Note: Descriptions are shown in the official language in which they were submitted.
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TRANSPORTERS AND ION CHANNELS
TECHNICAL FIELD
The invention relates to novel nucleic acids, transporters and ion channels
encoded by these
nucleic acids, and to the use of these nucleic acids and proteins in the
diagnosis, treatment, and
prevention of transport, neurological, muscle, immunological and cell
proliferative disorders. The
invention also relates to the assessment of the effects of exogenous compounds
on the expression of
nucleic acids and transporters and ion channels.
1o 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+~ ~+~ pl~ SOøa-~ 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.
Carrier proteins which transport a single solute from one side of the membrane
to the other
are called uniporters. In contrast, coupled transporters link the transfer of
one solute with
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 (SMVT). 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
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various thyroid pathologies because it is the molecular basis for radioiodide
thyroid-imaging techniques
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 carriers 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. GLUT1 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; GLUTS 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. Blsas (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"t values for certain
substrates, including stereoselectivity
for L- over D-lactate, and in their sensitivity to inhibitors. There are Na+-
monocarboxylate
2
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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
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 Tap1 and Tap2, the endoplasmic
reticulum based
major lustocompatibility (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
(Darks, D.M. (1986) J.
Med. Genet. 23:99-106).
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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 trausport 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 carriers 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)
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) anal 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.
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Similarly, apo D and another lipocalin, al-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 (rA2T~, 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 c1). Equ c1 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
charged metabolites between the cytosol and the mitochondrial matrix. Examples
include the ADP,
ATP carrier protein; the 2-oxoglutarate/malate carrier; the phosphate carrier
protein; the pyruvate
carrier; 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 (OM1M)
*275000 Graves Disease).
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This class of trausporters 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 trausporters 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 au
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) trausport of molecules into cells
against concentration
gradients, 3) initiation of muscle contraction, and 4) endocrine cell
secretion.
Ion Trausporters
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, Ca2+-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 Caz+ are low and
cytosolic concentration of
K~ is high. The vacuolar (V) class of ion trausporters includes H+ pumps on
intracellular organelles,
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 trausmembrane 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). The V-ATPases are composed of two functional
domains: the Vl
domain, a peripheral complex responsible for ATP hydrolysis; and the Vo
domain, an integral complex
responsible for proton trauslocation across the membrane. The F-ATPases are
structurally and
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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. Carrier 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 Caa+ 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,
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 Cl channels) open
their pores in response to changes in membrane potential; and ligand-gated
channels (e.g.,
acetylcholine-, serotonin-, and glutamate-gated canon 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.
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 Ca2+ 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
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channel proteins. The characteristic domain of these channel proteins
comprises six trausmembrane
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. LTSA 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 trausmembrane 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
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 Nay channels are heterotrimeric complexes composed of a 260 kDa
pore-
forming a subunit that associates with two smaller auxiliary subunits, (31 and
(32. The (32 subunit is a
integral membrane glycoprotein that contains an extracellular Ig domain, and
its association with a and
(31 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
trausmembrane 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 Nay
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
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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 Ca2+ 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 Cl- 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).
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-
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 dysrythtnia syndrome
(Curran, M.E. (1998)
Curr. Opin. Biotechnol. 9:565-572; Kaczorowski, G.J. and M.L. Garcia (1999)
Curr. Opin. Chem.
Biol. 3 :448-45 8).
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
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activity, seizures and epilepsy, and insulin regulation (Doupnik, C.A. et al.
(1995) Curr. Opin.
Neurobiol. 5:268-277; C~rran, supra).
The recently recognized TW1K 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 Ca 2+ channels have been classified into several subtypes
based upon their
electrophysiological and pharmacological characteristics. L-type Ca2+ 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 al 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 a1, 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 a+ channels that have been characterized
biochemically include
complexes of a pore-forming alphal subunit of approximately 190-250 kDa; a
transmembrane
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 Cav1 family of alphal subunits conduct L-
type Ca 2+ currents,
which initiate muscle contraction, endocrine secretion, and gene
transcription, and are regulated
primarily by second messenger-activated protein phosphorylation pathways. The
CadZ family of
alphal subunits conduct N-type, P/Q-type, and R-type Ca a+ 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 ~+
currents, which are activated and inactivated more rapidly and at more
negative membrane potentials
than other Ca 2+ 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).
to
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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 Caa+ influx into cells to
resupply Ca2+ 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 i~t 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. Clip. Oncol.
19:568-576).
Chloride channels are necessary in endocrine secretion and in regulation of
cytosolic and
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
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
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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
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, supf~a;
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
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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 (3 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 signaling 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 1I, 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,
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,
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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, supt~a).
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 I~vl.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).
Expression profiling
Microarrays are analytical tools used in bioanalysis. A microarray has a
plurality of molecules
spatially distributed over, and stably associated with, the surface of a solid
support. Microarrays of
polypeptides, polynucleotides, and/or antibodies have been developed and find
use in a variety of
applications, such as gene sequencing, monitoring gene expression, gene
mapping, bacterial
identification, drug discovery, and combinatorial chemistry.
One area in particular in which microarrays hnd use is in gene expression
analysis. Array
technology can provide a simple way to explore the expression of a single
polymorphic gene or the
expression profile of a large number of related or unrelated genes. When the
expression of a single
gene is examined, arrays are employed to detect the expression of a specific
gene or its variants.
When an expression profile is examined, arrays provide a platform for
identifying genes that are tissue
specific, are affected by a substance being tested in a toxicology assay, are
part of a signaling
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cascade, carry out housekeeping functions, or are specifically related to a
particular genetic
predisposition, condition, disease, or disorder.
The potential application of gene expression profiling is relevant to
improving the diagnosis,
prognosis, and treatment of cancers, such as breast cancer, lung cancer,
prostate cancer, ovarian
cancer, and bone cancer, as well as the treatment of vascular inflammation and
immune responses,
liver toxicity, and neurological disorders.
Breast cancer
More than 180,000 new cases of breast cancer are diagnosed each year, and the
mortality
rate for breast cancer approaches 10% of all deaths in females between the
ages of 45-54 (Gish, I~.
(1999) AWIS Magazine 28:7-10). However the survival rate based on early
diagnosis of localized
breast cancer is extremely high (97%), compared with the advanced stage of the
disease in which the
tumor has spread beyond the breast (22%). Current procedures for clinical
breast examination are
lacking in sensitivity and specificity, and efforts are underway to develop
comprehensive gene
expression profiles for breast cancer that may be used in conjunction with
conventional screening
methods to improve diagnosis and prognosis of this disease (Perou, C.M. et al.
(2000) Nature
406:747-752).
