75
Tyr Met Asn Glu Asn Thr Phe Lys Glu Arg Leu Thr Leu Phe Phe
80 85 90
Ile Thr Asn Gln Arg Ser Ser Leu Arg Ile Arg Leu Phe Asn Phe
95 100 105
Ser Leu Lys Leu Leu Ser Cys Leu Leu Tyr Ile Ile Arg Val Leu
110 115 120
Leu Glu Asn Pro Ser Gln Gly Asn Glu Trp Ser His Ile Phe Trp
125 130 135
Val Asn Arg Ser Leu Pro Leu Trp Gly Leu Gln Val Ser Val Ala
140 145 150
Leu Ile Ser Leu Phe Glu Thr Ile Leu Leu Gly Tyr Leu Ser Tyr
155 160 165
Lys Gly Asn Ile Trp Glu Gln Ile Leu Arg Ile Pro Phe Ile Leu
170 175 180
Glu Ile Ile Asn Ala Val Pro Phe Ile Ile Ser Ile Phe Trp Pro
185 190 195
Ser Leu Arg Asn Leu Phe Val Pro Val Phe Leu Asn Cys Trp Leu
200 205 210
Ala Lys His Ala Leu Glu Asn Met Ile Asn Asp Leu His Arg Ala
215 220 225
Ile Gln Arg Thr Gln Cys Cys Lys Cys Val Asn Gln Val Leu Ile
230 235 240
Val Ile Ser Thr Leu Leu Cys Leu Ile Phe Thr Cys Ile Cys Gly
245 250 255
Ile Gln His Leu Glu Arg Ile Gly Lys Lys Leu Asn Leu Phe Asp
260 265 270
Ser Leu Tyr Phe Cys Ile Val Thr Phe Ser Thr Val Gly Phe Gly
275 280 285
Asp Val Thr Pro Glu Thr Trp Ser Ser Lys Leu Phe Val Val Ala
290 295 300
Met Ile Cys Val Ala Leu Val Val Leu Pro Ile Gln Phe Glu Gln
305 310 315
Leu Ala Tyr Leu Trp Met Glu Arg Gln Lys Ser Gly Gly Asn Tyr
320 325 330
Ser Arg His Arg Ala Gln Thr Glu Lys His Val Val Leu Cys Val
335 340 345
Ser Ser Leu Lys Ile Asp Leu Leu Met Asp Phe Leu Asn Glu Phe
350 355 360
Tyr Ala His Pro Arg Leu Gln Asp Tyr Tyr Val Val Ile Leu Cys
365 370 375
Pro Thr Glu Met Asp Val Gln Val Arg Arg Val Leu Gln Ile Pro
380 385 390
Met Trp Ser Gln Arg Val Ile Tyr Leu Gln Gly Ser Ala Leu Lys
395 400 405
Asp Gln Asp Leu Leu Arg Ala Lys Met Asp Asp Ala Glu Ala Cys
410 415 420
Phe Ile Leu Ser Ser Arg Cys Glu Val Asp Arg Thr Ser Ser Asp
425 430 435
His Gln Thr Ile Leu Arg Ala Trp Ala Val Lys Asp Phe Ala Pro
440 445 450
43
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Asn Cys Pro Leu Tyr Val Gln Ile Leu Lys Pro Glu Asn Lys Phe
455 460 465
His Ile Lys Phe Ala Asp His Val Val Cys Glu Glu Glu Phe Lys
470 475 480
Tyr Ala Met Leu Ala Leu Asn Cys Ile Cys Pro Ala Thr Ser Thr
485 490 495
Leu Ile Thr Leu Leu Val His Thr Ser Arg Gly Gln Cys Val Cys
500 505 510
Leu Cys Cys Arg Glu Gly Gln Gln Ser Pro Glu Gln Trp Gln Lys
515 520 525
Met Tyr Gly Arg Cys Ser Gly Asn Glu Val Tyr His Ile Val Leu
530 535 540
Glu Glu Ser Thr Phe Phe Ala Glu Tyr Glu Gly Lys Ser Phe Thr
545 550 555
Tyr Ala Ser Phe His Ala His Lys Lys Phe Gly Val Cys Leu Ile
560 565 570
Gly Val Arg Arg Glu Asp Asn Lys Asn Ile Leu Leu Asn Pro Gly
575 580 585
Pro Arg Tyr Ile Met Asn Ser Thr Asp Ile Cys Phe Tyr Ile Asn
590 595 600
Ile Thr Lys Glu Glu Asn Ser Ala Phe Lys Asn Gln Asp Gln Gln
605 610 615
Arg Lys Ser Asn Val Ser Arg Ser Phe Tyr His Gly Pro Ser Arg
620 625 630
Leu Pro Val His Ser Ile Ile Ala Ser Met Gly Thr Val Ala Ile
635 640 645
Asp Leu Gln Asp Thr Ser Cys Arg Ser Ala Ser Gly Pro Thr Leu
650 655 660
Ser Leu Pro Thr Glu Gly Ser Lys Glu Ile Arg Arg Pro Ser Ile
665 670 675
Ala Pro Val Leu Glu Val Ala Asp Thr Ser Ser Ile Gln Thr Cys
680 685 690
Asp Leu Leu Ser Asp Gln Ser Glu Asp Glu Thr Thr Pro Asp Glu
695 700 705
Glu Met Ser Ser Asn Leu Glu Tyr Ala Lys Gly Tyr Pro Pro Tyr
710 715 720
Ser Pro Tyr Ile Gly Ser Ser Pro Thr Phe Cys His Leu Leu His
725 730 735
Glu Lys Val Pro Phe Cys Cys Leu Arg Leu Asp Lys Ser Cys Gln
740 745 750
His Asn Tyr Tyr Glu Asp Ala Lys Ala Tyr Gly Phe Lys Asn Lys
755 760 765
Leu Ile Ile Val Ala Ala Glu Thr Ala Gly Asn Gly Leu Tyr Asn
770 775 780
Phe Ile Val Pro Leu Arg Ala Tyr Tyr Arg Pro Lys Lys Glu Leu
785 790 795
Asn Pro Ile Val Leu Leu Leu Asp Asn Pro Pro Asp Met His Phe
800 805 810
Leu Asp Ala Ile Cys Trp Phe Pro Met Val Tyr Tyr Met Val Gly
815 820 825
Ser Ile Asp Asn Leu Asp Asp Leu Leu Arg Cys Gly Val Thr Phe
830 835 840
Ala Ala Asn Met Val Val Val Asp Lys Glu Ser Thr Met Ser Ala
845 850 855
Glu Glu Asp Tyr Met Ala Asp Ala Lys Thr Ile Val Asn Val Gln
860 865 870
44,
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
Thr Leu Phe Arg Leu Phe Ser Ser Leu Ser Ile Ile Thr Glu Leu
875 880 885
Thr His Pro Ala Asn Met Arg Phe Met Gln Phe Arg Ala Lys Asp
890 895 900
Cys Tyr Ser Leu Ala Leu Ser Lys Leu Glu Lys Lys Glu Arg Glu
905 910 915
Arg Gly Ser Asn Leu Ala Phe Met Phe Arg Leu Pro Phe Ala Ala
920 925 930
Gly Arg Val Phe Ser Ile Ser Met Leu Asp Thr Leu Leu Tyr Gln
935 940 945
Ser Phe Val Lys Asp Tyr Met Ile Ser Ile Thr Arg Leu Leu Leu
950 955 960
Gly Leu Asp Thr Thr Pro Gly Ser Gly Phe Leu Cys Ser Met Lys
965 970 975
Ile Thr Ala Asp Asp Leu Trp Ile Arg Thr Tyr Ala Arg Leu Tyr
980 985 990
Gln Lys Leu Cys Ser Ser Thr Gly Asp Val Pro Ile Gly Ile Tyr
995 1000 1005
Arg Thr Glu Ser Gln Lys Leu Thr Thr Ser Glu Ser Gln Ile Ser
1010 1015 1020
Ile Ser Val Glu Glu Trp Glu Asp Thr Lys Asp Ser Lys Glu Gln
1025 1030 1035
Gly His His Arg Ser Asn His Arg Asn Ser Thr Ser Ser Asp Gln
1040 1045 1050
Ser Asp His Pro Leu Leu Arg Arg Lys Ser Met Gln Trp Ala Arg
1055 1060 1065
Arg Leu Ser Arg Lys Gly Pro Lys His Ser Gly Lys Thr Ala Glu
1070 1075 1080
Lys Ile Thr Gln Gln Arg Leu Asn Leu Tyr Arg Arg Ser Glu Arg
1085 1090 1095
Gln Glu Leu Ala Glu Leu Val Lys Asn Arg Met Lys His Leu Gly
1100 1105 1110
Leu Ser Thr Val Gly Tyr Asp Glu Met Asn Asp His Gln Ser Thr
1115 1120 1125
Leu Ser Tyr Ile Leu Ile Asn Pro Ser Pro Asp Thr Arg Ile Glu
1130 1135 1140
Leu Asn Asp Val Val Tyr Leu Ile Arg Pro Asp Pro Leu Ala Tyr
1145 1150 1155
Leu Pro Asn Ser Glu Pro Ser Arg Arg Asn Ser Ile Cys Asn Val
1160 1165 1170
Thr Gly Gln Asp Ser Arg Glu Glu Thr Gln Leu
1175 1180
<210> 20
<211> 233
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7506408CD1
<400> 20
Met Leu Glu Gly Ala Glu Leu Tyr Phe Asn Val Asp His Gly Tyr
1 5 10 15
Leu Glu Gly Leu Val Arg Gly Cys Lys Ala Ser Leu Leu Thr Gln
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
20 25 30
Gln Asp Tyr Ile Asn Leu Val Gln Cys Glu Thr Leu Glu Ala Pro
35 40 45
Phe Phe Gln Asp Cys Met Ser Glu Asn Ala Leu Asp Glu Leu Asn
50 55 60
Ile Glu Leu Leu Arg Asn Lys Leu~Tyr Lys Ser Tyr Leu Glu Ala
65 70 75
Phe Tyr Lys Phe Cys Lys Asn His Gly Asp Val Thr Ala Glu Val
80 85 90
Met Cys Pro Ile Leu Glu Phe Glu Ala Asp Arg Arg Ala Phe Ile
95 100 105
Ile Thr Leu Asn Ser Phe Gly Thr Glu Leu Ser Lys Glu Asp Arg
110 115 120
Glu Thr Leu Tyr Pro Thr Phe Gly Lys Leu Tyr Pro Glu Gly Leu
125 130 135
Arg Leu Leu Ala Gln Ala Glu Asp Phe Asp Gln Met Lys Asn Val
140 145 150
Ala Asp His Tyr Gly Val Tyr Lys Pro Leu Phe Glu Ala Val Gly
155 160 165
Gly Ser Gly Gly Lys Thr Leu Glu Asp Val Phe Tyr Glu Arg Glu
170 175 180
Val Gln Met Asn Val Leu Ala Phe Asn Arg Gln Phe His Tyr Gly
185 190 195
Val Phe Tyr Ala Tyr Val Lys Leu Lys Glu Gln Glu Ile Arg Asn
200 205 210
Ile Val Trp Ile Ala Glu Cys Ile Ser Gln Arg His Arg Thr Lys
215 220 225
Ile Asn Ser Tyr Ile Pro Ile Leu
230
<210> 21
<211> 2232
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6911460CB1
<400> 21
attagctttg cccgaagttt ttccccacac tcttctttag catgctatta tggggaaagt 60
gaccactcct gggagcgggg gtggtcgggg cggtttggtg gcggggaagc ggctgtaact 120
tctacgtgac catggtacct gttgaaaaca ccgagggccc cagtctgctg aaccagaagg 180
ggacagccgt ggagacggag ggcagcggca gccggcatcc tccctgggcg agaggctgcg 240
gcatgtttac cttcctgtca tctgtcactg ctgctgtcag tggcctcctg gtgggttatg 300
aacttgggat catctctggg gctcttcttc agatcaaaac cttattagcc ctgagctgcc 360
atgagcagga aatggttgtg agctccctcg tcattggagc cctccttgcc tcactcaccg 420
gaggggtcct gatagacaga tatggaagaa ggacagcaat catcttgtca tcctgcctgc 480
ttggactcgg aagcttagtc ttgatcctca gtttatccta cacggttctt atagtgggac 540
gcattgccat aggggtctcc atctccctct cttccattgc cacttgtgtt tacatcgcag 600
agattgctcc tcaacacaga agaggccttc ttgtgtcact gaatgagctg atgattgtca 660
tcggcattct ttctgcctat atttcaaatt acgcatttgc caatgttttc catggctgga 720
agtacatgtt tggtcttgtg attcccttgg gagttttgca agcaattgca atgtattttc 780
ttcctccaag ccctcggttt ctggtgatga aaggacaaga gggagctgct agcaaggttc 840
ttggaaggtt aagagcactc tcagatacaa ctgaggaact cactgtgatc aaatcctccc 900
tgaaagatga atatcagtac agtttttggg atctgtttcg ttcaaaagac aacatgcgga 960
46
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
cccgaataat gataggacta acactagtat tttttgtaca aatcactggc caaccaaaca 1020
tattgttcta tgcatcaact gttttgaagt cagttggatt tcaaagcaat gaggcagcta 1080
gcctcgcctc cactggggtt ggagtcgtca aggtcattag caccatccct gccactcttc 1140
ttgtagacca tgtcggcagc aaaacattcc tctgcattgg ctcctctgtg atggcagctt 1200
cgttggtgac catgggcatc gtaaatctca acatccacat gaacttcacc catatctgca 1260
gaagccacaa ttctatcaac cagtccttgg atgagtctgt gatttatgga ccaggaaacc 1320
tgtcaaccaa caacaatact ctcagagacc acttcaaagg gatttcttcc catagcagaa 1380
gctcactcat gcccctgaga aatgatgtgg ataagagagg ggagacgacc tcagcatcct 1440
tgctaaatgc tggattaagc cacactgaat accagatagt cacagaccct ggggacgtcc 1500
cagctttttt gaaatggctg tccttagcca gcttgcttgt ttatgttgct gctttttcaa 1560
ttggtctagg accaatgccc tggctggtgc tcagcgagat ctttcctggt gggatcagag 1620
gacgagccat ggctttaact tctagcatga actggggcat caatctcctc atctcgctga 1680
catttttgac tgtaactgat cttattggcc tgccatgggt gtgctttata tatacaatca 1740
tgagtctagc atccctgctt tttgttgtta tgtttatacc tgagacaaag ggatgctctt 1800
tggaacaaat atcaatggag ctagcaaaag tgaactatgt gaaaaacaac atttgtttta 1860
tgagtcatca ccaagaagaa ttagtgccaa aacagcctca aaaaagaaaa ccccaggagc 1920
agctcttgga gtgtaacaag ctgtgtggta ggggccaatc caggcagctt tctccagaga 1980
cctaatggcc tcaacacctt ctgaacgtgg atagtgccag aacacttagg agggtgtctt 2040
tggaccaatg catagttgcg actcctgtgc tctcttttca gtgtcatgga actggttttg 2100
aagagacact ctgaaatgat aaagacagcc tttaatcccc ctcctcccca gaaggaacct 2160
caaaaggtag atgaggtaca aggtcctaag tgatctcttt ttctgagcag gatatcaggt 2220