Mutations in two genes, BRCA1 and BRCA2, are known to greatly predispose a
woman to
breast cancer and may be passed on from parents to children (Gish, supra).
However, this type of
hereditary breast cancer accounts for only about 5% to 9% of breast cancers,
while the vast majority
of breast cancer is due to non-inherited mutations that occur in breast
epithelial cells.
The relationship between expression of epidermal growth factor (EGF) and its
receptor,
EGFR, to human mammary carcinoma has been particularly well studied. (See
I~hazaie, K. et al.
(1993) Cancer and Metastasis Rev. 12:255-274, and references cited therein for
a review of this
area.) Overexpression of EGFR, particularly coupled with down-regulation of
the estrogen receptor,
is a marker of poor prognosis in breast cancer patients. In addition, EGFR
expression in breast tumor
metastases is frequently elevated relative to the primary tumor, suggesting
that EGFR is involved in
tumor progression and metastasis. This is supported by accumulating evidence
that EGF has effects
on cell functions related to metastatic potential, such as cell motility,
chemotaxis, secretion and
differentiation. Changes in expression of other members of the erbB receptor
family, of which EGFR
is one, have also been implicated in breast cancer. The abundance of erbB
receptors, such as HER-
2/neu, HER-3, and HER-4, and their ligands in breast cancer points to their
functional importance in
the pathogenesis of the disease, and may therefore provide targets for therapy
of the disease (Bacus,
S.S. et al. (1994) Am. J. Clin. Pathol. 102:513-S24). Other known markers of
breast cancer include a
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human secreted frizzled protein mRNA that is downregulated in breast tumors;
the matrix G1a protein
which is overexpressed in human breast carcinoma cells; Drg1 or RTP, a gene
whose expression is
diminished in colon, breast, and prostate tumors; maspin, a tumor suppressor
gene downregulated in
invasive breast carcinomas; and CaNl9, a member of the S 100 protein family,
all of which are down-
regulated in mammary carcinoma cells relative to normal mammary epithelial
cells (Zhou, Z. et al.
(1998) Int. J. Cancer 78:95-99; Chen, L. et al. (1990) Oncogene 5:1391-1395;
Ulrix, W. et al (1999)
FEBS Lett 455:23-26; Sager, R. et al. (1996) Curr. Top. Microbiol. Tmmunol.
213:51-64; and Lee,
S.W. et al. (1992) Proc. Natl. Acad. Sci. USA 89:2504-2508).
Cell lines derived from human mammary epithelial cells at various stages of
breast cancer
provide a useful model to study the process of malignant transformation and
tumor progression as it
has been shown that these cell lines retain many of the properties of their
parental tumors for lengthy
culture periods (Wistuba, LI. et al. (1998) Clip. Cancer Res. 4:2931-2938).
Such a model is
particularly useful for comparing phenotypic and molecular characteristics of
human mammary
epithelial cells at various stages of malignant transformation.
Lung cancer
Lung cancer is the leading cause of cancer death in the United States,
affecting more than
100,000 men and 50,000 women each year. Nearly 90% of the patients diagnosed
with lung cancer
are cigarette smokers. Tobacco smoke contains thousands of noxious substances
that induce
carcinogen metabolizing enzymes and covalent DNA adduct formation in the
exposed bronchial
epithelium. In nearly 80°l0 of patients diagnosed with lung cancer,
metastasis has already occurred.
Most commonly lung cancers metastasize to pleura, brain, bone, pericardium,
and liver. The decision
to treat with surgery, radiation therapy, or chemotherapy is made on the basis
of tumor histology,
response to growth factors or hormones, and sensitivity to inhibitors or
drugs. With current
treatments, most patients die within one year of diagnosis. Earlier diagnosis
and a systematic
approach to identification, staging, and treatment of lung cancer could
positively affect patient
outcome.
Lung cancers progress through a series of morphologically distinct stages from
hyperplasia to
invasive carcinoma. Malignant lung cancers are divided into two groups
comprising four
histopathological classes. The Non Small Cell Lung Carcinoma (NSCLC) group
includes squamous
cell carcinomas, adenocarcinomas, and large cell carcinomas and accounts for
about 70% of all lung
cancer cases. Adenocarcinomas typically arise in the peripheral airways and
often form mucin
secreting glands. Squamous cell carcinomas typically arise in proximal
airways. The histogenesis of
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squamous cell carcinomas may be related to chronic inflammation and injury to
the bronchial
epithelium, leading to squamous metaplasia. The Small Cell Lung Carcinoma
(SCLC) group accounts
for about 20% of lung cancer cases. SCLCs typically arise in proximal airways
and exhibit a number
of paraneoplastic syndromes including inappropriate production of
adrenocorticotropin and anti-diuretic
hormone.
Lung cancer cells accumulate numerous genetic lesions, many of which are
associated with
cytologically visible chromosomal aberrations. The high frequency of
chromosomal deletions
associated with lung cancer may reflect the role of multiple tumor suppressor
loci in the etiology of this
disease. Deletion of the short arm of chromosome 3 is found in over 90% of
cases and represents
one of the earliest genetic lesions leading to lung cancer. Deletions at
chromosome arms 9p and 17p
are also common. Other frequently observed genetic lesions include
overexpression of telomerase,
activation of oncogenes such as I~-ras and c-myc, and inactivation of tumor
suppressor genes such as
RB, p53 and CDKN2.
Genes differentially regulated in lung cancer have been identified by a
variety of methods.
Using mRNA differential display technology, Manda et al. (1999; Genomics 51:5-
14) identified five
genes differentially expressed in lung cancer cell lines compared to normal
bronchial epithelial cells.
Among the known genes, pulmonary surfactant apoprotein A and alpha 2
macroglobulin were down
regulated whereas nm23H1 was upregulated. Petersen et al.. (2000; Int J.
Cancer, 86:512-517) used
suppression subtractive hybridization to identify 552 clones differentially
expressed in lung tumor
derived cell lines, 205 of which represented known genes. Among the known
genes, throinbospondin-
1, fibronectin, intercellular adhesion molecule 1, and cytokeratins 6 and 18
were previously observed to
be differentially expressed in lung cancers. Wang et al. (2000; Oncogene
19:1519-1528) used a
combination of microarray analysis and subtractive hybridization to identify
17 genes differentially
overexpresssed in squamous cell carcinoma compared with normal lung
epithelium. Among the
known genes they identified were keratin isoform 6, KOC, SPRC, IGFb2, connexin
26, plakofillin 1
and cytokeratin 13.
Prostate Cancer
Prostate cancer is a common malignancy in men over the age of 50, and the
incidence
increases with age. In the US, there are approximately 132,000 newly diagnosed
cases of prostate
cancer and more than 33,000 deaths from the disorder each year.