taaaaaaaaa as 2232
<210> 22
<211> 4135
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 55138203CB1
<400> 22
acaaccccac aggccagctt tttcacatag ttgttaccag cacttggcca acagttgttt 60
ttcatcagtg ggtggagcag cttttcttgc ccccaaaaaa cagtcaacca ctcatttttc 120
attgggtata tgtattcggc aaacattggg tacctgctgt ttgttggcac tggtgttgag 180
aagatgaata acacaccctc tatggcccta gggagttccc attctggtag ggggaacctg 240
actcaggcag caacaaaacc ttctggttat gagaagacag atgatgtttc agagaagacc 300
tcactggctg accaggagga agtaaggact attttcatca accagcccca gctgacaaaa 360
ttctgcaata accatgtcag cactgcaaaa tacaacataa tcacattcct tccaagattt 420
ctctactctc agttcagaag agctgctaat tcattttttc tctttattgc actgctgcag 480
caaatacctg atgtgtcacc aacaggtcgt tatacaacac tggttcctct cttatttatt 540
ttagctgtgg cagctatcaa agagataata gaagatatta aacgacataa agctgataat 600
gcagtgaaca agaaacaaac gcaagttttg agaaatggtg cttgggaaat tgtccactgg 660
gaaaaggtaa atgttggaga tatagttata ataaaaggca aagagtatat acctgctgac 720
actgtacttc tctcatcaag tgagccccaa gccatgtgct acattgaaac atccaactta 780
gatggtgaaa caaacttgaa aattagacag ggcttaccag caacatcaga tatcaaagac 840
gttgacagtt tgatgaggat ttctggcaga attgagtgtg aaagtccaaa cagacatctc 900
tacgattttg ttggaaacat aaggcttgat ggacatggca ccgttccact gggagcagat 960
cagattcttc ttcgaggagc tcagttgaga aatacacagt gggttcatgg aatagttgtc 1020
tacactggac atgacaccaa gctgatgcag aattcaacaa gtccaccact taagctctca 1080
aatgtggaac ggattacaaa tgtacaaatt ttgattttat tttgtatctt aattgccatg 1140
tctcttgtct gttctgtggg ctcagccatt tggaatcgaa ggcattctgg aaaagactgg 1200
tatctcaatc taaactatgg tggcgctagt aattttggac tgaatttctt gaccttcatc 1260
atccttttca acaatctcat tcctatcagc ttattggtta cattagaagt tgtgaaattt 1320
acccaggcat acttcataaa ttgggatctt gacatgcact atgaacccac agacactgct 1380
47
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
gctatggctc gaacatctaa tctgaatgag gaacttggcc aggttaaata catattttct 1440
gacaaaactg gtactctgac atgcaatgta atgcagttta agaagtgcac catagcggga 1500
gttgcttatg gccatgtccc tgaacctgag gattatggct gctctcctga tgaatggcag 1560
aactcacagt ttggagatga aaaaacattt agtgattcat cattgctgga aaatctccaa 1620
aataatcatc ccaccgcacc tataatatgt gaatttctta caatgatggc agtctgtcac 1680
acagcagtgc cagagcgaga aggtgacaag attatttatc aagcagcatc tccagatgag 1740
ggagcattgg tcagagcagc caagcaattg aattttgttt tcactggaag aacacccgac 1800
tcggtgatta tagattcact ggggcaggaa gaaagatatg aattgctcaa tgtcttggag 1860
tttaccagtg ctaggaaaag aatgtcagtg attgttcgca ctccatctgg aaagttacga 1920
ctctactgca aaggagctga cactgtaatt tatgatcgac tggcagagac gtcaaaatac 1980
aaagaaatta ccctaaaaca tttagagcag tttgctacag aagggttaag aactttatgt 2040
tttgctgtgg ctgagatttc agagagcgac tttcaggagt ggcgagcagt ctatcagcga 2100
gcatctacat ctgtgcagaa caggctactc aaactcgaag agagttatga gttgattgaa 2160
aagaatcttc agctacttgg agcaacagcc attgaggata aattacaaga tcaagtgcct 2220
gaaaccatag aaacgctaat gaaagcagac atcaaaatct ggatccttac aggggacaag 2280
caagaaactg ccattaacat cggacactcc tgcaaactgt tgaagaagaa catgggaatg 2340
attgttataa atgaaggctc tcttgatgga acaagggaaa ctctcagtcg tcactgtact 2400
acccttggtg atgctctccg gaaagagaat gattttgctc ttataattga tgggaaaacc 2460
ctcaaatatg ccttaacctt tggagtacga cagtatttcc tggacttagc tttgtcatgc 2520
aaagctgtca tttgctgtcg ggtttctcct cttcaaaaat ctgaagttgt tgagatggtt 2580
aagaaacaag tcaaagtcgt aacgcttgca atcggtgatg gagcaaatga tgtcagcatg 2640
atacagacag cgcacgttgg tgttggtatc agtggcaatg aaggcctgca ggcagctaat 2700
tcctctgact actccatagc tcagttcaaa tatttgaaga atttactgat gattcatggt 2760
gcctggaact ataacagagt ctccaagtgc atcttatact gcttctacaa gaatatagtg 2820
ctctatatta tcgagatctg gtttgccttt gttaatggct tttctggaca gatcctcttt 2880
gaaagatggt gtataggtct ctataacgtg atgtttacag caatgcctcc tttaactctt 2940
ggaatatttg agagatcatg cagaaaagag aacatgttga agtaccctga attatacaaa 3000
acatctcaga atgccctgga cttcaacacc aaggttttct gggttcattg tttaaatggc 3060
ctcttccact cagttattct gttttggttt ccactaaaag cccttcagta tggtactgca 3120
tttggaaatg ggaaaacctc ggattatctg ctactgggaa actttgtgta cacttttgtg 3180
gtgataactg tgtgtttgaa agctggattg gagacatcat attggacatg gttcagccac 3240
atagcgatat gggggagcat cgcactctgg gtggtgtttt tgggaatcta ctcatctctg 3300
tggcctgcca ttccgatggc ccctgatatg tcaggagagg cagccatgtt gttcagttct 3360
ggagtctttt ggatgggctt gttattcatc cctgtggcat ctctgctcct tgatgtggtg 3420
tacaaggtta tcaagaggac tgcttttaaa acattggtcg atgaagttca ggagctggag 3480
gcaaaatctc aagacccagg agcagttgta cttggaaaaa gcctgaccga gagggcgcaa 3540
ctgctcaaga acgtctttaa gaagaaccac gtgaacttgt accgctctga atccttgcaa 3600
caaaatctgc tccatgggta tgcgttctct caagatgaaa atggaatcgt ttcacagtct 3660
gaagtgataa gagcatatga taccacgaaa cagaggcccg acgaatggtg atggggagag 3720
cctgaaaggc aggctctgtt acctctctaa ggagagctac caggttgtca ccgcagtctg 3780
ctaaccaatt ccagtctggt ccatgaagag gaaaggtaga tctgagctca tctcgctgat 3840
ggacattcag attcatgtat attatagaca taagcactgt gcaactgtac tgtaacacca 3900
tctcttttgg atttttttaa ggtatttgct aagtctttgt aaacggaaat tgaaaatgac 3960
ctggtatctt gccagagggc tttcttaaac ggagaataag tcagtattct tatgccatta 4020
ctgtggggct gtaactgact gtcagtttat tggctgtacc acaaggtaac caaccattaa 4080
aaaactctaa atgatattta gttaaaggga ctctgtggta tccagactta gattt 4135
<210> 23
<211> 2970
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7478871CB1
48
CA 02438206 2003-08-06
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<400> 23
atgcaaccag ccagagggcc cctggcttca gaacctagga ctgtactggt tctgagattc 60
tgtgcaagcc tcatggaaat gaagctgcca ggccaggaag ggtttgaagc ctccagtgct 120
cctagaaata ttccttcagg ggagctggac agcaaccctg accctggcac cggccccagc 180
cctgatggcc cctcagacac agagagcaag gaactgggag tacccaaaga ccctctgctc 240
ttcattcagc tgaatgagct gctgggctgg ccccaggcgc tggagtggag agagacaggc 300
acgtgggtac tgtttgagga gaagttggag gtggctgcag gccggtggag tgccccccac 360
gtgcccaccc tggcactgcc cagcctccag aagctccgca gcctgctggc cgagggcctt 420
gtactgctgg actgcccagc tcagagcctc ctggagctcg tggagcaggt gaccagggtg 480
gagtcgctga gcccagagct gagagggcag ttgcaggcct tgctgctgca gagaccccag 540
cattacaacc agaccacagg caccaggccc tgctggggtg agagcccctc cctgggccca 600
ggaccaagac cctgtacaac cagaccacag gcaccaggcc ctgctgggca gtgtcagaac 660
cccctgagac agaagctacc tccaggagct gaggcaggga ctgtgctggc aggggagctg 720
ggcttcctgg cacagccact gggagccttt gttcgactgc ggaaccctgt ggtactgggg 780
tcccttactg aggtgtccct cccaagcagg tttttctgcc ttctcctggg cccctgtatg 840
ctgggaaagg gctaccatga gatgggacgg gcagcagctg tcctcctcag tgacccgcaa 900
ttccagtggt cagttcgtcg ggccagcaac cttcatgacc ttctggcagc cctggatgca 960
ttcctagagg aggtgacagt gcttccccca ggtcggtggg acccaacagc ccggattccc 1020
ccgcccaaat gtctgccatc tcagcacaaa aggcttccct cgcaacagcg ggagatcaga 1080
ggtcccgccg tcccgcgcct gacctcggct gaggacaggc accgccatgg gccacacgca 1140
cacagcccgg agttgcagcg gaccggcagg ctgtttgggg gccttatcca ggacgtgcgc 1200
aggaaggtcc cgtggtaccc cagcgatttc ttggacgccc tgcatctcca gtgcttctcg 1260
gccgtactct acatttacct ggccactgtc actaatgcca tcacttttgg gggtctgctg 1320
ggagatgcca ctgatggtgc ccagggagtg ctggaaagtt tcctgggcac agcagtggct 1380
ggagctgcct tctgcctgat ggcaggccag cccctcacca ttctgagcag cacggggcca 1440
gtgctggtct ttgagcgcct gctcttctct ttcagcagag attacagcct ggactacctg 1500
cccttccgcc tatgggtggg catctgggtg gctacctttt gcctggtgct ggtggccaca 1560
gaggccagtg tgctggtgcg ctacttcacc cgcttcactg aggaaggttt ctgtgccctc 1620
atcagcctca tcttcatcta cgatgctgtg ggcaaaatgc tgaacttgac ccatacctat 1680
cctatccaga agcctgggtc ctctgcctac gggtgcctct gccaataccc aggcccagga 1740
ggaaatgagt ctcaatggat aaggacaagg ccaaaagaca gagacgacat tgtaagcatg 1800
gacttaggcc tgatcaatgc atccttgctg ccgccacctg agtgcacccg gcagggaggc 1860
caccctcgtg gccctggctg tcatacagtc ccagacattg ccttcttctc ccttctcctc 1920
ttccttactt ctttcttctt tgctatggcc ctcaagtgtg taaagaccag ccgcttcttc 1980
ccctctgtgg tgcgcaaagg gctcagcgac ttctcctcag tcctggccat cctgctcggc 2040
tgtggccttg atgctttcct gggcctagcc acaccaaagc tcatggtacc cagagagttc 2100
aagcccacac tccctgggcg tggctggctg gtgtcacctt ttggagccaa cccctggtgg 2160
tggagtgtgg cagctgccct gcctgccctg ctgctgtcta tcctcatctt catggaccaa 2220
cagatcacag cagtcatcct caaccgcatg gaatacagac tgcagaaggg agctggcttc 2280
cacctggacc tcttctgtgt ggctgtgctg atgctactca catcagcgct tggactgcct 2340
tggtatgtct cagccactgt catctccctg gctcacatgg acagtcttcg gagagagagc 2400
agagcctgtg cccccgggga gcgccccaac ttcctgggta tcagggaaca gaggctgaca 2460
ggcctggtgg tgttcatcct tacaggagcc tccatcttcc tggcacctgt gctcaagttc 2520
attccaatgc ctgtgctcta tggcatcttc ctgtatatgg gggtggcagc gctcagcagc 2580
attcagttca ctaatagggt gaagctgttg ttgatgccag caaaacacca gccagacctg 2640
ctactcttgc ggcatgtgcc tctgaccagg gtccacctct tcacagccat ccagcttgcc 2700
tgtctggggc tgctttggat aatcaagtct acccctgcag ccatcatctt ccccctcatg 2760
ttgctgggcc ttgtgggggt ccgaaaggcc ctggagaggg tcttctcacc acaggaactc 2820
ctctggctgg atgagctgat gccagaggag gagagaagca tccctgagaa ggggctggag 2880
ccagaacact cattcagtgg aagtgacagt gaagattcag agctgatgta tcagccaaag 2940
gctccagaaa tcaacatttc tgtgaattag 2970
<210> 24
<211> 1835
<212> DNA
<213> Homo Sapiens
49
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
<220>
<221> misc_feature
<223> Incyte ID No: 7483601CB1
<400> 24
atggatcatg ctgaagaaaa tgaaatcctt gcagcaaccc agaggtacta tgtggaaagg 60
cctatcttta gtcatccggt cctccaggaa agactacaca caaaggacaa ggttcctgat 120
tccattgcgg ataagctgaa acaggcattc acatgtactc ctaaaaaaat aagaaatatc 180
atttatatgt tcctacccat aactaaatgg ctgccagcat acaaattcaa ggaatatgtg 240
ttgggtgact tggtctcagg cataagcaca ggggtgcttc agcttcctca aggcttagcc 300
tttgcaatgc tggcagctgt gcctccaata tttggcctgt acccttcatt ttaccctgtt 360
atcatgtatt gttttcttgg aacctccaga cacatatcca taggtccttt tgctgttatt 420
agcctgatga ttggtggtgt agctgttcga ttagtaccag atgatatagt cattccagga 480
ggagtaaatg caaccaatgg cacagaggcc agagatgcct tgagagtgaa agtcgccatg 540
tctgtgacct tactttcagg aatcattcag ttttgcctag gtgtctgtag gtttggattt 600
gtggccatat atctcacaga gcctctggtc cgtgggttta ccaccgcagc agctgtgcat 660
gtcttcacct ccatgttaaa atatctgttt ggagttaaaa caaagcggta cagtggaatc 720
ttttccgtgg tgtatagtac agttgctgtg ttgcagaatg ttaaaaacct caacgtgtgt 780
tccctaggcg tcgggctgat ggtttttggt ttgctgttgg gtggcaagga gtttaatgag 840
agatttaaag agaaattgcc ggcgcctatt cctttagagt tctttgcggt cgtaatggga 900
actggcattt cagctgggtt taacttgaaa gaatcataca atgtggatgt cgttggaaca 960
cttcctctag ggctgctacc tccagccaat ccggacacca gcctcttcca ccttgtgtac 1020
gtagatgcca ttgccatagc catcgttgga ttttcagtga ccatctccat ggccaagacc 1080
ttagcaaata aacatggcta ccaggttgac ggcaatcagg agctcattgc cctgggactg 1140
tgcaattcca ttggctcact cttccagacc ttttcaattt catgctcctt gtctcgaagc 1200
cttgttcagg agggaaccgg tgggaagaca cagcttgcag gttgtttggc ctcattaatg 1260
attctgctgg tcatattagc aactggattc ctctttgaat cattgcccca ggctgtgctg 1320
tcggccattg tgattgtcaa cctgaaggga atgtttatgc agttctcaga tctccccttt 1380
ttctggagaa ccagcaaaat agagctgacc atctggctta ccacttttgt gtcctccttg 1440
ttcctgggat tggactatgg tttgatcact gctgtgatca ttgctctgct gactgtgatt 1500
tacagaacac agaggtgaaa gaaattcctg gaataaaaat atttcaaata aatgccccaa 1560
tttactatgc aaatagggac tgtatagcca agcttaaaag aaagactggg gtgaacccag 1620
cagtcatcat ggggacaggg gaaaggcgtg gggaatacgc taagggagtc ggaatggaaa 1680
tgggcacggc atgtggtaag cgatgcggag tatgggggtt aacaagcgaa aaggggtgga 1740
gaaaattccc aatgtaaaag attttggaag gagaatgacc cggaagacac aagttgggtt 1800
tacaataggt tggggagacg gcggaaagag ggtta 1835
<210> 25
<211> 2220
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7487851CB1
<400> 25
caaggcagca tgagccgatc acccctcaat cccagccaac tccgatcagt gggctcccag 60
gatgccctgg cccccttgcc tccacctgct ccccagaatc cctccaccca ctcttgggac 120
cctttgtgtg gatctctgcc ttggggcctc agctgtcttc tggctctgca gcatgtcttg 180
gtcatggctt ctctgctctg tgtctcccac ctgctcctgc tttgcagtct ctccccagga 240
ggactctctt actccccttc tcagctcctg gcctccagct tcttttcacg tggtatgtct 300
accatcctgc aaacttggat gggcagcagg ctgcctcttg tccaggctcc atccttagag 360
ttccttatcc ctgctctggt gctgaccagc cagaagctac cccgggccat ccagacacct 420
ggaaactgtg agcacagagc aagggcaagg gcctccctca tgctgcacct ttgtagggga 480
cctagctgcc atggcctggg gcactggaac acttctctcc aggaggtgtc cggggcagtg 540
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
gtagtatctg ggctgctgca gggcatgatg gggctgctgg ggagtcccgg ccacgtgttc 600
ccccactgtg ggcccctggt gctggctccc agcctggttg tggcagggct ctctgcccac 660
agggaggtag cccagttctg cttcacacac tgggggttgg ccttgctggt tatcctgctc 720
atggtggtct gttctcagca cctgggctcc tgccagtttc atgtgtgccc ctggaggcga 780
gcttcaacgt catcaactca cactcctctc cctgtcttcc ggctcctttc ggtgctgatc 840
ccagtggcct gtgtgtggat tgtttctgcc tttgtgggat tcagtgttat cccccaggaa 900
ctgtctgccc ccaccaaggc accatggatt tggctgcctc acccaggtga gtggaattgg 960
cctttgctga cgcccagagc tctggctgca ggcatctcca tggccttggc agcctccacc 1020
agttccctgg gctgctatgc cctgtgtggc cggctgctgc atttgcctcc cccacctcca 1080
catgcctgca gtcgagggct gagcctggag gggctgggca gtgtgctggc cgggctgctg 1140
ggaagcccca tgggcactgc atccagcttc cccaacgtgg gcaaagtggg tcttatccag 1200
gctggatctc agcaagtggc tcacttagtg gggctactct gcgtggggct tggactctcc 1260
cccaggttgg ctcagctcct caccaccatc ccactgcctg ttgttggtgg ggtgctgggg 1320
gtgacccagg ctgtggtttt gtctgctgga ttctccagct tctacctggc tgacatagac 1380
tctgggcgaa atatcttcat tgtgggcttc tccatcttca tggccttgct gctgccaaga 1440
tggtttcggg aagccccagt cctgttcagc acaggctgga gccccttgga tgtattactg 1500
cactcactgc tgacacagcc catcttcctg gctggactct caggcttcct actagagaac 1560
acgattcctg gcacacagct tgagcgaggc ctaggtcaag ggctaccatc tcctttcact 1620
gcccaagagg ctcgaatgcc tcagaagccc agggagaagg ctgctcaagt gtacagactt 1680
cctttcccca tccaaaacct ctgtccctgc atcccccagc ctctccactg cctctgccca 1740
ctgcctgaag accctgggga tgaggaagga ggctcctctg agccagaaga gatggcagac 1800
ttgctgcctg gctcagggga gccatgccct gaatctagca gagaagggtt taggtcccag 1860
aaatgaccag aacgcctact tctgccttgg ttaatttagc cctaactctc atctgctgga 1920
gagtcagctc ccaaactgtt ctttcttgta ggcagaggat atgtgtgtgt gtattacatg 1980
ggactgtcta gaggttccat ttcccaatag ggtgggttgc ctttccttgt cttaattagg 2040
cctaactgtt ccagagcaga ggccatgatt tagtggacca tgaatgattg agattttgcc 2100
tgtgtactat caatgccact tgaacccaag cattcacttt aatacttact gagcatctcc 2160
catgtgcaag gtcctggaac tacagggata agacagggtc catgccgtct caaggcattt 2220
<210> 26
<211> 1517
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472881CB1
<400> 26
taagaacaga agtggaaagc cttacttacc acagtttatt atatgtttca tgcccgtgat 60
aattactttt ataatgccac ttgtgaaaaa attgatcaga ttaggatgaa tcaccttgct 120
ggccaacagt tattggaatg attctccatg tgtgacttcg ttgcactatt acaaaatgtg 180
gcaggataga cctgcccagc cattgttgcc gatgttcatt tgtaatgctg ccttaaggag 240
atgaggagat gagagccaat tgttccagca gctcagcctg ccctgccaac agttcagagg 300
aggagctgcc agtgggactg gaggcgcatg gaaacctgga gctcgttttc acagtggtgc 360
ccactgtgat gatggggctg ctcatgttct ctttgggatg ttccgtggag atccggaagc 420
tgtggtcgca catcaggaga ccctggggca ttgctgtggg actgctctgc cagtttgggc 480
tcatgccttt tacagcttat ctcctggcca ttagcttttc tctgaagcca gtccaagcta 540
ttgctgttct catcatgggc tgctgcccgg ggggcaccat ctctaacatt ttcaccttct 600
gggttgatgg agatatggat ctcagcatca gtatgacaac ctgttccacc gtggccgccc 660
tgggaatgat gccactctgc atttatctct acacctggtc ctggagtctt cagcagaatc 720
tcaccattcc ttatcagaac ataggaatta cccttgtgtg cctgaccatt cctgtggcct 780
ttggtgtcta tgtgaattac agatggccaa aacaatccaa aatcattctc aagattgggg 840
ccgttgttgg tggggtcctc cttctggtgg tcgcagttgc tggtgtggtc ctggcgaaag 900
gatcttggaa ttcagacatc acccttctga ccatcagttt catctttcct ttgattggcc 960
atgtcacggg ttttctgctg gcacttttta cccaccagtc ttggcaaagg tgcaggacaa 1020
51
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
tttccttaga aactggagct cagaatattc agatgtgcat caccatgctc cagttatctt 1080
tcactgctga gcacttggtc cagatgttga gtttcccact ggcctatgga ctcttccagc 1140
tgatagatgg atttcttatt gttgcagcat atcagacgta caagaggaga ttgaagaaca 1200
aacatggaaa aaagaactca ggttgcacag aagtctgcca tacgaggaaa tcgacttctt 1260
ccagagagac caatgccttc ttggaggtga atgaagaagg tgccatcact cctgggccac 1320
cagggccaat ggattgccac agggctctcg agccagttgg ccacatcact tcatgtgaat 1380
agcagggact agctggctgg actggccccc ttctttttca gtggccagta aagacagtgt 1440
gcagctgaca catgaatctt gttggtaggg ccagtgtgaa tatttaagtg ttcaatgtta 1500
gaatatttat attttca 1517
<210> 27
<211> 2142
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7612560CB1
<400> 27
ggtgtacatc tacactagac accttcctgc ttccctcctt ccagagcaga cctctttgtc 60
accccgagct ccttgtttct taagcagtca tgtctgtgac aaaaagtact gagggtcccc 120
agggagccgt tgccatcaaa ttggacctta tgtcgcctcc tgaaagtgcc aagaagttgg 180
agaacaagga ctctacattc ttggatgaaa gtccttcaga gtcagcaggc ttgaagaaga 240
ccaagggcat aacagtgttc caggccttga ttcacctggt gaaaggcaac atgggcacag 300
ggatcctggg actacccctc gctgtgaaga acgcgggcat cctgatgggc ccactcagtc 360
tgctggtgat gggcttcatt gcctgccact gtatgcacat cctggtcaag tgtgcccagc 420
gcttctgtaa gaggcttaac aagcccttta tggactatgg ggacacggtg atgcatggac 480
tagaagccaa ccccaacgcc tggctccaga atcacgctca ctggggaagg catatcgtga 540
gcttcttcct tattatcacc caacttggct tctgctgtgt gtacattgtg tttttggctg 600
ataatttaaa acaggtagtg gaagctgtta atagcacaac caacaactgc tattccaatg 660
agacggtgat tctgaccccc accatggact cgcgactcta catgctctcc ttcctgccct 720
tcctggtgct gctggtcctc atccggaacc tcaggatctt gaccatcttc tccatgctgg 780
ccaacatcag catgctggtc agcttggtca tcatcataca gtacattacc caggaaatcc 840
cagaccccag ccggttgcca ctggtagcaa gctggaagac ctaccctctc ttcttcggaa 900
cagccatttt ttcttttgaa agcattggtg tggttctgcc tctggaaaac aagatgaaga 960
atgcccgcca cttcccagcc atcctgtctt tgggaatgtc catcgtcact tccctataca 1020
ttggcatggc ggctctgggc tacctgcggt ttggagatga catcaaggcc agcataagcc 1080
ttaacctgcc taactgctgg ctgtaccagt ctgtcaagct tctctacatt gccggcatcc 1140
tgtgcaccta tgccctgcag ttctacgtcc ctgcagaaat catcatcccc tttgccatct 1200
cccgggtgtc aacacgctgg gcactgcctc tggatctgtc cattcgcctc gtcatggtct 1260
gcctgacatg cctcctggcc atcctcatcc cccgcctgga cctggtcatc tccctggtgg 1320
gctccgtgag tggcaccgcc ctggccctca tcatcccacc gctcctggag gtcaccacgt 1380
tctactcaga gggcatgagc cccctcacca tcttcaagga cgtcctgatc agcatcctgg 1440
gcttcgtggg ctttgtggtg gggacctacc aggccctgga cgagctgctc aagtcagaag 1500
actctcaccc cttttccaac tccaccactt ttgttcgcgt ggagctatgc aagaagcagc 1560
caccagaggg ccccaagtgg cagcaactgg ccaaaggaga tgcagccagc taagactgtc 1620
cacactttgg cagacaaccg gttttccctt ttctgggtct gttcaaaaag caaacattaa 1680
gggtgggcac ataatccaca agccagaaag ttgtgcacgg ctccagtgtt gagatgggta 1740
gggccaagat gaccagtgtg aaaactctca gatagaaagg agccatgcat attaaatgag 1800
gggcaacaaa catttcaaac gattagataa cattttctcc caactcaaag atcccaacaa 1860
tgaataggag gcatggaagt agatgtgcca atggggaggg atgaggagtg aacatgaata 1920
ttatttgaat agactttacc tcttaattct tgcaacatgc attcttgatt acctactgtg 1980
tgccaaacaa gattttgtag aatattgcaa aaatgaccat aaattcctcg tgataatgtg 2040
actttgcacc tgctcctatg aaaagatgaa gtctgtatct gtatccctta aatttttttg 2100
cttgtttgtc ggttttgttt tgtgttttgt ttttttgaga tg 2142
52
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
<210> 28
<211> 1661
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2880370C81
<400> 28
gacactaagc tttaaattca agtaaatagg aggctttttt tttttcgcat aagcagaaat 60
gaggaaatca agaggaagag attagatttc tgttgtgata aatcgaatct gttaaatgcc 120
atgacttttt aattgtctta atcacaagtt aaaccggttg tgttgctgct tagatggcta 180
tatatttgtt taaaagtaca gcagtccctc ctactggact ttgatcctac aaaaacaact 240
gttatctaac tcaccctcag actgtcactg gaacacctgc atgaagaatg ttctttcatt 300