Once cancer cells arise in the prostate, they are stimulated by testosterone
to a more rapid
growth. Thus, removal of the testes can indirectly reduce both rapid growth
and metastasis of the
cancer. Over 95 percent of prostatic cancers are adenocarcinomas which
originate in the prostatic
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acini. The remaining 5 percent are divided between squamous cell and
transitional cell carcinomas,
both of which arise in the prostatic ducts or other parts of the prostate
gland.
As with most tumors, prostate cancer develops through a multistage progression
ultimately
resulting in an aggressive tumor phenotype. The initial step in tumor
progression involves the
hyperproliferation of normal luminal and/or basal epithelial cells. Androgen
responsive cells become
hyperplastic and evolve into early-stage tumors. Although early-stage tumors
are often androgen
sensitive and respond to androgen ablation, a population of androgen
independent cells evolve from the
hyperplastic population. These cells represent a more advanced form of
prostate tumor that may
become invasive and potentially become metastatic to the bone, brain, or lung.
A variety of genes
may be differentially expressed during tumor progression. For example, loss of
heterozygosity (LOH)
is frequently observed on chromosome 8p in prostate cancer. Fluorescence in
situ hybridization
(FISH) revealed a deletion for at least 1 locus on 8p in 29 (69%) tumors, with
a significantly higher
frequency of the deletion on 8p21.2-p21.1 in advanced prostate cancer than in
localized prostate
cancer, implying that deletions on 8p22-p21.3 play an important role in tumor
differentiation, while
8p21.2-p21.1 deletion plays a role in progression of prostate cancer (Oba, K.
et al. (2001) Cancer
Genet. Cytogenet. 124: 20-26).
A primary diagnostic marker for prostate cancer is prostate specific antigen
(PSA). PSA is a
tissue-specific serine protease almost exclusively produced by prostatic
epithelial cells. The quantity
of PSA correlates with the number and volume of the prostatic epithelial
cells, and consequently, the
levels of PSA are an excellent indicator of abnormal prostate growth. Men with
prostate cancer
exhibit an early linear increase in PSA levels followed by an exponential
increase prior to diagnosis.
However, since PSA levels are also influenced by factors such as inflammation,
androgen and other
growth factors, some scientists maintain that changes in PSA levels are not
useful in detecting
individual cases of prostate cancer.
Current areas of cancer research provide additional prospects for markers as
well as potential
therapeutic targets for prostate cancer. Several growth factors have been
shown to play a critical role
in tumor development, growth, and progression. The growth factors Epidermal
Growth Factor (EGF),
Fibroblast Growth Factor (FGF), and Tumor Growth Factor alpha (TGFa) are
important in the growth
of normal as well as hyperproliferative prostate epithelial cells,
particularly at early stages of tumor
development and progression, and affect signaling pathways in these cells in
various ways (Lin, J. et
al. (1999) Cancer Res. 59:2891-2897; Putz, T. et al. (1999) Cancer Res. 59:227-
233). The TGF-(3
family of growth factors are generally expressed at increased levels in human
cancers and the high
expression levels in many cases.correlates with advanced stages of malignancy
and poor survival
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(Gold, L.I. (1999) Crit. Rev. Oncog. 10:303-360). Finally, there are human
cell lines representing both
the androgen-dependent stage of prostate cancer (LNCap) as well as the
androgen-independent,
hormone refractory stage of the disease (PC3 and DU-145) that have proved
useful in studying gene
expression patterns associated with the progression of prostate cancer, and
the effects of cell
treatments on these expressed genes (Chung, T.D. (1999) Prostate 15:199-207).
Ovarian cancer
Ovarian cancer is the leading cause of death from a gynecologic cancer. The
majority of
ovarian cancers are derived from epithelial cells, and 70% of patients with
epithelial ovarian cancers
present with late-stage disease. As a result, the long-term survival rate for
this disease is very low.
Identification of early-stage markers for ovarian cancer would significantly
increase the survival rate.
Genetic variations involved in ovarian cancer development include mutation of
p53 and microsatellite
instability. Gene expression patterns likely vary when normal ovary is
compared to ovarian tumors.
Bone cancer
Osteosarcoma is the most common malignant bone tumor in children.
Approximately 80% of
patients present with non-metastatic disease. After the diagnosis is made by
an initial biopsy,
treatment involves the use of 3-4 courses of neoadjuvant chemotherapy before
definitive surgery,
followed by post-operative chemotherapy. With currently available treatment
regimens, approximately
30-4.0% of patients with non-metastatic disease relapse after therapy.
Currently, there is no
prognostic factor that can be used at the time of initial diagnosis to predict
which patients will have a
high risk of relapse. The only significant prognostic factor predicting the
outcome in a patient with
non-metastatic osteosarcoma is the histopathologic response of the primary
tumor resected at the time
of definitive surgery. The degree of necrosis in the primary tumor is a
reflection of the tumor
response to neoadjuvant chemotherapy. A higher degree of necrosis (good or
favorable response) is
associated with a lower risk of relapse and a better outcome. Patients with a
lower degree of
necrosis (poor or unfavorable response) have a much higher risk of relapse and
poor outcome even
after complete resection of the primary tumor. Unfortunately, poor outcome
cannot be altered despite
modification of post-operative chemotherapy to account for the resistance of
the primary tumor to
neoadjuvant chemotherapy. Thus, there is an urgent need to identify prognostic
factors that can be
used at the time of diagnosis to recognize the subtypes of osteosarcomas that
have various risks of
relapse, so that more appropriate chemotherapy can be used at the outset to
improve the outcome.
Inflammation and immune responses
Atherosclerosis is a pathological condition characterized by a chronic local
inflammatory
response within the vessel wall of major arteries. Disease progression results
in the formation of
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atherosclerotic lesions, unstable plaques which occasionally rupture;
precipitating a catastrophic
thrombotic occlusion of the vessel lumen. Atherosclerosis and the associated
coronary artery disease
and cerebral stroke represent the most common causes of death in
industrialized nations. Although
certain key risk factors have been identified, a full molecular
characterization that elucidates the
causes and identifies all potential therapeutic targets for this complex
disease has not been achieved.
Molecular characterization of atherosclerosis requires identification of the
genes that contribute to
lesion growth, stability, dissolution, rupture and induction of occlusive
vessel thrombi.
Blood vessel walls are composed of two tissue layers: au endothelial cell (EC)
layer which
comprises the lumenal surface of the vessel, and an underlying vascular smooth
muscle cell (VSMC)
layer. Through dynamic interactions with each other and with surrounding
tissues, the vascular
endothelium and smooth muscle tissues maintain vascular tone, control
selective permeability of the
vascular wall, direct vessel remodeling and angiogenesis, and modulate
inflammatory and immune
responses.