ttttaaaaac gattttgcat atatgattta tttcagcttt caaaatgatt agaaaacttt 360
ttattgttct acttttgttg cttgtgacta tagaagaagc aaggatgtca tcgctcagtt 420
ttctgaatat agagaagact gaaatactat ttttcacaaa gactgaagaa accatccttg 480
taagttcaag ctacgaaaat aaacggccta attccagcca cctctttgtg aaaatagaag 540
atcctaaaat actacaaatg gtgaatgtgg ccaagaagat ctcatcagat gctacaaact 600
ttaccataaa tctggtgact gatgaagaag gagaaacaaa tgtgactatt caactctggg 660
attctgaagg taggcaagaa agactcattg aagaaatcaa gaatgtgaaa gtcaaagtgc 720
tcaaacaaaa agacagtcta ctccaggcac caatgcatat tgatagaaat atcctaatgc 780
ttattttacc actaatacta ttgaataagt gtgcatttgg ttgtaagatt gaattacagc 840
tgtttcaaac agtatggaag agacctttgc cagtaattct tggggcagtt acacagtttt 900
ttctgatgcc attttgcggg tttcttttgt ctcagattgt ggcattgcct gaggcgcaag 960
cttttggagt tgtaatgacc tgcacgtgcc caggaggggg tgggggctat ctctttgctc 1020
tgcttctaga tggagatttc acattggcca ttttgatgac ttgcacatca acattattgg 1080
ctctgatcat gatgcctgtc aattcttata tatacagtag gatattaggg ttgtcaggta 1140
cattccatat tcctgtttct aaaattgtgt caacactcct tttcatactt gtgccagtat 1200
caattggaat agtcatcaag catagaatac ctgaaaaagc aagcttctta gagagaataa 1260
ttagacctct gagttttatt ttaatgttcg taggaattta tttgactttc acagtgggat 1320
tagtgttctt aaaaacagat aatctagagg tgattctgtt gggtctctta gttcctgctt 1380
tgggtttgct gtttgggtac tcctttgcta aagtttgtac gctgcctctt cctgtttgta 1440
aaactgttgc tattgaaagt gggatgttaa atagtttctt agctcttgcc gttattcagc 1500
tgtcttttcc acagtccaag gccaatttag cttctgtggc tccttttaca gtagccatgt 1560
gttctggatg tgaaatgtta ctgatcattc tagtttacaa ggctaagaaa agatgtatct 1620
ttttcttaca agataaaagg aaaagaaatt tcctaatcta a 1661
<210> 29
<211> 1501
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6267489CB1
<400> 29
ccagaggaaa ctagtcacaa aaaccctgac tatcacctga tagattgctt gtgctgcctg 60
ataattactc gcacttttcc caggctagtg caaatcttca ggggccgtcc aggactacag 120
agctgtttca ccctaccttg gcttcaatct cttcccccat gctcgaaggt gcggagctgt 180
acttcaacgt ggaccatggc tacctggagg gcctggttcg aggatgcaag gccagcctcc 240
tgacccagca agactatatc aacctggtcc agtgtgagac cctagaagac ctgaaaattc 300
atctccagac tactgattat ggtaactttt tggctaatca cacaaatcct cttactgttt 360
ccaaaattga cactgagatg aggaaaagac tatgtggaga atttgagtat ttccggaatc 420
53
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
attccctgga gcccctcagc acatttctca cctatatgac gtgcagttat atgatagaca 480
atgtgattct gctgatgaat ggtgcattgc agaaaaaatc tgtgaaagaa attctgggga 540
agtgccaccc cttgggccgt ttcacagaaa tggaagctgt caacattgca gagacacctt 600
cagatctctt taatgccatt ctgatcgaaa cgccattagc tccattcttc caagactgca 660
tgtctgaaaa tgctctagat gaactgaata ttgaattgct acgcaataaa ctatacaagt 720
cttaccttga ggcattctat aaattctgta agaatcatgg tgatgtcaca gcagaagtta 780
tgtgtcccat tcttgagttt gaggccgaca gacgtgcttt tatcatcact cttaactcct 840
ttggcactga attgagcaaa gaagaccgag agaccctcta tccaaccttc ggcaaactct 900
atcctgaggg gttgcggctg ttggctcaag cagaagactt tgaccagatg aagaacgtag 960
cggatcatta cggagtatac aaacctttat ttgaagctgt aggtggcagt gggggaaaga 1020
cattggagga cgtgttttac gagcgtgagg tacaaatgaa tgtgctggca ttcaacagac 1080
agttccacta cggtgtgttt tatgcatatg taaagctgaa ggaacaggaa attagaaata 1140
ttgtgtggat agcagaatgt atttcacaga ggcatcgaac taaaatcaac agttacattc 1200
caattttata acccaagtaa ggttctcaaa tgtagaaaat tataaatgtt aaaaggaagt 1260
tattgaagaa aataaaagaa attatgttat attatctaga ctacacaaaa gtaagccaca 1320
ctatatcttc atgagttgca aatccatgga aacacagtaa accagccctg aaacaaagca 1380
tttccttgtt ttcagtggta ttagatcttg tttccacatg tctgtctcat tcttcactgg 1440
gccttacagg ttagttttaa ttaactctat ggtatttttc tattcttgtc tgatcatgtt 1500
a 1501
<210> 30
<211> 5526
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7484777CB1
<400> 30
caggctgttt tgtgcaggct gtccctcttc ttcaaaatcg tgcatcccct ccccgaagca 60
gcaggcagtg tgcctccatt cagccacatt tggtatgcat gagcacggct gcagagagag 120
gggaggtggc tgttttaaga aggttcaggg gctcaggcaa ggctacttga ctagtcttcc 180
aagttccagg aagcctctgc cctaatggaa tttgcaggtg tggagatgac catgggatgc 240
cagagccgtg ggggaccgtt tattttctag gcattgctca ggttttcagt ttcttgtttt 300
cctggtggaa tttggaaggg gtcatgaatc aggctgatgc tcctcgaccc ctaaactgga 360
ccatccggaa gctgtgccac gcagcctttc ttccatctgt cagacttctg aaggctcaga 420
aatcctggat agaaagagca ttttataaaa gagaatgtgt ccacatcata cccagcacca 480
aagaccccca taggtgttgc tgtgggcgtc tgataggcca gcatgttggc ctcaccccca 540
gtatctccgt gcttcagaat gagaaaaatg aaagtcgcct ctcccgaaat gacatccagt 600
ctgaaaagtg gtccatcagc aaacacactc aactcagccc tacggatgct tttgggacca 660
ttgagttcca aggaggtggc cattccaaca aagccatgta tgtgcgagta tcttttgata 720
caaaacctga tctcctctta cacctgatga ccaaggaatg gcagttggag cttcccaagc 780
ttctcatctc tgtccatggg ggcctgcaga actttgaact ccagccaaaa ctcaagcaag 840
tctttgggaa agggctcatc aaagcagcta tgacaactgg agcgtggata ttcactggag 900
gggttaacac aggtgttatt cgtcatgttg gcgatgcctt gaaggatcat gcctctaagt 960
ctcgaggaaa gatatgcacc ataggtattg ccccctgggg aattgtggaa aaccaggagg 1020
acctcattgg aagagatgtt gtccggccat accagaccat gtccaatccc atgagcaagc 1080
tcactgttct caacagcatg cattcccact tcattctggc tgacaacggg accactggaa 1140
aatatggagc agaggtgaaa cttcgaagac aactggaaaa gcatatttca ctccagaaga 1200
taaacacaag aatcggtcaa ggtgttcctg tggtggcact catagtggaa ggaggaccca 1260
atgtgatctc gattgttttg gagtaccttc gagacacccc tcccgtgcca gtggttgtct 1320
gtgatgggag tggacgggca tcggacatcc tggcctttgg gcataaatac tcagaagaag 1380
gcggactgat aaatgaatct ttgagggacc agctgttggt gactatacag aagactttca 1440
catacactcg aacccaagct cagcatctgt tcatcatcct catggagtgc atgaagaaga 1500
aggaattgat tacggtattt cggatgggat cagaaggaca ccaggacatt gatttggcta 1560
54
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
tcctgacagc tttactcaaa ggagccaatg cctcggcccc agaccaactg agcttagctt 1620
tagcctggaa cagagtcgac atcgctcgca gccagatctt tatttacggg caacagtggc 1680
cggtgggatc tctggagcaa gccatgttgg atgccttagt tctggacaga gtggattttg 1740
tgaaattact catagagaat ggagtaagca tgcaccgttt tctcaccatc tccagactag 1800
aggaattgta caatacgaga catgggccct caaatacatt gtaccacttg gtcagggatg 1860
tcaaaaaggg gaacctgccc ccagactaca gaatcagcct gattgacatc ggcctggtga 1920
tcgagtacct gatgggcggg gcttatcgct gcaactacac gcgcaagcgc ttccggaccc 1980
tctaccacaa cctcttcggc cccaagaggc ccaaagcctt gaaactgctg ggaatggagg 2040
atgatattcc cttgaggcga ggaagaaaga caaccaagaa acgtgaagaa gaggtggaca 2100
ttgacttgga tgatcctgag atcaaccact tccccttccc tttccatgag ctcatggtgt 2160
gggctgttct catgaagcgg cagaagatgg ccctgttctt ctggcagcac ggtgaggagg 2220
ccatggccaa ggccctggtg gcctgcaagc tctgcaaagc catggctcat gaggcctctg 2280
agaacgacat ggttgacgac atttcccagg agctgaatca caattccaga gactttggcc 2340
agctggctgt ggagctcctg gaccagtcct acaagcagga cgaacagctg gccatgaaac 2400
tgctgacgta tgagctgaag aactggagca acgccacgtg cctgcagctt gccgtggctg 2460
ccaaacaccg cgacttcatc gcgcacacgt gcagccagat gctgctcacc gacatgtgga 2520
tgggccggct ccgcatgcgc aagaactcag gcctcaaggt aattctggga attctacttc 2580
ctccttcaat tctcagcttg gagttcaaga acaaagacga catgccctat atgtctcagg 2640
cccaggaaat ccacctccaa gagaaggagg cagaagaacc agagaagccc acaaaggaaa 2700
aagaggaaga ggacatggag ctcacagcaa tgttgggacg aaacaacggg gagtcctcca 2760
ggaagaagga tgaagaggaa gttcagagca agcaccggtt aatccccctc ggcagaaaaa 2820
tctatgaatt ctacaatgca cccatcgtga agttctggtt ctacacactg gcgtatatcg 2880
gatacctgat gctcttcaac tatatcgtgt tagtgaagat ggaacgctgg ccgtccaccc 2940
aggaatggat cgtaatctcc tatattttca ccctgggaat agaaaagatg agagagattc 3000
tgatgtcaga gccagggaag ttgctacaga aagtgaaggt atggctgcag gagtactgga 3060
atgtcacgga cctcatcgcc atccttctgt tttctgtcgg aatgatcctt cgtctccaag 3120
accagccctt caggagtgac gggagggtca tctactgcgt gaacatcatt tactggtata 3180
tccgtctcct agacatcttc ggcgtgaaca agtatttggg cccgtatgta atgatgattg 3240
gaaaaatgat gatagacatg atgtactttg tcatcattat gctggtggtt ctgatgagct 3300
ttggggtcgc caggcaagcc atcctttttc ccaatgagga gccatcatgg aaactggcca 3360
agaacatctt ctacatgccc tattggatga tttatgggga agtgtttgcg gaccagatag 3420
accctccctg tggacagaat gagacccgag aggatggtaa aataatccag ctgcctccct 3480
gcaagacagg agcttggatc gtgccggcca tcatggcctg ctacctctta gtggcaaaca 3540
tcttgctggt caacctcctc attgctgtct ttaacaatac attttttgaa gtaaaatcga 3600
tatccaacca agtctggaag tttcagaggt atcagctcat catgactttc catgaaaggc 3660
cagttctgcc cccaccactg atcatcttca gccacatgac catgatattc cagcacctgt 3720
gctgccgatg gaggaaacac gagagcgacc cggatgaaag ggactacggc ctgaaactct 3780
tcataaccga tgatgagctc aagaaagtac atgactttga agagcaatgc atagaagaat 3840
acttcagaga aaaggatgat cggttcaact catctaatga tgagaggata cgggtgactt 3900
cagaaagggt ggagaacatg tctatgcggc tggaggaagt caacgagaga gagcactcca 3960
tgaaggcttc actccagacc gtggacatcc ggctggcgca gctggaagac cttatcgggc 4020
gcatggccac ggccctggag cgcctgacag gtctggagcg ggccgagtcc aacaaaatcc 4080
gctcgaggac ctcgtcagac tgcacggacg ccgcctacat tgtccgtcag agcagcttca 4140
acagccagga agggaacacc ttcaagctcc aagagagtat agaccctgca ggtgaggaga 4200
ccatgtcccc aacttctcca accttaatgc cccgtatgcg aagccattct ttctattcag 4260
tcaatatgaa agacaaaggt ggtatagaaa agttggaaag tatttttaaa gaaaggtccc 4320
tgagcctaca ccgggctact agttcccact ctgtagcaaa agaacccaaa gctcctgcag 4380
cccctgccaa caccttggcc attgttcctg attccagaag accatcatcg tgtatagaca 4440
tctatgtctc tgctatggat gagctccact gtgatataga ccctctggac aattccgtga 4500
acatccttgg gctaggcgag ccaagctttt caactccagt accttccaca gccccttcaa 4560
gtagtgccta tgcaacactt gcacccacag acagacctcc aagccggagc attgattttg 4620
aggacatcac ctccatggac actagatctt tttcttcaga ctacacccac ctcccagaat 4680
gccaaaaccc ctgggactca gagcctccga tgtaccacac cattgagcgt tccaaaagta 4740
gccgctacct agccaccaca ccctttcttc tagaagaggc tcccattgtg aaatctcata 4800
gctttatgtt ttccccctca aggagctatt atgccaactt tggggtgcct gtaaaaacag 4860
cagaatacac aagtattaca gactgtattg acacaaggtg tgtcaatgcc cctcaagcaa 4920
55.