The inflammatory response is a complex vascular reaction mediated by numerous
cytokines,
chemokines, growth factors, and other signaling molecules expressed by
activated ECs, VSMCs and
leukocytes. Inflammation protects the organism during trauma and infection,
but can also lead to
pathological conditions such as atherosclerosis. Activation of vascular
endothelium is a central event
in a wide range of physiological and disease processes such as vascular tone
regulation, coagulation
and thrombosis, atherosclerosis, inflammation and some infectious diseases.
The pro-inflammatory cytokines, interleukin (IL)-1 and tumor necrosis factor
(TNF), are
secreted by a small number of activated macrophages or other cells and can set
off a cascade of
vascular changes, largely through their ability to alter gene expression
patterns in ECs and VSMCs.
These vascular changes include vasodilation and increased permeability of
microvasculature, edema,
and leukocyte extravasation and transmigration across the vessel wall.
Ultimately, leukocytes,
particularly neutrophils and monocytes/macrophages, accumulate iu the
extravascular space, where
they remove injurious agents by phagocytosis and oxidative killing, a process
accompanied by release
of toxic factors, such as proteases and reactive oxygen species.
IL-1 and TNF induce pro-inflammatory, thrombotic, and anti-apoptotic changes
in gene
expression by signaling through receptors on the surface of ECs and VSMCs;
these receptors activate
transcription factors such as NFkB as well as AP-1, IRF-1, and NF-GMa, leading
to alterations in
gene expression. Genes known to be differentially regulated in EC by 1L-1 and
TNF include E
selectin, VCAM-1, ICAM-1, PAF, IkBa, IAP-1, MCP-1, eotaxin, ENA-78, G-CSF,
A20, ICE, and
complement C3 component. A key event in inflammation, adhesion and
transmigration of blood
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leukocytes across the vascular endothelium, for example, is mediated by
increased expression of E
selectin, P selectin, ICAM-1, and VCAM-1 on activated endothelium.
Several investigators have examined changes in vascular cell gene expression
associated with
various inflammatory diseases or model systems. Examining human umbilical vein
endothelial cells
(IIUVEC) activated by recombinant TNF-a or conditioned medium from activated
human primary
monocytes, Horrevoets et al. (1999; Blood 93:3418-3431) identified 106
differentially regulated genes.
In a similar approach, deVries et al. (2000; JBC 275:23939-23947) identified
40 differentially regulated
genes in umbilical cord artery-derived smooth muscle cells activated by
conditioned media from
cultured macrophages after stimulation with oxidized LDL particles. In both
studies, many of the
identified genes were already known to be involved in inflammation. Comparing
expression profiles
from inflammatory diseased tissues, cultured macrophages, chondrocyte cell
lines, primary
chondrocytes, and synoviocytes, Heller et al. (1997; Proc Natl Acad Sci USA
94:2150-2155) identifed
candidate genes involved in inflammatory responses, including TNF, IL-1 IL-6,
IL-8 G-CSF,
RANTES, and V-CAM. From this candidate gene set, tissue inhibitor of
metalloproteinase 1, ferritin
light chain, and manganese superoxide dismutase were found to be
differentially expressed in
rheumatoid arthritis (RA) relative to inflammatory bowel disease (IBD).
Further, IL-3, chemokine
Groa, and metalloproteinase matrix metallo-elastase were expressed in both RA
and 1BD. Most
recently, in an analysis of cultured aortic smooth muscle cells treated with
TNF-a, Haley et a1. (2000;
Circulation 102:2185-2189) found a 20-fold increase in eotaxin, an eosinophil
chemotactic factor. The
overexpression of eotaxin and its receptor CCR3 in atherosclerotic lesions was
confirmed by northern
analysis.
Human coronary artery endothelial cells (HCAECs) are primary cells derived
from the
endothelium of a human coronary artery. HCAECs are used as an experimental
model for
investigating the role of the endothelium in human vascular biology itt vitro.
Human umbilical artery
endothelial cells (HUAECs) are primary cells derived from the endothelium of
an umbilical artery.
Human uterine myometrium microvascular endothelial cells (UtMVECs) are primary
cells derived
from the uterine myometrium microvasculature. Human Iliac Artery Endothelial
Cells (HIAECs) are
primary cells derived from the endothelium of an iliac artery. Human umbilical
vein endothelial cells
(HWECs) are a primary cell line derived from the endothelium of the human
umbilical vein.
ECV304 is a human endothelial line.
Neurological disorders
Characterization of region-specific gene expression in the human brain
provides a context and
background for molecular neurobiology on a variety of neurological disorders.
For example,
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Alzheimer's disease (AD) is a progressive, neurodestructive process of the
human neocortex,
characterized by the deterioration of memory and higher cognitive function. A
progressive and
irreversible brain disorder, AD is characterized by three major pathogenic
episodes involving (a) an
aberrant processing and deposition of beta-amyloid precursor protein (betaAPP)
to form neurotoxic
beta-amyloid (betaA) peptides and an aggregated insoluble polymer of betaA
that forms the senile
plaque, (b) the establishment of intraneuronal neuritic tan pathology yielding
widespread deposits of
agyrophilic neurofibrillary tangles (NFT) and (c) the initiation and
proliferation of a brain-specific
inflammatory response. These three seemingly disperse attributes of AD
etiopathogenesis are licked
by the fact that proinflammatory microglia, reactive astrocytes and their
associated cytokines and
chemokines are associated with the biology of the microtubule associated
protein tan, betaA speciation
and aggregation. Missense mutations in the presenilin genes PS 1 and PS2,
implicated in early onset
familial AD, cause abnormal betaAPP processing with resultant overproduction
of betaA42 and
related neurotoxic peptides. Specific betaA fragments such as betaA42 can
further potentiate
proinflammatory mechanisms. Expression of the inducible oxidoreductase
cyclooxygenase-2 and
cytosolic phospholipase A2 (cPLA2) is strongly activated during cerebral
ischemia and trauma,
epilepsy and AD, indicating the induction of proinflammatory gene pathways as
a response to brain
injury. Neurotoxic metals such as aluminum and zinc, both implicated in AD
etiopathogenesis, and
arachidonic acid, a major metabolite of brain cPLA2 activity, each polymerize
hyperphosphorylated
tan to form NFT-like bundles. Studies have identified a reduced risk for AD in
patients aged over 70
2o years who were previously treated with non-steroidal anti-inflammatory
drugs for non-CNS afflictions
that include arthritis. (For a review of the interrelationships between the
mechanisms of PS 1, PS2 and
betaAPP gene expression, tan and betaA deposition and the induction,
regulation and proliferation in
AD of the neuroinflammatory response, see Lukiw, W.J, and Bazan, N.G. (2000)
Neurochem. Res.