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
ttgcggacag agctgccttc cctggaggtc ttggagacaa agtggaggac ttaacttgct 4980
gccatccaga gcgagaagca gaactgagtc accccagctc tgacagtgag gagaatgagg 5040
ccaaaggccg cagagccacc attgcaatat cctcccagga gggtgataac tcagagagaa 5100
ccctgtccaa caacatcact gttcccaaga tagagcgcgc caacagctac tcggcagagg 5160
agccaagtgc gccatatgca cacaccagga agagcttctc catcagtgac aaactcgaca 5220
ggcagcggaa cacagcaagc ctgcgaaatc ccttccagag aagcaagtcc tccaagccgg 5280
agggccgagg ggacagcctg tccatgagga aactgtccag aacatcggct ttccaaagct 5340
ttgaaagcaa gcacacctaa accttcttaa tatccgccac agaaggctca agaatccagc 5400
cctaaaattc tctccaactc cagtttttcc cctttccttg aatcatacct gctttattct 5460
tagctgagca aaacaagcaa tgctttggga ggtgttaact caaaggtgac ttctgggcca 5520
cagatc 5526
<210> 31
<211> 2739
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2493969CB1
<400> 31
gcgcagtaag tgcggactgc cagccaccag ccttggcagc cagctcgtcg cctccagccc 60
cgaccccgac attcatgccc aggagaaggc tgcactgggt ccctctgggc ctttcctaaa 120
agggagatcc ctgttcacta gatgagttcc agaaccatcc actaaggctt tgtagccccc 180
ttccatcagc tgaccttcac tgcatcccct atcgctcaag atgagtggct tcttcacctc 240
gctggacccc cggcgggtgc agtggggagc tgcctggtat gcaatgcact ccaggatcct 300
acgcaccaaa ccagtggagt ccatgctaga gggaactggg accaccacgg cacatggaac 360
taagctagcc caggtactca ccacagtgga cctcatctct cttggcgttg gcagctgtgt 420
gggcactggc atgtatgtgg tctctggcct ggtggccaag gaaatggcag gacctggtgt 480
cattgtgtcc ttcatcattg cagccgtcgc atccatatta tcaggcgtct gctatgcaga 540
gtttggagtt cgagtcccca agaccacagg atctgcctac acctacagct atgtcactgt 600
tggggaattt gtggcatttt tcattggctg gaacctgatc ctggagtacc tgattggcac 660
tgcggccgga gccagtgctc tgagcagcat gtttgactca ctagccaacc acaccatcag 720
ccgctggatg gcggacagcg tgggaaccct caatggcctg gggaaaggtg aagaatcata 780
cccagacctt ctggctctgt tgatcgcggt catcgtgacc atcattgttg ctctgggggt 840
gaagaattcc ataggcttca acaatgttct caatgtgctg aacctggcag tatgggtgtt 900
catcatgatc gcaggcctct tcttcatcaa tgggaaatac tgggcggagg gccagttctt 960
gccccacggc tggtcagggg tgctgcaagg agcagcaaca tgcttctacg ctttcattgg 1020
ctttgacatc atcgccacca ctggagagga agccaagaat cccaacacgt ccatccctta 1080
tgctatcact gcctccctgg tcatctgcct gacagcatat gtgtctgtga gcgtgatctt 1140
aactctgatg gtgccatatt ataccattga cacggaatcc ccactcatgg agatgtttgt 1200
ggctcatggg ttctatgctg ccaaattcgt agtggccatt gggtcggttg caggactgac 1260
agtcagcttg ctggggtccc tcttcccgat gccgagggtc atttatgcca tggctggtga 1320
cgggctcctt ttcaggttcc tggctcacgt cagctcctac acagagacac cagtggtggc 1380
ctgcatcgtg tcggggttcc tggcagcgct cctcgcactg ttggtcagct tgagagacct 1440
gatagagatg atgtctatcg gcacgctcct ggcctacacc ttggtctctg tctgtgtctt 1500
gctccttcga taccaacctg agagtgacat tgatggtttt gtcaagttct tgtctgagga 1560
gcacaccaag aagaaggagg gcattctggc tgactgtgag aaggaagctt gttctcctgt 1620
gagtgagggg gatgagtttt ctggcccagc caccaacaca tgtggggcca agaacttacc 1680
atccttggga gacaatgaga tgctcatagg gaaatcagac aagtcaacct acaacgtcaa 1740
ccaccccaat tacggcaccg tggacatgac cacaggcata gaagctgatg aatccgaaaa 1800
tatttatctc atcaagttaa agaagctgat tgggcctcat tattacacca tgagaatccg 1860
gctgggcctt ccaggcaaaa tggaccggcc cacagcagcg acggggcaca cggtgaccat 1920
ctgcgtgctc ctgctcttca tcctcatgtt catcttctgc tccttcatca tctttggttc 1980
tgactacatc tcagagcaga gctggtgggc catccttctg gttgttctga tggtgctgct 2040
56
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
gatcagcacc ctggtgtttg tgatcctgca gcagccagag aaccccaaga agctgcccta 2100
catggcccct tgcctcccct ttgtgcctgc ctttgccatg ctggtgaaca tctatctcat 2160
gctaaagctc tccaccatca catggatccg gtttgcggtc tggtgctttg tgggtctgct 2220
catttatttt ggatatggca tctggaacag caccctggaa atcagcgctc gagaagaggc 2280
cctgcaccaa agcacgtacc aacgctacga cgtggatgac cccttctcag tggaggaggg 2340
tttctcctac gccacagagg gcgagagcca ggaggactgg ggcgggccca ctgaagacaa 2400
aggcttctat taccaacaga tgtcagatgc gaaggcaaac ggccggacaa gtagcaaagc 2460
gaagagcaaa agcaaacaca aacagaactc agaggccctg attgcaaatg atgagttaga 2520
ttactctcca gagtaggaga aacacacaag tgggtagaaa tggtgatgac tgattttcag 2580
taacttaacc tgtgggctag aaggtgaaaa cttttttggc tctcatttca caaatccagc 2640
cttccccaaa ttcaatccct agtcatagcc tgtcatttgc tacttttgct cttcaggata 2700
gttctgttga agggcttaac ctgggtcccc taactggtc 2739
<210> 32
<211> 4321
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3244593CB1
<400> 32
atggtgggtg aaggacccta ccttatctca gatctggacc agcgaggccg gcggagatcc 60
tttgcagaaa gatatgaccc cagcctgaag accatgatcc cagtgcgacc ctgtgcaagg 120
ttagcaccca acccggtgga tgatgccggg ctactctcct tcgccacatt ttcctggctc 180
acgccggtga tggtgaaagg ctaccggcaa aggctgaccg tagacaccct gcccccattg 240
tcgacatatg actcatctga caccaatgcc aaaagatttc gagtcctttg ggatgaagag 300
gtagcaaggg tgggtcctga gaaggcctct ctgagccacg tggtgtggaa attccagagg 360
acacgcgtgt tgatggacat cgtggccaac atcctgtgca tcatcatggc agccataggg 420
ccgacagttc tcattcacca aatcctccag cagactgaga ggacctctgg gaaagtctgg 480
gttggcattg gactgtgcat agcccttttt gccaccgagt ttaccaaagt cttcttttgg 540
gcccttgcct gggccatcaa ctaccgcacg gccatccggt tgaaggtggc gctctccacc 600
ttggtttttg aaaacctagt gtccttcaag acattgaccc acatctctgt tggcgaggtg 660
ctcaatatac tgtcaagtga tagctattct ttgtttgaag ctgccttgtt ttgtcctttg 720
ccagccacca tcccgatcct aatggtcttt tgtgcggcgt acgccttttt cattctgggg 780
cccacagctc tcatcgggat atcagtgtat gtcatattca tacccgtcca gatgtttatg 840
gccaagctca attcagcttt ccgaaggtca gcaattttgg tgacagacaa gcgagttcag 900
acaatgaatg agtttctgac ctgcatcagg ctgatcaaaa tgtatgcctg ggagaaatct 960
tttaccaaca ctatccaaga tataagaagg agggaaagaa aattactgga aaaagctgga 1020
tttgtccaaa gtggaaactc tgccctggcc cccatcgtgt ccaccatagc catcgtgctg 1080
acattatcct gccacatcct cctgagacgc aaactcaccg cacccgtggc atttagtgtg 1140
attgccatgt ttaatgtaat gaagttttcc attgcaatct tgcccttctc catcaaagca 1200
atggctgaag cgaatgtctc tctaaggaga atgaagaaaa ttctcataga taaaagcccc 1260
ccatcttaca tcacccaacc agaagaccca gatactgtct tgcttttagc aaatgccacc 1320
ttgacatggg agcatgaagc cagcaggaaa agtaccccaa agaaattgca gaaccagaaa 1380
aggcatttat gcaagaaaca gaggtcagag gcatacagtg agaggagtcc accagccaag 1440
ggagccactg gcccagagga gcaaagtgac agcctcaaat cggttctgca cagcataagc 1500
tttgtggtga gaaaggggaa gatcttggga atatgtggga atgtgggaag tggaaagagc 1560
tccctccttg cagctctcct aggacagatg cagctgcaga aaggggtggt ggcagtcaat 1620
ggaactttgg cctacgtttc acagcaggca tggatctttc atggaaatgt gagagaaaac 1680
atactctttg gagaaaagta tgatcaccaa aggtatcagc acacagtccg cgtctgtggc 1740
ctccagaagg acctgagcaa cctcccctat ggagacctga ctgagattgg ggagcggggc 1800
ctcaacctct ctggggggca gaggcagagg attagcctgg cccgcgctgt ctactccgac 1860
cgtcagctct acctgctgga cgaccccctg tcggccgtgg acgcccacgt ggggaagcac 1920
gtctttgagg agtgcattaa gaagacgctc aggggaaaga cagtcgtcct ggtgacccac 1980
57
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
cagctacagt tcttagagtc ttgtgatgaa gttattttat tagaagatgg agagatttgt 2040
gaaaagggaa cccacaagga gttaatggag gagagagggc gctatgcaaa actgattcac 2100
aacctgcgag gattgcagtt caaggatcct gaacaccttt acaatgcagc aatggtggaa 2160
gccttcaagg agagccctgc tgagagagag gaagatgctg gtataatcgt tttggctcca 2220
ggaaatgaga aagatgaagg aaaagaatct gaaacaggct cagaatttgt agacacaaaa 2280
gggtacctcc tttctctctt cactgtgttc ctcttcctcc tgatgattgg cagcgctgcc 2340
ttcagcaact ggtggctggg tctctggttg gacaagggct cacggatgac ctgtgggccc 2400
cagggcaaca ggaccatgtg tgaggtcggc gcggtgctgg cagacatcgg tcagcatgtg 2460
taccagtggg tgtacactgc aagcatggtg ttcatgctgg tgtttggcgt caccaaaggc 2520
ttcgtcttca ccaagaccac actgatggca tcctcctctc tgcatgacac ggtgtttgat 2580
aagatcttaa agagcccaat gagtttcttt gacacgactc ccactggcag gctaatgaac 2640
cgtttttcca aggatatgga cgagctggat gtgaggctgc cgtttcacgc agagaacttt 2700
ctgcagcagt tttttatggt ggtgtttatt ctcgtgatct tggctgctgt gtttcctgct 2760
gtccttttag tcgtggccag ccttgctgta ggcttcttca ttctgttacg cattttccac 2820
agaggagtcc aggagctcaa gaaggtggag aatgtcagcc ggtcaccctg gttcacccac 2880
atcacctcct ccatgcaggg cctgggcatc attcacgcct atggcaagaa ggagagctgc 2940
atcacctatc acctcctcta ctttaactgt gctctcaggt ggtttgcgct gagaatggat 3000
gtcctcatga acatccttac cttcactgtg gccttgttgg tgaccctgag tttctcctcc 3060
atcagtactt catccaaagg cctgtcattg tcatacatca tccagctgag cggactgctc 3120
caagtgtgtg tgcgaacggg aacagagacg caagccaaat tcacctccgt ggagctgctc 3180
agggaataca tttcgacctg tgttcctgaa tgcactcatc ccctcaaagt ggggacctgt 3240
cccaaggact ggcccagctg tggggagatc accttcagag actatcagat gagatacaga 3300
gacaacaccc cccttgttct cgacagcctg aacttgaaca tacaaagtgg gcagacagtc 3360
gggattgttg gaagaacagg ttccggaaag tcatcgttag gaatggcttt gtttcgtctg 3420
gtggagccag ccagtggcac aatctttatt gatgaggtgg atatctgcat tctcagcttg 3480
gaagacctca gaaccaagct gactgtgatc ccacaggatc ctgtcctgtt tgtaggtaca 3540
gtaaggtaca acttggatcc ctttgagagt cacaccgatg agatgctctg gcaggttctg 3600
gagagaacat tcatgagaga cacaataatg aaactcccag aaaaattaca ggcagaagtc 3660
acagaaaatg gagaaaactt ctcagtaggg gaacgtcagc tgctttgtgt ggcccgagct 3720
cttctccgta attcaaagat cattctcctt gatgaagcca ccgcctctat ggactccaag 3780
actgacaccc tggttcagaa caccatcaaa gatgccttca agggctgcac tgtgctgacc 3840
atcgcccacc gcctcaacac agttctcaac tgcgatcacg tcctggttat ggaaaatggg 3900
aaggtgattg agtttgacaa gcctgaagtc cttgcagaga agccagattc tgcatttgcg 3960
atgttactag cagcagaagt cagattgtag aggtcctggc ggctgattct agaggaggaa 4020
gaggctctgt gagatgaata ggaggagtct tcaggaggag gggctgtcct ctccgcaggc 4080
agccctggtc ttcagcccct cccatccacg gagtgagctg gggctgaagt tgtccccact 4140
gccatactca gtccatgtca ccccacttgg tgggcttggg gttggttctg ggtggtgaac 4200
cggggcagac ccagctaatg gattaaaaaa ctgcccttca cctcccaaat ccccaagggt 4260
tcctcatgtg ttttcaccaa aaccacccca gtgcctgaga ttgaaaatat tgtaactttc 4320
a 4321
<210> 33
<211> 4519
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4921451CB1
<400> 33
ttcaggaccg ttggcaccgg gctaacggtt ccaccacgtc cgccgccctg gacgcccgcg 60
gcctgccccc ccctgcctct cctgcgccga tacacttcga gtggattctg gccatttgag 120
cattctctcc aactctccaa tccccagtct gcccccacgg gggtctcccc cacctctccc 180
ccgtcccaca gcctaaaccc ctcttcgccc tgaacctccc ttttcctcat gcggtgaatg 240
ggcactggcc ccgctcagac tcccaggagc accagagctg gccctgagcc aagccctgcc 300
58
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
ccaccaggac ctggggacac gggtgactca gacgtgactc aggaaggctc aggtcctgct 360
ggcatccgcg gagccccacc agcatgggca gcctcggcca gagagaagat ctccgagatg 420
aggacaggaa ctcaggtgct gatcctgggc ggagggggcg gtgcagcatt cacctggaag 480
gtccaggcca acaaccgtgc ctacaacggg cagttcaagg agaaggtgat cctgtgctgg 540
caaaggaaga aatacaagac caatgtcatc cgcacggcca agtacaactt ctactcgttc 600
ctgccgctga acctgtacga gcagttccac cgcgtgtcca acctgttctt cctcatcatc 660
atcatcctgc agagcattcc cgacatctcc acgctgccct ggttctcgct cagtacccct 720
atggtctgcc tcctcttcat ccgtgccacc cgggacctgg tggacgacat ggggagacac 780
aagagtgaca gagccatcaa caacagaccc tgccagattc tgatggggaa gagcttcaag 840
cagaagaaat ggcaggatct gtgcgtgggg gatgtggtct gtctccgcaa ggacaacatc 900
gtcccagtga gctggggtgg accccgaggt cccagaacca cgcgccccct caccgagagc 960
acccctccca gggtggggag ggctgccgca cccccaattt gtcttgcatc ccctcttgca 1020
acgctgcccc ccactccaca ccaggccgac atgctcttgc tggccagcac ggagcccagc 1080
agcctgtgct atgtggagac ggtggacatt gacggggaga ccaacttgaa gttcagacag 1140
gccctgatgg tcacccacaa agaactggcc actataaaga agatggcgtc ctttcaaggc 1200
acagtgacgt gtgaggcgcc taacagtcgg atgcaccact tcgtggggtg cctggaatgg 1260
aatgacaaga aatactccct ggacattggc aacctcctcc tccgaggctg caggattcgc 1320
aacacagaca cctgctatgg actggtcatt tatgctggtt ttgacacaaa aattatgaag 1380
aactgtggca agatccattt gaagagaacc aagctggacc tcctgatgaa caagctggtg 1440
gttgtgatct tcatctccgt ggtgcttgtc tgcctggtgt tggccttcgg cttcggtttc 1500
tcagtcaaag aattcaaaga ccaccactac tacctctcgg gggtgcatgg gagcagcgtg 1560
gccgcagagt ccttcttcgt cttctggagc ttcctcatcc tgctcagcgt caccatcccg 1620
atgtccatgt tcatcctgtc cgagttcatc tacctgggga acagcgtctt catcgactgg 1680
gacgtgcaga tgtactacaa gccgcaggac gtgcctgcca aggcccgcag caccagcctc 1740
aacgaccacc tgggccaggt ggaatacatc ttctcggaca agacgggcac gctcacgcag 1800
aacatcttga ccttcaacaa gtgctgcatc agc.ggccgcg tctatggaga acccctacct 1860
ctggaacaag ttcgccgacg ggaagctgct cttccacaat gcggccctgc tgcacctcgt 1920
gcggaccaac ggggacgagg ccgtgcggga gttctggcgc ctgctggcca tctgccacac 1980
ggtgatgacc agctgttgta ccaggcggcc tcccccgacg agggggcgct ggtcaccgca 2040
gcccggaact tcggctacgt gttcctgtcc cgcacccagg acaccgtcac gatcatggag 2100
ctgggggagg aacgggtcta ccaggtcctg gccataatgg acttcaacag cacgcgcaaa 2160
cggatgtcgg tgctggttcg aaagccagag ggcgccatct gcctgtacac caagggcgcc 2220
gacacggtca tcttcgaacg cttgcacagg aggggggcaa tggaatttgc cacagaggag 2280
gccttggctg cctttgccca ggagaccctg cggacactgt gcctggccta cagggaggtg 2340
gctgaggaca tttacgagga ctggcagcag cgccaccagg aggccagcct cctgctgcag 2400
aaccgggcac aggccctgca acaggtgtac aacgagatgg agcaggacct caggctgctg 2460
ggagccacag ccatcgagga cagactccag gacggtgtcc ctgaaaccat caaatgtctc 2520
aagaagagca acatcaaaat atgggtgctc accggggaca agcaggaaac ggctgtgaac 2580
atcggcttcg cctgcgagct gctgtcagag aatatgctca ttctggagga gaaggagatt 2640
agccgcatcc tggagaccta ctgggaaaac agtaacaacc ttctaaccag ggagtccctg 2700
tcgcaggtca agctggcctt ggtcattaac ggagacttcc tggacaaact gctggtgtcc 2760
ctgcggaagg agccgcgcgc cctggcgcag aacgtgaaca tggacgaggc gtggcaggag 2820
ctcggccagt ccaggaggga tttcctctac gccaggcgcc tgtccctgct gtgccggagg 2880
ttcgggctcc cgctggctgc accgccagcc caggactcca gagcccgccg tagctccgag 2940
gtgctgcagg agcgcgcctt cgtggacctg gcgtccaagt gccaggcggt catctgctgc 3000
cgcgtgacgc ccaagcagaa ggccctgatc gtggccctgg tcaagaagta ccaccaggtg 3060
gtgaccctgg ccatcgggga cggtgccaac gacatcaaca tgatcaagac cgcggacgtg 3120
ggcgtggggc tggcgggcca ggagggcatg caggcagttc agaacagcga cttcgtgctc 3180
ggccagttct gcttcctgca gcgcctcctg ctggtgcacg gccgctggtc ctacgtgcgg 3240
atctgcaagt tcctgcgcta cttcttctac aagagcatgg ccagcatgat ggtgcaggtc 3300
tggtttgcct gctacaacgg cttcaccggc caggacgtga gcgcagagca gagcctggag 3360
aagccggagc tgtacgtggt ggggcagaag gacgagctct tcaactactg ggtcttcgtc 3420
caagccatcg cccatggtgt gaccacctct ctggtcaact tcttcatgac actgtggatc 3480
agccgcgaca cggcgggacc cgccagcttc agcgaccacc agtcctttgc ggtcgtggtg 3540
gccctgtctt gcctgctgtc catcaccatg gaggtcattc ttatcatcaa gtactggacc 3600
gccctgtgcg tggcgaccat cctcctcagc cttggtttct acgccatcat gactaccacc 3660
59
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
acccagagct tctggctctt cagagtatcc cccacgacct tcccgtttct gtatgccgac 3720
ctcagcgtga tgtcctctcc ctccatcctg ctggtggtcc tgctgagtgt gtccataaac 3780
accttccctg tcctggccct ccgagtcatc ttcccagccc tcaaggagct acgtgccaag 3840
gaggagaagg tggaggaggg ccccagcgag gagattttca ccatggagcc cttgcctcat 3900
gtacaccggg agtctcgtgc ccgccgttcc agctatgctt tctcccaccg ccagctgacg 3960
ttggagagcc agccagactc ctcggaggag aagtcagcat ttttgaagcc ctccacaccg 4020
ttccggaaga gctggcaaaa ggagcctcac acccccaagg aggggacggt gccacttcca 4080
gacaagaccc acaaatctca ggtggagact ctgccaccaa gtctggaaga atcgtccacg 4140
tccacgagcg agcagcctat ggaggtggag ctgtggcccg cggagaagca gtcatcatca 4200
tccatggagt ggctgctggt gcccggggag gagcagctat ccttgccccc agaggagcag 4260
tcattgccct ctgcggaggg gaccagggtt cagcagtgac gtagcatctg aatccctaga 4320
cccatctgat gaagaggcat cttcgagccc aaaggagtca cgctggcata tcaggaagat 4380
gtccttcctg ggaagaagaa gctccagcca gttctgctgc aagtcaacca gcatgcaggg 4440
ggccttcctc taaagacaag gactccacat gcttttcttt ttctaataaa ccagggtcca 4500
tctgacccca gcgctaaaa 4519
<210> 34
<211> 2922
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5547443CB1
<400> 34
gaggagtctg gcatggctca tgaatcagca gaggacttgt ttcatttcaa cgtagggggc 60
tggcatttct cagttcccag aagcaaactc tctcagtttc cagactccct gctgtggaaa 120
gaggcttcag ccttgacctc ttcagaaagc cagaggctat ttatcgacag agatggttcc 180
acatttaggc acgtgcacta ttacctctac acctccaaac tctccttctc cagttgtgca 240
gaactgaact tgctgtatga gcaagcattg ggtttgcagc tgatgccttt gctgcagact 300
ctagataacc tgaaggaagg gaaacaccat ctacgcgtac ggcctgcaga cctacctgtt 360
gctgagagag catctctgaa ctactggcgt acatggaagt gtattagcaa accctcagaa 420
tttccaatta aaagcccagc ctttacaggc ctacatgata aggcacctct ggggctcatg 480
gacacacccc tgttagacac agaagaggag gtgcactact gcttcctgcc cctagacctg 540
gtggccaaat atcccagcct agtgactgaa gacaacctgc tgtggctggc tgagacggtg 600
gccctcatcg agtgcgagtg cagcgagttc cgcttcattg tgaattttct tcgctcacag 660
aagattttac taccggataa tttctccaac attgatgtat tagaagcaga agtggaaatt 720
ctggaaatcc ctgcactcac tgaagccgta aggtggtacc ggatgaacat gggtggctgt 780
tccccgacca cctgttctcc cctgagcccc gggaaggggg cccgcacagc cagcctggag 840
tccgtgaaac cgctctacac aatggccctg ggtctgctgg tcaagtaccc ggactctgcg 900
ctgggccagc ttcgcatcga gagcacgcta gacggaagcc gactgtacat cacagggaat 960
ggcgtcctct ttcagcacgt caagaactgg ctggggactt gccggctgcc cctgacagag 1020
accatttccg aggtatatga gctctgtgcc ttcctagaca aaagggacat cacctacgag 1080
ccaatcaaag ttgctttgaa gactcatctg gagccaagga ctttggcacc catggatgtg 1140
ctcaatgagt ggacggcaga gatcactgtg tattccccac aacagatcat caaagtgtat 1200
gttggaagcc actggtacgc aaccaccctg cagacactgc tgaagtatcc agaactgctg 1260
tccaaccctc agagagtgta ctggatcaca tatggacaaa ccctgctcat ccacggggat 1320
ggccagatgt tccgacacat tctcaacttc ctgagacttg gcaaactgtt tttaccatct 1380
gaatttaagg aatggcccct cttctgccag gaggtggagg aataccacat tccatccctc 1440
tcagaagccc ttgcacaatg tgaagcatac aagtcatgga ctcaggagaa agaatctgaa 1500
aatgaagaag ctttttccat caggaggctg catgtggtga cagaagggcc agggtcactg 1560
gtggagttca gtagagacac taaagaaacc acagcctaca tgcctgtgga cttcgaagac 1620
tgcagtgaca ggactccatg gaacaaggct aagggaaacc tggtcaggtc caaccagatg 1680
gatgaggctg agcagtacac tcggcccatc caggtgtccc tatgccgaaa tgccaagagg 1740
gctggcaacc ctagcacata ctcacactgc cgtggcttgt gtaccaatcc tggacactgg 1800
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
gggagccacc ctgagagccc cccaaagaag aaatgcacca caatcaacct cacacagaaa 1860
tctgaaacca aagaccctcc cgccactccc atgcaaaaac tcatctccct ggtgagagaa 1920
tgggacatgg tcaattgcaa acagtgggaa ttccagccac tgacagccac acggagcagc 1980
cccttggagg aggccaccct gcagctcccc ttgggaagcg aggctgcttc ccagcccagc 2040
acctcagctg cctggaaagc ccattccaca gcctcagaga aggatccagg accacaggca 2100
ggggctggag ctggagcgaa agacaagggg ccagagccaa ccttcaagcc atacttaccc 2160
ccaaaaagag ctggcaccct gaaggactgg agcaagcaga ggaccaagga gagagaaagc 2220
cctgcccctg agcagcctct gcccgaggcc agtgaggtgg acagcctagg ggttatcctc 2280
aaagtgactc acccccccgt ggtgggcagc gatggcttct gcatgttctt tgaggacagc 2340
atcatctata ccacggagat ggacaacctc aggcacacaa cacccacagc cagtccccag 2400
ccccaagaag tgactttcct gagtttctct ctgtcctggg aagagatgtt ttatgcacag 2460
aaatgtcact gcttcctggc tgacatcatc atggattcca tcaggcaaaa ggaccccaaa 2520
gccatcacag ccaaggtggt ctccctggcc aatcggctgt ggaccctgca catcagcccc 2580
aagcagtttg tggtagattt gctggccatc accggcttca aggatgaccg gcacacccag 2640
gagcgcctgt acagctgggt ggagcttaca ctgcccttcg ccaggaaata tggccgatgc 2700
atggacctgc tcatccagag gggcctgtct aggtctgtct cttactccat cctgggaaag 2760
tacctacaag aggactaggg tgcccagaga tgcagcccct catgccccac ccgccaagtc 2820
tcattttaat tggagatagc ccagaatgca tgtgcccatc agagggtaca tatcagtcta 2880
ttttttaata taaacaaata aaagattaaa tcacacatca as 2922
<210> 35
<211> 2763
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 56008413CB1
<400> 35
ggaccccagg ccgggccggg ccgagaggct gccatgggct ccgtggggag ccagcgcctt 60
gaggagccca gcgtggcagg cacaccagac ccgggcgtag tgatgagctt caccttcgac 120
agtcaccagc tggaggaggc ggcggaggcg gctcagggcc agggccttag ggccaggggc 180
gtcccagctt tcacggatac tacattggac gagccagtgc ccgatgaccg ttatcacgcc 240
atctactttg cgatgctgct ggctggcgtg ggcttcctgc tgccatacaa cagcttcatc 300
acggacgtgg actacctgca tcacaagtac ccagggacct ccatcgtgtt tgacatgagc 360
ctcacctaca tcttggtggc actggcagct gtcctcctga acaacgtcct ggtggagaga 420
ctgaccctgc acaccaggat caccgcaggc tacctcttag ccttgggccc tctccttttt 480
atcagcatct gcgacgtgtg gctgcagctc ttctctcggg accaggccta cgccatcaac 540
ctggccgctg tgggcaccgt ggccttcggc tgcacagtgc agcaatccag cttctacggg 600
tacacgggga tgctgcccaa gcggtacacg cagggggtga tgaccgggga gagcacggcg .660
ggcgtgatga tctctctgag ccgcatcctc acgaagctgc tgctgcccga cgagcgcgcc 720
agcacgctca tcttcttcct ggtgtcggtg gcgctggagc tgctgtgttt cctgctgcac 780
ctgttagtgc ggcgcagccg cttcgtgctc ttctatacca cacggccgcg tgacagccac 840
cggggcaggc caggcctggg caggggctat ggctaccgcg tgcaccacga cgttgtcgcc 900
ggggacgtcc acttcgagca cccagccccg gccctggccc ccaacgagtc cccaaaggac 960
agcccagccc acgaggtgac cggcagcggc ggggcctaca tgcgctttga cgtgccgcgg 1020
ccaagggtcc agcgcagctg gcccaccttc agagccctgt tactgcaccg ctacgtggtg 1080
gcgcgggtga tctgggccga catgctctcc atcgccgtga cctacttcat cacgctgtgc 1140
ctgttccccg gcctcgagtc tgagatccgc cactgcatcc tgggcgagtg gctgcccatc 1200
ctcatcatgg ctgtgttcaa cctgtcagac ttcgtgggca agatcctggc agccctgccc 1260
gtggactggc ggggcaccca cctgctggcc tgctcctgcc tgcgtgtggt cttcatcccc 1320
ctcttcatcc tgtgcgtcta ccccagcggc atgcccgccc tccgtcaccc cgcctggccc 1380
tgcatcttct cactgctcat gggcatcagc aacggctact tcggcagcgt gcccatgatc 1440
ctggcggcag gcaaagtgag ccccaagcag cgggagctgg cagggaacac catgaccgtg 1500
tcctacatgt cagggctgac gctggggtcc gccgtggcct actgcaccta cagcctcacc 1560
61
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
cgcgacgctc acggcagctg cctgcacgcc tccaccgcca atggttccat cctcgcaggc 1620
ctctgagcca gccccgccca ctgccaggga cgccgagggc ctgaccaggg gccccgaggc 1680
ctgagggccc ctcccctgtc cccacctcag tgcctgcggg gccctgagcc tccccctgtg 1740
ccagcagccc cactccctca gggtccagcc atgccccacc ctggactgaa gttctgcaaa 1800
gtcctccgag gaccggaaca cgtttctgcg acccggggct ctggccagca ctgtgttctg 1860
cgtttggtct catacctgcg tctaccttcc atctgtgtcc agcggccccg gctccagccc 1920
agccagcact ctgcagggtc acacgcaccg tgtccccacc caggacagca gacacccgcc 1980
agagtgtgcg cgcccagtga ctgcaccccg gccctcatca cccaccggca ctgatcgggg 2040
caccgcctgg cccagcctcc accagggacc cctcctcatg aactctggag ccctgagagg 2100
agaggggcag ccccccacct tgtcaccctc agggcttccc cttctgtcct cattcttaga 2160
gactgcttct cccaaacata acgcgttagc catgaaggag tcggagccct gggtccgaat 2220
ggacccgcct gcggtctgca tcagcctctg ggaaaccaca gcagtgatgc cagctgggca 2280
cgtcaggacc tccccacaca cccacacgat gccacaggtc agggggctgt gcctgactag 2340
ggagccctcc cattgccttc ctggcccggg atagaagagg ggaggtaagt ctgggggcta 2400
cgaagccggg cccccacacc ctggctgaag tcagcttgac ctaggtcttg accctcatcc 2460
agcaagggac tcgacagacc caagggtccc tggaacgtag ggaggggctg ggggtcactc 2520
cagcccgggc ctcccagaac accaggcccg tgtgggtggc accctgaggt caggggatcc 2580
taagggtgtc cttccagaga cggtgtttcc agggggagga ccgcccccgc ttccagatcc 2640
ccggccccgg ctgtgactgc cctgtttcac ccctgctgtg tcccatcccc cgtctgtcca 2700
ctaactgtac cgcaccggcc atttaaagat gaaggcagac cgctgccaaa aaaaaaaaaa 2760