2000 25:1173-1184).
Tumor necrosis factor-alpha (TNF-a) is a pleiotropic cytokine that plays a
central role in
mediation of the inflammatory response through activation of multiple signal
transduction pathways.
TNF-a is produced by activated lymphocytes, macrophages, and other white blood
cells, and activates
endothelial cells. Interferon-gamma (IFN~y), also known as Type II interferon
or immune interferon, is
a cytokine produced primarily by T-lymphocytes and natural killer cells.
Mature IFNy exists as
noncovalently- licked homodimers. IFNy displays antiviral, antiproliferative,
immunoregulatory, and
proinflammatory activities and is important in host defense mechanisms. 1FN-y
induces the production
of cytokines; upregulates the expression of class I and 1I MHC antigens, Fc
receptor, and leukocyte
adhesion molecule; modulates macrophage effector functions; influences isotype
switching; potentiates
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the secretion of immunoglobulins by B cells; augments TH1 cell expansion; and
may be required for
TH1 cell differentiation. IFNy exerts its biological activities by binding to
specific cell surface
receptors, which display high affinity binding sites. The IFNy receptor is
present on almost all cell
types except mature erythrocytes. Upon binding to its receptor, IFNy triggers
the activation of JAK-1
and JAK-2 kinases resulting in the phosphorylation of STAT1. Both IFNY and TNF-
a are considered
proinflammatory cytokines. Cross-talk can exist between the signal
transduction pathways of two
cytokines; for example, signal transduction cascades initiated by two
different cytokines lead to the
activation of NfkB.
Liver toxicity
The human C3A cell line is a clonal derivative of HepG2/C3 (hepatoma cell
line, isolated from
a 15-year-old male with liver tumor), which was selected for strong contact
inhibition of growth. The
use of a clonal population enhances the reproducibility of the cells. C3A
cells have many
characteristics of primary human hepatocytes in culture: i) expression of
insulin receptor and insulin-
like growth factor II receptor; ii) secretion of a high ratio of serum albumin
compared with a-
fetoprotein,; iii) conversion of ammonia to urea and glutamine; iv) metabolism
of aromatic amino
acids; and v) proliferation in glucose-free and insulin-free medium. The C3A
cell lice is now well
established as an in vitro model of the mature human liver (Mickelson et al.
(1995) Hepatology
22:866-875; Nagendra et al. (1997) Am. J. Physiol. 272:6408-6416).
The potential application of gene expression profiling is relevant to
measuring the toxic
response to potential therapeutic compounds and of the metabolic response to
therapeutic agents. For
instance, diseases treated with steroids and disorders caused by the metabolic
response to treatment
with steroids include adenomatosis, cholestasis, cirrhosis, hemangioma, Henoch-
Schonlein purpura,
hepatitis, hepatocellular and metastatic carcinomas, idiopathic
thrombocytopenic purpura, porphyria,
sarcoidosis, and Wilson disease. It is desirable to measure the toxic response
to potential therapeutic
compounds and of the metabolic response to therapeutic agents.
Steroids are a class of lipid-soluble molecules, including cholesterol, bile
acids, vitamin D, and
hormones, that share a common four-ring structure based on
cyclopentanoperhydrophenanthrene and
that carrry out a wide variety of functions. Steroid hormones, produced by the
adrenal cortex, ovaries,
and testes, include glucocorticoids, mineralocorticoids, androgens, and
estrogens. Steroid hormones
3o are widely used for fertility control and in anti-inflammatory treatments
fox physical injuries and
diseases such as arthritis, asthma, and auto-immune disorders. Progesterone, a
naturally occurring
progestin, is primarily used to treat amenorrhea, abnormal uterine bleeding,
or as a contraceptive.
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Medroxyprogesterone (MAH), also known as 6 a -methyl-17 hydroxyprogesterone,
is a synthetic
progestin with a pharmacological activity about 15 times greater than
progesterone. MAH is usually
used for the treatment of renal and endometrial carcinomas, amenorrhea,
abnormal uterine bleeding,
and endometriosis associated with hormonal imbalance. The primary
contraceptive effect of
exogenous progestins involves the suppression of the midcycle surge of LH. The
exact mechanism of
action, however, is unknown. At the cellular level, progestins diffuse freely
into target cells and bind
to the progesterone receptor. Target cells include the female reproductive
tract, mammary gland,
hypothalamus, and pituitary. Once bound to the receptor, progestins slow the
frequency of release of
gonadotropin releasing hormone (GnRH) from the hypothalamus and blunt the pre-
ovulatory LH surge,
thereby preventing follicular maturation and ovulation. Interestingly, the MAH
stimulatory effect on
the respiratory centers has been used clinically to treat low blood
oxygenation due to sleep apnea,
chronic obstructive pulmonary disease, or hypercapnia (excess of CO 2 in
blood). Beclomethasone is
a synthetic glucocorticoid that is used for treating steroid-dependent asthma,
relieving symptoms
associated with allergic or nonallergic (vasomotor) rhinitis, or for
preventing recurrent nasal polyps
following surgical removal. The anti-inflammatory and vasoconstrictive effects
of intranasal
beclomethasone are 5,000 times greater than those produced by hydrocortisone.
Budesonide is a
corticosteroid used to control symptoms associated with allergic rhinitis or
asthma. Dexamethasone is
a synthetic glucocorticoid used in anti-inflammatory or immunosuppressive
compositions. Prednisone
is metabolized in the liver to its active form, prednisolone, a glucocorticoid
with anti-inflammatory
properties. Betamethasone is a synthetic glucocorticoid with antiinflammatory
and immunosuppressive
activity and is used to treat psoriasis and fungal infections, such as
athlete's foot and ringworm. By
comparing both the levels and sequences expressed in tissues from subjects
exposed to or treated with
steroid compounds with the levels and sequences expressed in normal untreated
tissue it is possible to
determine tissue responses to steroids. Budesonide (Bude) is a corticosteroid
used to control
symptoms associated with allergic rhinitis or asthma. Budesonide has high
topical anti-inflammatory
activity but low systemic activity. Prednisone is a corticosteroid that is
metabolized in the liver to its
active form, prednisolone. Prednisone is roughly four times more potent as a
glucocorticoid than
hydrocortisone. Prednisone is intermediate between hydrocortisone and
dexamethasone in duration of
action. Prednisone is used in conditions such as allograft rejection, asthma,
systemic lupus
erythematosus, and many other inflammatory states.
Glucocorticoids are naturally occurring hormones that prevent or suppress
inflammation and
immune responses when administered at pharmacological doses. At the molecular
level, unbound
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glucocorticoids readily cross cell membranes and bind with high affinity to
specific cytoplasmic
receptors. Subsequent to binding, transcription and, ultimately, protein
synthesis are affected. The
result can include inhibition of leukocyte infiltration at the site of
inflammation, interference in the
function of mediators of inflammatory response, and suppression of humoral
immune responses. The
anti-inflammatory actions of corticosteroids are thought to involve
phospholipase A2 inhibitory proteins,
collectively called lipocortins. Lipocortins, in turn, control the
biosynthesis of potent mediators of
inflammation such as prostaglandins and leukotrienes by inhibiting the release
of the precursor
molecule arachidonic.
There is a need in the art for new compositions, including nucleic acids and
proteins, for the
diagnosis, prevention, and treatment of transport, neurological, muscle,
immunological and cell
proliferative disorders.
SUMMARY OF THE INVENTION
Various embodiments of the invention provide purified polypeptides,
transporters and ion
channels, referred to collectively as 'TRICH' and individually as 'TRICH-1,'
'TRICH-2,' 'TRICH-3,'
'TRICH-4,' 'TRICH-S,' '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,' 'TRICH-20,' 'TRICH-21,' 'TRICH-22,' 'TRICH-23,' 'TRICH-24,' 'TRICH-
25,' and
'TRICH-2f and methods for using these proteins and their encoding
polynucleotides for the detection,
diagnosis, and treatment of diseases and medical conditions. Embodiments also
provide methods.for
utilizing the purified transporters and ion channels and/or their encoding
polynucleotides for facilitating
the drug discovery process, including determination of efficacy, dosage,
toxicity, and pharmacology.
Related embodiments provide methods for utilizing the purified transporters
and ion channels and/or
their encoding polynucleotides for investigating the pathogenesis of diseases
and medical conditions.
An embodiment 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 N0:1-
26, b) a polypeptide comprising a naturally occurring amino acid sequence at
least 90% identical or at
least about 90% identical to an amino acid sequence selected from the group
consisting of SEQ 1D
N0:1-26, c) a biologically active fragment of a polypeptide having an amino
acid sequence selected
from the group consisting of SEQ D7 N0:1-26, and d) an immunogenic fragment of
a polypeptide
having an amino acid sequence selected from the group consisting of SEQ D7
NO:l-26. Another
embodiment provides an isolated polypeptide comprising an amino acid sequence
of SEQ ID NO:1-26:
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Still another embodiment 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 >D N0:1-26, b) a polypeptide comprising a naturally
occurring amino acid
sequence at least 90% identical or at least about 90% identical to an amino
acid sequence selected
from the group consisting of SEQ m N0:1-26, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ D7
N0:1-26, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ )D N0:1-26. In another embodiment, the polynucleotide
encodes a polypeptide
selected from the group consisting of SEQ )D N0:1-26. In an alternative
embodiment, the
polynucleotide is selected from the group consisting of SEQ )D N0:27-52.
Still another embodiment 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 N0:1-26, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical or at least about 90% identical to an amino acid sequence
selected from the group
consisting of SEQ m N0:1-26, c) a biologically active fragment of a
polypeptide having an amino acid
sequence selected from the group consisting of SEQ m N0:1-26, and d) an
immunogenic fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ )D N0:1-26.
Another embodiment provides a cell transformed with the recombinant
polynucleotide. Yet another
embodiment provides a transgenic organism comprising the recombinant
polynucleotide.
Another embodiment 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 )D N0:1-26, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical or at least about 90% identical to an amino acid sequence
selected from the group
consisting of SEQ >l7 NO:1-26, c) a biologically active fragment of a
polypeptide having an amino acid
sequence selected from the group consisting of SEQ 117 N0:1-26, and d) an
immunogenic fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ JD N0:1-26.
'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.
Yet another embodiment provides an isolated antibody which specifically binds
to a
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polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid sequence
selected from the group consisting of SEQ ~ N0:1-26, b) a polypeptide
comprising a naturally
occurring amino acid sequence at least 90% identical or at least about 90%
identical to an amino acid
sequence selected from the group consisting of SEQ 117 N0:1-26, c) a
biologically active fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ )D N0:1-26,
and d) an immunogenic fragment of a polypeptide having au amino acid sequence
selected from the
group consisting of SEQ D7 NO:1-26.
Still yet another embodiment 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 N0:27-52, b) a polynucleotide comprising a naturally
occurring polynucleotide
sequence at least 90% identical or at least about 90% identical to a
polynucleotide sequence selected
from the group consisting of SEQ ll~ NO:27-52, 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 other embodiments, the polynucleotide can comprise at
least about 20, 30, 40,
60, 80, or 100 contiguous nucleotides.
1'et another embodiment provides a method for detecting a target
polynucleotide in a sample,
said target polynucleotide being selected from the group consisting of.a) a
polynucleotide comprising a
polynucleotide sequence selected from the group consisting of SEQ m N0:27-52,
b) a polynucleotide
comprising a naturally occurring polynucleotide sequence at least 90%
identical or at least about 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:27-52, 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). T'he 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. In a related embodiment, the method can include
detecting the amount of
the hybridization complex. In still other embodiments, the probe can comprise
at least about 20, 30,
40, 60, 80, or 100 contiguous nucleotides.
Still yet another embodiment provides a method for detecting a target
polynucleotide in a
sample, said target polynucleotide being selected from the group consisting of
a) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:27-52, b) a
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polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical or at
least about 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:27-52, 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. In a related embodiment, the method can include detecting
the amount of the
amplified target polynucleotide or fragment thereof.
Another embodiment 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 N0:1-26, b) a polypeptide comprising a
naturally occurring
amino acid sequence at least 90% identical or at least about 90% identical to
an amino acid sequence
selected from the group consisting of SEQ ID N0:1-26, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID N0:1-26,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID N0:1-26, and a pharmaceutically acceptable
excipient. In one
embodiment, the composition can comprise an amino acid sequence selected from
the group consisting
of SEQ ID N0:1-26. Other embodiments provide a method of treating a disease or
condition
associated with decreased or abnormal expression of functional TRICH,
comprising administering to a
patient in need of such treatment the composition.
Yet another embodiment 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 117 N0:1-26, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical or at least
about 90% identical to an
amino acid sequence selected from the group consisting of SEQ >D N0:1-26, c) a
biologically active
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
ID N0:1-26, and d) an immunogenic fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ )D N0:1-26. The method comprises a)
exposing a sample
comprising the polypeptide to a compound, and b) detecting agonist activity in
the sample. Another
embodiment provides a composition comprising an agonist compound identified by
the method and a
pharmaceutically acceptable excipient. Yet another embodiment provides a
method of treating a
disease or condition associated with decreased expression of functional TRICH,
comprising
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administering to a patient in need of such.treatment the composition.