aaa 2763
<210> 36
<211> 5211
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6127911CB1
<400> 36
aagagctgct ggagtaggca cccatttaaa gaaaaaatga agaagcagca ataaagaagt 60
tgtaatcgtt acctagacaa acagagaact ggttttgaca gtgtttctag agtgcttttt 120
attattttcc tgacagttgt gttccaccat gattactttc tccttcagcg aataggctaa 180
atgaatatga aacagaaaag cgtgtatcag caaaccaaag cacttctgtg caagaatttt 240
cttaagaaat ggaggatgaa aagagagagc ttattggaat ggggcctctc aatacttcta 300
ggactgtgta ttgctctgtt ttccagttcc atgagaaatg tccagtttcc tggaatggct 360
cctcagaatc tgggaagggt agataaattt aatagctctt ctttaatggt tgtgtataca 420
ccaatatcta atttaaccca gcagataatg aataaaacag cacttgctcc tcttttgaaa 480
ggaacaagtg tcattggggc accaaataaa acacacatgg acgaaatact tctggaaaat 540
ttaccatatg ctatgggaat catctttaat gaaactttct cttataagtt aatatttttc 600
cagggatata acagtccact ttggaaagaa gatttctcag ctcattgctg ggatggatat 660
ggtgagtttt catgtacatt gaccaaatac tggaatagag gatttgtggc tttacaaaca 720
gctattaata ctgccattat agaaatcaca accaatcacc ctgtgatgga ggagttgatg 780
tcagttactg ctataactat gaagacatta cctttcataa ctaaaaatct tcttcacaat 840
gagatgttta ttttattctt cttgcttcat ttctccccac ttgtatattt tatatcactc 900
aatgtaacaa aagagagaaa aaagtctaag aatttgatga aaatgatggg tctccaagat 960
tcagcattct ggctctcctg gggtctaatc tatgctggct tcatctttat tatttccata 1020
ttcattacaa ttatcataac attcacccaa attatagtca tgactggctt catggtcata 1080
tttatactct tttttttata tggcttatct ttggtagctt tggtgttcct gatgagtgtg 1140
ctgttaaaga aagctgtcct caccaatttg gttgtgtttc tccttaccct cttttgggga 1200
tgtctgggat tcactgtatt ttatgaacaa cttccttcat ctctggagtg gattttgaat 1260
atttgtagcc cttttgcctt tactactgga atgattcaga ttatcaaact ggattataac 1320
ttgaatggtg taatttttcc tgacccttca ggagactcat acacaatgat agcaactttt 1380
tctatgttgc ttttggatgg tctcatctac ttgctattgg cattatactt tgacaaaatt 1440
62
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
ttaccctatg gagatgagcg ccattattct cctttatttt tcttgaattc atcatcttgt 1500
ttccaacacc aaaggactaa tgctaaggtt attgagaaag aaatcgatgc tgagcatccc 1560
tctgatgatt attttgaacc agtagctcct gaattccaag gaaaagaagc catcagaatc 1620
agaaatgtta agaaggaata taaaggaaaa tctggaaaag tggaagcatt gaaaggcttg 1680
ctctttgaca tatatgaagg tcaaatcacg gcaatcctgg gtcacagtgg agctggcaaa 1740
tcttcactgc taaatattct taatggattg tctgttccaa cagaaggatc agttaccatc 1800
tataataaaa atctctctga aatgcaagac ttggaggaaa tcagaaagat aactggcgtc 1860
tgtcctcaat tcaatgttca atttgacata ctcaccgtga aggaaaacct cagcctgttt 1920
gctaaaataa aagggattca tctaaaggaa gtggaacaag aggtacaacg aatattattg 1980
gaattggaca tgcaaaacat tcaagataac cttgctaaac atttaagtga aggacagaaa 2040
agaaagctga cttttgggat taccatttta ggagatcctc aaattttgct tttagatgaa 2100
ccaactactg gattggatcc cttttccaga gatcaagtgt ggagcctcct gagagagcgt 2160
agagcagatc atgtgatcct tttcagtacc cagtccatgg atgaggctga catcctggct 2220
gatagaaaag tgatcatgtc caatgggaga ctgaagtgtg caggttcttc tatgtttttg 2280
aaaagaaggt ggggtcttgg atatcaccta agtttacata ggaatgaaat atgtaaccca 2340
gaacaaataa catccttcat tactcatcac atccccgatg ctaaattaaa aacagaaaac 2400
aaagaaaagc ttgtatatac tttgccactg gaaaggacaa atacatttcc agatcttttc 2460
agtgatctgg ataagtgttc tgaccaggga gtgacaggtt atgacatttc catgtcaact 2520
ctaaatgaag tctttatgaa actggaagga cagtcaacta tcgaacaaga tttcgaacaa 2580
gtggagatga taagagactc agaaagcctc aatgaaatgg agctggctca ctcttccttc 2640
tctgaaatgc agacagctgt gagtgacatg ggcctctgga gaatgcaagt ctttgccatg 2700
gcacggctcc gtttcttaaa gttaaaacgt caaactaaag tgttattgac cctattattg 2760
gtatttggaa tcgcaatatt ccctttgatt gttgaaaata taatatatgc tatgttaaat 2820
gaaaagatcg attgggaatt taaaaacgaa ttgtattttc tctctcctgg acaacttccc 2880
caggaacccc gtaccagcct gttgatcatc aataacacag aatcaaatat tgaagatttt 2940
ataaaatcac tgaagcatca aaatatactt ttggaagtag atgactttga aaacagaaat 3000
ggtactgatg gcctctcata caatggagct atcatagttt ctggtaaaca aaaggattat 3060
agattttcag ttgtgtgtaa taccaagaga ttgcactgtt ttccaattct tatgaatatt 3120
atcagcaatg ggctacttca aatgtttaat cacacacaac atattcgaat tgagtcaagc 3180
ccatttcctc ttagccacat aggactctgg actgggttgc cggatggttc ctttttctta 3240
tttttggttc tatgtagcat ttctccttat atcaccatgg gcagcatcag tgattacaag 3300
aaaaatgcta agtcccagct atggatttca ggcctctaca cttctgctta ctggtgtggg 3360
caggcactag tggacgtcag cttcttcatt ttaattctcc ttttaatgta tttaattttc 3420
tacatagaaa acatgcagta ccttcttatt acaagccaaa ttgtgtttgc tttggttata 3480
gttactcctg gttatgcagc ttctcttgtc ttcttcatat atatgatatc atttattttt 3540
cgcaaaagga gaaaaaacag tggcctttgg tcattttact tcttttttgc ctccaccatc 3600
atgttttcca tcactttaat caatcatttt gacctaagta tattgattac caccatggta 3660
ttggttcctt catatacctt gcttggattt aaaacttttt tggaagtgag agaccaggag 3720
cactacagag aatttccaga ggcaaatttt gaattgagtg ccactgattt tctagtctgc 3780
ttcataccct actttcagac tttgctattc gtttttgttc taagatgcat ggaactaaaa 3840
tgtggaaaga aaagaatgcg aaaagatcct gttttcagaa tttcccccca aagtagagat 3900
gctaagccaa atccagaaga acccatagat gaagatgaag atattcaaac agaaagaata 3960
agaacagcca ctgctctgac cacttcaatc ttagatgaga aacctgttat aattgccagc 4020
tgtctacaca aagaatatgc aggccagaag aaaagttgct tttcaaagag gaagaagaaa 4080
atagcagcaa gaaatatctc tttctgtgtt caagaaggtg aaattttggg attgctagga 4140
cccagtggtg ctggaaaaag ttcatctatt agaatgatat ctgggatcac aaagccaact 4200
gctggagagg tggaactgaa aggctgcagt tcagttttgg gccacctggg gtactgccct 4260
caagagaacg tgctgtggcc catgctgacg ttgagggaac acctggaggt gtatgctgcc 4320
gtcaaggggc tcaggaaagc ggacgcgagg ctcgccatcg caagattagt gagtgctttc 4380
aaactgcatg agcagctgaa tgttcctgtg cagaaattaa cagcaggaat cacgagaaag 4440
ttgtgttttg tgctgagcct cctgggaaac tcacctgtct tgctcctgga tgaaccatct 4500
acgggcatag accccacagg gcagcagcaa atgtggcagg caatccaggc agtcgttaaa 4560
aacacagaga gaggtgtcct cctgaccacc cataacctgg ctgaggcgga agccttgtgt 4620
gaccgtgtgg ccatcatggt gtctggaagg cttagatgca ttggctccat ccaacacctg 4680
aaaaacaaac ttggcaagga ttacattcta gagctaaaag tgaaggaaac gtctcaagtg 4740
actttggtcc acactgagat tctgaagctt ttcccacagg ctgcagggca ggaaaggtat 4800
63
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
tcctctttgt taacctataa gctgcccgtg gcagacgttt accctctatc acagaccttt 4860
cacaaattag aagcagtgaa gcataacttt aacctggaag aatacagcct ttctcagtgc 4920
acactggaga aggtattctt agagctttct aaagaacagg aagtaggaaa ttttgatgaa 4980
gaaattgata caacaatgag atggaaactc ctccctcatt cagatgaacc ttaaaacctc 5040
aaacctagta attttttgtt gatctcctat aaacttatgt tttatgtaat aattaatagt 5100
atgtttaatt ttaaagatca tttaaaatta acatcaggta tattttgtaa atttagttaa 5160
caaatacata aattttaaaa ttattcttcc tctcaacata ggggtgatag c 5211
<210> 37
<211> 5701
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6427133CB1
<400> 37
gctcccaagg ctgagattac tctgcttcat ctggatcgcc catctctggg gtctcatggc 60
tgagtttcag ttccccaatc ctacctgctc ctcagggggc cagcactggg gctgcaggta 120
ggccacctgt tgagacctgg tgaaagatca ggtataataa tgttctgcag tgaaaagaaa 180
ttgcgtgaag tggaacggat agtgaaagcc aatgaccgtg aatataatga aaagttccag 240
tatgcggata atcgtatcca cacatcgaaa tataatattc tcaccttctt gccaattaat 300
ttatttgaac agttccaaag agtggcaaat gcctattttc tttgccttct gattttacag 360
ctaattccag aaatttcctc cttgacctgg tttaccacca ttgtgccttt ggtcctggtg 420
ataactatga cagctgtcaa agatgccaca gatgactatt ttcgccacaa gagtgataat 480
caagtgaata atcggcagtc tgaagtgctc atcaacagca aactgcagaa tgaaaaatgg 540
atgaatgtca aagtgggaga catcattaaa ttagaaaata accaatttgt tgctgctgat 600
ttacttctcc tatcaagtag tgagccacat ggtctctgtt atgttgaaac tgctgagctt 660
gatggggaaa cgaacctaaa agtccgccat gcactatcag ttacttcaga acttggagca 720
gatatcagca gacttgcagg gtttgatggg attgttgtct gtgaggtgcc taacaacaag 780
ttagataaat tcatgggaat cctttcttgg aaagacagca agcattccct caacaatgag 840
aagataatcc cgagaggctg catcctgaga aataccagct ggtgttttgg aatggttatt 900
tttgcaggtc ctgacactaa actaatgcag aatagtggta agacaaagtt taaaaggaca 960
agcattgata gattgatgaa tactctagta ctatggattt ttgggtttct gatatgcttg 1020
ggaattattc ttgcaatagg aaattcaatc tgggagagtc aaactgggga ccaattcaga 1080
actttcctct tttggaatga aggagagaag agctctgtgt tctccggatt cttaacattc 1140
tggtcatata ttattattct caatacagtt gtacccattt ccttatatgt gagtgtggaa 1200
gtaattcgtc taggacacag ttattttata aactgggacc ggaagatgta ttattctcga 1260
aaagcaatac ctgcagtggc tcgaacgacc acgctcaatg aggaactggg gcagattgag 1320
tacattttct ccgacaaaac gggtaccctc actcaaaaca tcatgacctt taaaagatgt 1380
tccattaatg ggagaatcta tggtgaagta catgatgacc tggatcagaa gacagaaata 1440
actcaggaaa aagagcctgt ggatttctca gtcaaatctc aagcggatag agaatttcag 1500
ttctttgacc acaatctgat ggaatccatt aaaatgggtg atcccaaagt tcatgaattc 1560
cttaggttac ttgctctctg ccacactgta atgtcagaag agaatagcgc aggagagctg 1620
atttaccaag ttcagtcacc tgatgaaggg gctctagtga ctgccgctag aaattttggg 1680
ttcattttta aatcccggac cccagagacc ataacaatag aagaattggg aacactagtt 1740
acttatcaat tacttgcctt tttggatttc aacaacacca gaaaaaggat gtctgtcata 1800
gttc.gaaacc cagaaggaca gataaagctt tattccaaag gagcagatac tattctgttt 1860
gaaaaacttc atccttccaa tgaagtcctt ttgtctttga cgtcagacca cctcagtgaa 1920
tttgcagggg aaggccttcg gaccttggcc atcgcataca gagacctgga tgacaagtac 1980
tttaaagagt ggcataagat gcttgaagat gcgaatgctg ccacagaaga gagggatgaa 2040
cgaatagctg ggctatatga agaaattgaa agagatttga tgctactagg tgccactgct 2100
gtagaagata agttacagga gggtgttatt gaaacagtta caagtttatc actagccaat 2160
attaagatct gggtcctaac aggagacaaa caagaaactg ccatcaacat cggttatgcc 2220
tgcaacatgc tgactgacga catgaatgat gtgtttgtga tagcagggaa taatgctgtg 2280
64
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
gaagtgagag aagaactcag gaaagcaaaa caaaatttgt ttggacaaaa cagaaatttt 2340
tccaatggcc atgtagtttg tgaaaaaaag cagcagctgg agttggattc tattgtagaa 2400
gaaaccataa caggagatta tgccttaatc ataaatggcc acagtttggc tcatgcccta 2460
gaaagtgatg tcaagaatga tctcctagaa cttgcttgca tgtgtaagac tgtaatttgc 2520
tgcagggtca ctccactcca gaaagcccaa gtggtagagc tggtgaagaa gtacagaaat 2580
gctgttactt tggccattgg tgatggagcc aatgatgtca gcatgattaa aagtgctcac 2640
attggtgttg gcatcagcgg ccaggaagga ttgcaagcag tcttagccag cgactattca 2700
tttgcacagt ttagatatct ccaaaggctt ctccttgttc atggaaggtg gtcttatttc 2760
cgaatgtgca aattcttatg ctatttcttc tataagaatt ttgcatttac acttgtgcat 2820
ttctggtttg gtttcttctg tggtttctca gcccagactg tttatgacca gtggttcatc 2880
acccttttta acattgttta cacatcactg cctgttttag ccatggggat ttttgaccag 2940
gatgtgagtg accagaacag cgtggactgt ccccagctct acaaaccagg acagctgaat 3000
ctgcttttta acaagcgtaa atttttcatt tgcgtgttgc atggaatcta cacctcatta 3060
gtccttttct tcatccccta tggggccttt tacaacgtgg ctggagaaga tgggcaacat 3120
attgctgact accagtcctt tgcagttacc atggccacat ctttggtcat tgtggtcagt 3180
gtgcagatag ccttggatac cagttactgg actttcatta atcacgtctt catctggggg 3240
agcattgcca tttatttctc cattttattt acaatgcaca gtaatggcat ctttggcatc 3300
ttcccaaacc agtttccatt tgttggtaat gcacgacatt ccctgaccca gaagtgcatc 3360
tggcttgtaa ttctcttaac aacagtggct tcagttatgc cagtggtggc attcagattt 3420
ttgaaggtgg atttataccc aaccctgagt gatcagatcc gccggtggca gaaggctcaa 3480
aagaaggcaa ggcctccaag tagccgaagg cctcggaccc gcaggtcaag ctcaagaagg 3540
tctggatatg cttttgctca ccaagaaggc tatggagagc ttatcacatc tggaaaaaat 3600
atgcgagcta aaaatccacc cccaacatca gggctggaaa agacacatta taatagcact 3660
agctggattg aaaatttatg taagaaaacc acagacaccg tgagcagctt tagccaggat 3720