Still yet another embodiment 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 N0:1-26, b) a
polypeptide
comprising a naturally occurring amino acid sequence at least 90% identical or
at least about 90%
identical to an amino acid sequence selected from the group consisting of SEQ
)D NO:1-26, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-26, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ID N0:1-26. The method
comprises a)
exposing a sample comprising the polypeptide to a compound, and b) detecting
antagonist activity in
the sample. Another embodiment provides a composition comprising an antagonist
compound
identified by the method and a pharmaceutically acceptable excipient. Yet
another embodiment
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.
Another embodiment provides a method of screenitng 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 N0:1-26, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical or at least
about 90% identical to an
amino acid sequence selected from the group consisting of SEQ D7 N0:1-26, c) a
biologically active
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
)D N0:1-26, and d) an immunogenic fragment of a polypeptide having an amino
acid sequence
selected from the,group consisting of SEQ ~ NO:1-26. 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.
Yet another embodiment 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 D7 N0:1-26, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical or at least
about 90% identical to an
amino acid sequence selected from the group consisting of SEQ ID N0:1-26, c) a
biologically active
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
ID N0:1-26, and d) an immunogenic fragment of a polypeptide having an amino
acid sequence
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selected from the group consisting of SEQ ID N0:1-26. 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.
Still yet another embodiment 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 ~ N0:27-52,
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.
Another embodiment 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 N0:27-52, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical or at
least about 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:27-52, 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
ID N0:27-52, ii) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least
90% identical or at least about 90% identical to a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:27-52, 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 can comprise a fragment of a
polynucleotide selected from the
group consisting of i)-v) above; c) quantifying the amount of hybridization
complex; and d) comparing
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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 full length polynucleotide and
polypeptide
embodiments of the invention.
Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog, and the PROTEOIV>E database identification numbers and annotations of
PROTEOME
database homologs, for polypeptide embodiments 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 embodiments, 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 embodiments, along with selected fragments of the
polynucleotides.
Table 5 shows representative cDNA libraries for polynucleotide embodiments.
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
polynucleotides and
polypeptides, along withapplicable descriptions, references, and threshold
parameters.
Table ~ shows single nucleotide polymorphisms found in polynucleotide
sequences of the
invention, along with allele frequencies in different human populations.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleic acids, and methods are described, it is
understood that
embodiments of the invention are not limited to the particular machines,
instruments, 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
invention.
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
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host cell" includes a plurality of such host cells, and a reference to "an
antibody' is a reference to one
or more antibodies and equivalents thereof known to those skilled in the art,
and so forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods are now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing the
cell lines, protocols, reagents and vectors which are reported in the
publications and which might be
used in connection with various embodiments of the invention. Nothing herein
is to be construed as an
admission that the invention is not entitled to antedate such disclosure by
virtue of prior invention.
DEFINITIONS
"TRICIT' 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
encoding TRICH. The
encoded protein may also be "altered," and may contain deletions, insertions,
or substitutions of amino
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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 one or more similarities
in polarity, charge,
solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of
the residues, as long as the
biological 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" can refer to an oligopeptide,
a peptide, a
polypeptide, or a protein sequence, or a fragment of any of these, and to
naturally occurring or
synthetic molecules. Where "amino acid sequence" is recited to refer to a
sequence of a naturally
occurring protein molecule, "amino acid sequence" and like terms are not meant
to limit the amino acid
sequence to the complete native amino acid sequence associated with the
recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid. Amplification
may be carried out using polymerase chain reaction (PCR) technologies or other
nucleic acid
amplification 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, anticalins,
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')2, 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 .
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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
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 carn'er to slow clearance of the aptamer from
the circulatory system.
Aptamers maybe specifically cross-linked to their cognate ligands, e.g., by
photo-activation of a
cross-linker (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).
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 occurring 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 polynucleotide having a specific nucleic acid sequence.
Antisense compositions
may include DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having
modified backbone
linkages such as phosphorothioates, rnethylphosphonates, 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
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naturally occurring nucleic acid sequence produced by the cell to form
duplexes which block either
transcription or translation. The designation "negative" or "minus" can refer
to the antisense strand,
and the designation "positive" or "plus" can refer to the sense strand of a
reference DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic TRICH, or
of any oligopeptide thereof,
to induce a specific immune response in appropriate animals or cells and to
bind with specific
antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
A "composition comprising a given polynucleotide" and a "composition
comprising a given
polypeptide" can refer to any composition containing the given polynucleotide
or polypeptide. The
composition may comprise a dry formulation or an aqueous solution.
Compositions comprising
polynucleotides 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 o 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.
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Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
~ His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr S er, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Tle, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
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, aryl, 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.
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"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 a polynucleotide encoding TRICH
which can
be 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 about 5 to about 1000 contiguous
nucleotides or amino acid
residues. A fragment used as a probe, primer, antigen, therapeutic molecule,
or for other purposes,
to may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or
at least 500 contiguous
nucleotides or amino acid residues in length. Fragments may be preferentially
selected from certain
regions of a molecule. For example, a polypeptide fragment may comprise a
certain length of
contiguous amino acids selected from the first 250 or 500 amino acids (or
first 25% or 50%) of a
polypeptide as shown in a certain defined sequence. Clearly these lengths are
exemplary, and any
15 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 117 NO:27-52 can comprise a region of unique polynucleotide
sequence
that specifically identifies SEQ ID N0:27-52, for example, as distinct from
any other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:27-52 can
be employed
20 in one or more embodiments of methods of the invention,. for example, in
hybridization and
amplification technologies and in analogous methods that distinguish SEQ ID
N0:27-52 from related
polynucleotides. The precise length. of a fragment of SEQ m N0:27-52 and the
region of SEQ ll~
NO:27-52 to which the fragment corresponds are routinely determinable by one
of ordinary skill in the
art based on the intended purpose for the fragment.
25 A fragment of SEQ ID N0:1-26 is encoded by a fragment of SEQ ID N0:27-52. A
fragment of SEQ m N0:1-26 can comprise a region of unique amino acid sequence
that specifically
identifies SEQ 117 N0:1-26. For example, a fragment of SEQ ID N0:1-26 can be
used as an
immunogenic peptide for the development of antibodies that specifically
recognize SEQ ID NO:1-26.
The precise length of a fragment of SEQ ID NO:1-26 and the region of SEQ m
NO:1-26 to which
30 the fragment corresponds can be determined based on the intended purpose
for the fragment using
one or more analytical methods described herein or otherwise known in the art.