aaaacagtga aactgtgagt caatatgaat ttaaaccacg tagttatctt ttcacttcag 3780
gtggagctga aattctgctg gctccagagt ttgagatttg aggcaagagg tggggcaggc 3840
agattgcctc acttaactta aatctgcggc agacaactgc cagtgcccat caaacaggag 3900
tgtgcgctat ggaaaaccag gccagagggt cactgtctgg tttgtgattt ggtggacaaa 3960
acactcgctg ttacaagtac agattttttt tttttttaaa tcaacctaga taccaattga 4020
cctgaacttt agaatcttat ttatggagaa aaacttgtaa agctgcatat tcactgaatg 4080
gatcctcagg cggataaaag ggtgcatttt aaaggtatat atccaagctg aaaagcatgc 4140
ctattgacag ataaacatgt atctgtaaga tcagcctttc ccaaggtata cttttaaaat 4200
ttaaagcgtg tactgtgttg ctttcagact gagttgcatg tcactcttta gtcttgatat 4260
ctacctgtct gttcagccag gacaacaaat ggcttccaag cctgaagaat acaaaagtgt 4320
gcttgtgttt ctcattttta taccagtcta gggacaaagg agactgaaca tctttgcagc 4380
aggataggct ggtaatttga tcaaatttat tcaaaaagct ctcagtctgt gtcatgtaag 4440
gacatgctta tgaaatgtga gagaggctcg ccactaagta ttctaaatac ttttcaatgg 4500
cttttctaac aacctcagta gtaatttgct gagcatcatc cagaccatta atagaatcag 4560
caaagcactg gaatttcaca ctttaatgat aatattccac atagtctatg ggcaaatatt 4620
ttcaacattt ccaattttta aagcttcaga attgaagcca aacaaattaa taaataattg 4680
ttttaattac tatttaaaaa ctcaggttta gattgtttaa aattagttgc ttttgatact 4740
cagctgtcat gtttataatt caaacatgta gtaaacatat gtaggtaagg ttgttttttt 4800
ggagatgttg cagctcaaat ttcagtccac atatgaatca tcagtgtatt ttccataaag 4860
tgattcgggc atatttgtgt gaaaacctca gttctgtcac ttcttacctc tataaacttg 4920
gacgataatg tgccttctct gagactcagt ttcttcctct gtaaaatgag gacatactac 4980
ctacctcatg tggttggttg atgattgtct gtcaaagcac aaactctgaa attattaaaa 5040
acataattat ttcataaaca gatgagttaa gttccagtta actcaacatc agtataacag 5100
agcaattgga agagaatatg aaaaaactgg aatctaaata gtcagtgagg aaggctttga 5160
taaaatgaaa ttgccagaaa gatataaaac tggttagggt cctacaggga aataaaatta 5220
taaccgtgga ggtacatttc tctaccagaa agcaaaaata aagcatcatg tcttaatggt 5280
tttctacaaa tcaacttcta attctacaga gtccttaatc tggtccctat taaattcttg 5340
gtcagacaaa gttacatttc ccaagagagt caggtgacac ttgagtgagt ttgatggata 5400
atgagctaat gtgatatcta taggtcacaa ttttttaaaa ccaaaatttt caagtctggg 5460
ataatctttc ctaaatggga tcaaatgaaa taatatgtgt aaaagagtca aatgcagtcc 5520
tttaccatag taactgccta tggacgttgt ctttccctta catgcctgcc tacacttaac 5580
cagatgttgg ttttcaatgt ctaatttgtc attagtttca ccacatttgc tcactttttg 5640
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
taacattttt gcaagatttg aaaactttca gtaaatgttt tggcactatt ggtaaaaaaa 5700
a 5701
<210> 38
<211> 1990
<212> DNA
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7472932CB1
<400> 38
atggctcatg ccccagaacc agacccggcc gccagcgacc tcggggatga gaggcccaag 60
tgggacaaca aggcccagta cctcctgagc tgcatcgggt ttgccgtggg gctggggaac 120
atttggcggt tcccatacct gtgccagacc tatggaggag gtgccttcct catcccctac 180
gtcatcgcgc tggtcttcga ggggatcccc attttccacg tcgagctcgc catcggccag 240
cggctgcgga agggcagcgt cggcgtgtgg acggccatct ccccgtacct cagtggagta 300
ggtctgggct gtgtcacgct gtccttcctg atcagcctgt actacaacac catcgtggcg 360
tgggtgctgt ggtacctcct caactccttc cagcacccgc tgccctggag ctcctgccca 420
ccggacctca acagaacagg ttttgtggag gagtgccagg gcagcagcgc cgtgagctac 480
ttctggtacc ggcagacact gaacatcaca gccgacatca atgacagtgg ctccatccag 540
tggtggctgc tcatctgctt ggcagcctcc tgggcagtcg tgtacatgtg tgtcatcagg 600
ggcattgaga ctacagggaa ggtgatttac ttcacagctt tgttccctta cctggtcctg 660
accatctttc tcatcagagg gctgaccctg ccaggggcaa caaaaggact catctacttg 720
ttcactccca acatgcacat tctccagaac ccccgggtgt ggctggacgc agccacccag 780
atattcttct ctctgtccct ggccttcgga ggacacatcg cttttgcaag ttacaactcg 840
cccaggaatg actgccagaa ggatgcggtg gtcatcgccc tggtcaacag gatgacctcc 900
ctgtacgcgt ccatcgctgt cttctctgtc ctggggttca aagcaactaa tgactgtccc 960
cgcagaaaca tcctcagcct catcaacgac tttgacttcc cagagcagag catctccagg 1020
gacgactacc cagccgtcct catgcacctg aacgccacct ggcccaagag ggtggcccag 1080
ctccccctga aggcctgcct cctggaagac tttctggata agagtgcctc gggcccgggc 1140
ctggccttcg tcgtcttcac ggagaccgac ctccacatgc cgggggctcc tgtgtgggcc 1200
atgctcttct tcgggatgct gttcaccttg gggctatcga ccatgttcgg gaccgtggag 1260
gcggtcatca cacccctgct ggacgtgggg gtcctgccta gatgggtccc caaggaggcc 1320
ctgactgggc tggtctgcct ggtctgcttc ctctccgcca cctgcttcac gctgcagtct 1380
gggaactact ggctggagat tttcgacaat tttgccgctt ccctgaacct gctcatgttg 1440
gcctttctcg aggttgtggg tgtcgtttat gtttatggaa tgaaacggtt ctgcgatgac 1500
attgcgtgga tgaccgggag gcggcccagc ccctactggc ggctgacctg gagggtggtc 1560
agtcccctgc tgctgaccat ctttgtggct tacatcatcc tcctgttctg gaagccactg 1620
agatacaagg cctggaaccc caaatacgag ctgttcccct cgcgtcagga gaagctctac 1680
ccgggctggg cgcgcgccgc ctgtgtgctg ctgtccttgc tgcccgtgct gtgggtcccg 1740
gtggccgcgc ttgctcagct gctcacccgg cggaggcgga cgtggaggga cagggacgcg 1800
cgcccagaca cggacatgcg cccggacacg gacacgcgcc cagacacgga catgcgcccg 1860
gacacggaca tgcgctgaag ccggccggag cggggcctgc atgggcgggt ctgtgggggg 1920
gcttggcctg atggtgggcg gggccccgcc cacagggccg accccaatac accagcgact 1980
caaccttgaa 1990
<210> 39
<211> 3760
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 8463147CB1
66
ctacctcatg tggttggttg atgattgtct gtcaaagcac aaactctga
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
<400> 39
atgacacagg catatcagaa atatattcta gaaaagttac ctaaaagccc tggagacaaa 60
ggcagagcat ggcctgggtc aactccatct gggaatttgc tgtccccatt catggcagct 120
tctaactcct ttcctgagct gtgtagccag gtttccagaa gagagtactg ggacctgcat 180
ggaataccgt ctgaccactt ttctgtgagg gtacaagttg aattctatat gaatgaaaat 240
acatttaaag aaagactaac attatttttc ataacaaacc agagatcaag tctaaggata 300
cgcctgttca atttttctct caaattacta agctgcttat tatacataat ccgagtacta 360
ctagaaaacc cttcacaagg aaatgaatgg tctcatatct tttgggtgaa cagaagtcta 420
cctttgtggg gcttacaggt ttcagtggca ttgataagtc tgtttgaaac aatattactt 480
ggttatctta gttataaggg aaacatctgg gaacagattt tacgaatacc cttcatcttg 540
gaaataatta atgcagttcc cttcattatc tcaatattct ggccttcctt aaggaatcta 600
tttgtcccag tctttctgaa ctgttggctt gccaaacatg ccttggaaaa tatgattaat 660
gatctacaca gagccattca gcgtacacag tgctgcaaat gtgttaatca agttttgatt 720
gtaatatcta cattactatg ccttatcttc acctgcattt gtgggatcca acatctggaa 780
cgaataggaa agaagctgaa tctctttgac tccctttatt tctgcattgt gacgttttct 840
actgtgggct tcggggatgt cactcctgaa acatggtcct ccaagctttt tgtagttgct 900
atgatttgtg ttgctcttgt ggttctaccc atacagtttg aacagctggc ttatttgtgg 960
atggagagac aaaagtcagg aggaaactat agtcgacata gagctcaaac tgaaaagcat 1020
gtcgtcctgt gtgtcagctc actgaagatt gatttactta tggatttttt aaatgaattc 1080
tatgctcatc ctaggctcca ggattattat gtggtgattt tgtgtcctac tgaaatggat 1140
gtacaggttc gaagggtact gcagattcca atgtggtccc aacgagttat ctaccttcaa 1200
ggttcagccc ttaaagatca agacctattg agagcaaaga tggatgacgc tgaggcctgt 1260
tttattctca gtagccgttg tgaagtggat aggacatcat ctgatcacca aacaattttg 1320
agagcatggg ctgtgaaaga ttttgctcca aattgtcctt tgtatgtcca gatattaaag 1380
cctgaaaata aatttcacat caaatttgct gatcatgttg tttgtgaaga agagtttaaa 1440
tacgccatgt tagctttaaa ctgtatatgc ccagcaacat ctacacttat tacactactg 1500
gttcatacct ctagagggca gtgtgtgtgc ctgtgttgca gagaaggcca gcaatcgcca 1560
gaacaatggc agaagatgta cggtagatgc tccgggaatg aagtctacca cattgttttg 1620
gaagaaagta cattttttgc tgaatatgaa ggaaagagtt ttacatatgc ctctttccat 1680
gcacacaaaa agtttggcgt ctgcttgatt ggtgttagga gggaggataa taaaaacatt 1740
ttgctgaatc caggtcctcg atacattatg aattctacag acatatgctt ttatattaat 1800
attaccaaag aagagaattc agcatttaaa aaccaagacc agcagagaaa aagcaatgtg 1860
tccaggtcgt tttatcatgg accttccaga ttacctgtac atagcataat tgccagcatg 1920
ggtactgtgg ctatagactt gcaagataca agctgtagat cagcaagtgg ccctaccctg 1980
tctcttccta cagagggaag caaagaaata agaagaccta gcattgctcc tgttttagag 2040
gttgcagata catcatcgat tcaaacatgt gatcttctaa gtgaccaatc agaagatgaa 2100
actacaccag atgaagaaat gtcttcaaac ttagagtatg ctaaaggtta cccaccttat 2160
tctccatata taggaagttc acccactttt tgtcatctcc ttcatgaaaa agtaccattt 2220
tgctgcttaa gattagacaa gagttgccaa cataactact atgaggatgc aaaagcctat 2280
ggattcaaaa ataaactaat tatagttgca gctgaaacag ctggaaatgg attatataac 2340
tttattgttc ctctcagggc atattataga ccaaagaaag aacttaatcc catagtactg 2400
ctattggata acccgccaga tatgcatttt ctggatgcaa tctgttggtt tccaatggtt 2460
tactacatgg tgggctctat tgacaaccta gatgacttac tcaggtgtgg agtgactttt 2520
gctgctaata tggtggttgt ggataaagag agcaccatga gtgccgagga agactacatg 2580
gcagatgcca aaaccattgt gaacgtgcag acactcttca ggttgttttc cagtctcagt 2640
attatcacag agctaactca ccccgccaac atgagattca tgcaattcag agccaaagac 2700
tgttactctc ttgctctttc aaaactggaa aagaaagaac gggagagagg ctctaacttg 2760
gcctttatgt ttcgactgcc ttttgctgct gggagggtgt ttagcatcag tatgttggac 2820
actctgctgt atcagtcatt tgtgaaggat tatatgattt ctatcacgag acttctgttg 2880
ggactggaca ctacaccagg atctgggttt ctttgttcta tgaaaatcac tgcagatgac 2940
ttatggatca gaacttatgc cagactttat cagaagttgt gttcttctac tggagatgtt 3000
cccattggaa tctacaggac tgagtctcag aaacttacta catctgagtc tcaaatatct 3060
atcagtgtag aagagtggga agacaccaaa gactccaaag aacaagggca ccaccgcagc 3120
aaccaccgca actcaacatc cagtgaccag tcggaccatc ccttgctgcg gagaaaaagc 3180
atgcagtggg cccgaagact gagcagaaaa ggcccaaaac actctggtaa aacagctgaa 3240
aaaataaccc agcagcgact gaacctctac aggaggtcag aaagacaaga gcttgctgaa 3300
67
CA 02438206 2003-08-06
WO 02/077237 PCT/US02/03657
cttgtgaaaa atagaatgaa acacttgggt ctttctacag tgggatatga tgaaatgaat 3360
gatcatcaaa gtaccctctc ctacatcctg attaacccat ctccagatac cagaatagag 3420
ctgaatgatg ttgtatactt aattcgacca gatccactgg cctaccttcc aaacagtgag 3480
cccagtcgaa gaaacagcat ctgcaatgtc actggtcaag attctcggga ggaaactcaa 3540
ctttgataaa aataaaatga gaaacttttt tcctacaaag accttgcttg aaaccacaaa 3600
agttttgctg gcacgaaaga aactagatgg aaatatatgt aattctctca tatttaaaaa 3660
cgtaatctct tctcttagaa gtatagatca ttttgaaact taatgtacta cttactggta 3720
ctctccctat taatatttga aggacctcaa tggaaagcgg 3760
<210> 40
<211> 1150
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7506408CB1
<400> 40
ccagaggaaa ctagtcacaa aaaccctgac tatcacctga tagattgctt gtgctgcctg 60
ataattactc gcacttttcc caggctagtg caaatcttca ggggccgtcc aggactacag 120
agctgtttca ccctaccttg gcttcaatct cttcccccat gctcgaaggt gcggagctgt 180
acttcaacgt ggaccatggc tacctggagg gcctggttcg aggatgcaag gccagcctcc 240
tgacccagca agactatatc aacctggtcc agtgtgagac cctagaagct ccattcttcc 300
aagactgcat gtctgaaaat gctctagatg aactgaatat tgaattgcta cgcaataaac 360
tatacaagtc ttaccttgag gcattctata aattctgtaa gaatcatggt gatgtcacag 420
cagaagttat gtgtcccatt cttgagtttg aggccgacag acgtgctttt atcatcactc 480
ttaactcctt tggcactgaa ttgagcaaag aagaccgaga gaccctctat ccaaccttcg 540
gcaaactcta tcctgagggg ttgcggctgt tggctcaagc agaagacttt gaccagatga 600
agaacgtagc ggatcattac ggagtataca aacctttatt tgaagctgta ggtggcagtg 660
ggggaaagac attggaggac gtgttttacg agcgtgaggt acaaatgaat gtgctggcat 720,
tcaacagaca gttccactac ggtgtgtttt atgcatatgt aaagctgaag gaacaggaaa 780
ttagaaatat tgtgtggata gcagaatgta tttcacagag gcatcgaact aaaatcaaca 840
gttacattcc aattttataa cccaagtaag gttctcaaat gtagaaaatt ataaatgtta 900
aaaggaagtt attgaagaaa ataaaagaaa ttatgttata ttatctagac tacacaaaag 960
taagccacac tatatcttca tgagttgcaa atccatggaa acacagtaaa ccagccctga 1020
aacaaagcat ttccttgttt tcagtggtat tagatcttgt ttccacatgt ctgtctcatt 1080
cttcactggg ccttacaggt tagttttaat taactctatg gtatttttct attcttgtct 1140
1150
gatcatgtta
68