A "full length" polynucleotide is one containing at least a translation
initiation codon (e.g.,
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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 one
or more
computer algorithms or programs known in the art or described herein. For
example, percent identity
can be determined using the default parameters of the CLUSTAL V algorithm as
incorporated into
the MEGALIGN version 3.12e sequence alignment program. This program is part of
the
LASERGENE software package, a suite of molecular biological analysis programs
(DNASTAR,
Madison WI). CLUSTAL V is described in Higgins, D.G. and P.M. Sharp (1989;
CABIOS 5:151-
153) and in Higgins, D.G. et al. (1992; CABIOS 8:189-191). For pairwise
alignments of
polynucleotide sequences, the default parameters are set as follows: Ktuple=2,
gap penalty=5,
window=4, and "diagonals saved"=4. The "weighted" residue weight table is
selected as the default.
Percent identity is reported by CLUSTAL V as the "percent similarity" between
aligned
polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
which can be used 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.govBLAST/. The BLAST software suite includes various
sequence analysis
programs including "blastn," that is used to align a known polynucleotide
sequence with other
polynucleotide sequences from a variety of databases. Also available is a tool
called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The
"BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
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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 S0, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length supported
by the sequences shown herein, in the tables, figures, or Sequence Listing,
may be used to describe a
length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some alignment
methods take into account conservative amino acid substitutions. Such
conservative substitutions,
explained in more detail above, generally preserve the charge and
hydrophobicity at the site of
substitution, thus preserving the structure (and therefore function) of the
polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e ~.
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: I~tuple=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
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CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for'a
paiiwise
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: 11 and Extension Gap: 1 penalties
Gap x drop-off.' S0
Expect: 10
1o Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ ID number, or may be measured over
a shorter length,
for example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, afragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size and which. contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibodya' 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
CA 02458625 2004-02-16
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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 ~tg/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
S°C to 20°C lower than the thermal melting point (T"~ for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions 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
Press, Plaiuview 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, SS°C, or
42°C may be used. SSC concentration may
be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.
Typically, blocking
reagents are used to block non-specific hybridization. Such blocking reagents
include, for instance,
sheared and denatured salmon sperm DNA at about 100-200 ~.g/ml. Organic
solvent, such as
formamide at a concentration of about 35-50% v/v, may also be used under
particular circumstances,
such as for RNA:DNA hybridizations. Useful variations on these wash conditions
will be readily
apparent to those of ordinary skill in the art. Hybridization, particularly
under high stringency
conditions, may be suggestive of evolutionary similarity between the
nucleotides. Such similarity is
strongly indicative of a similar role for the nucleotides and their encoded
polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acids 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 present in
solution and another nucleic acid 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
polynucleotide
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
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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 "microarra~' refers to an arrangement of a plurality of
polynucleotides,
polypeptides, antibodies, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, antibody, 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
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
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by cell type depending on the enzymatic milieu of TRICH.
"Probe" refers to nucleic acids encoding TRICH, their complements, or
fragments thereof,
which are used to detect identical, allelic or related nucleic acids. Probes
are isolated oligonucleotides
or polynucleotides attached to a detectable label or reporter molecule.
Typical labels include
radioactive isotopes, ligands, chemiluminescent agents, and enzymes. "Primers"
are short nucleic
acids, usually DNA oligonucleotides, which may be annealed to a target
polynucleotide by
complementary base-pairing. The primer may then be extended along the target
DNA strand by a
DNA polymerase enzyme. Primex pairs can be used for amplification (and
identification) of a nucleic
acid, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook, 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. (1999; Short
Protocols in Molecular
Biolo , 4t'' ed., John Wiley & Sons, New York NY), and Innis, M. et al. (1990;
PCR Protocols, A
Guide to Methods and Applications, Academic Press, San Diego CA). PCR primer
pairs can be
derived from a known sequence, for example, by using computer programs
intended for that purpose
such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to 5,000
nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection
programs have incorporated additional features for expanded capabilities. For
example, the PrimOU
primer selection program (available to the public from the Genome Center at
University of Texas
South West Medical Center, Dallas TX) is capable of choosing specific primers
from megabase
sequences and is thus useful for designing primers on a genomc-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
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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 nucleic acid 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 maybe part of a viral vector,
e.g., based on a,
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions
(UTRs). Regulatory elements interact with host or viral proteins which control
transcription,
translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
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An "RNA equivalent," in reference to a DNA molecule, is composed of the same
linear
sequence of nucleotides as the reference DNA molecule 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,
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 about 60%
free, preferably at least about 75% free, and most preferably at least about
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
sequences into a prokaryotic or eukaryotic host cell. The method for
transformation is selected based
on tlae type of host cell being transformed and may include, but is not
limited to, bacteriophage or viral
CA 02458625 2004-02-16
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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 trausiently
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. In another embodiment, the nucleic acid can be introduced
by infection with a
recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002)
Science 295:868-872). The
erm 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 polynucleotides 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
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individuals of a given species. Polymorphic variants also may encompass
"single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide. sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 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 lengthof one of the polypeptides..
THE INVENTION
Various embodiments of the invention include 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
embodiments of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to
a single Incyte project identification number (Iucyte Project ID). Each
polypeptide sequence is
denoted by both a polypeptide sequence identification number (Polypeptide SEQ
ID NO:) and an
Incyte polypeptide sequence number (Iucyte 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.
Column 6 shows the Incyte ID numbers of physical, full length clones
corresponding to the polypeptide
and polynucleotide sequences of the invention. The full length clones encode
polypeptides which have
at least 95 % sequence identity to the polypeptide sequences shown in column
3.
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 ZD 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
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GenBank homolog and the PROTEOME database identification numbers (PROTEOME )D
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 117 NO:) and the
corresponding Incyte
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 N0:1 is 49% identical, from residue S11 to residue K626, to human CTL1
protein (GenBank
D7 g6996442) as determined by the Basic Local Alignment Search Tool (BLAST).
(See Table 2.)
The BLAST probability score is 9.0e-168, which indicates the probability of
obtaining the observed
polypeptide sequence alignment by chance. SEQ m N0:1 also contains an eight
transmembrane
helices regions as determined by using a hidden Markov model for the
prediction of txansmembrane
helices. (See Table 3.) In an alternative example, SEQ 117 N0:3 is 57%
identical, from residue E10
to residue V115, to human SLC11A3 iron transporter (GenBank )D g8895485) as
determined by the
Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST
probability score is 4.7e-
25, which indicates the probability of obtaining the observed polypeptide
sequence alignment by
chance. In an alternative example, SEQ >D N0:6 is 88% identical, from residue
M1 to residue 5944,
to rat potassium channel (GenBank D7 g2745729) 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 >l7 N0:6
also contains a
PAC motif, a PAS domain, a cyclic nucleotide-binding domain, and an ion
transport protein domain as
determined by searching for statistically significant matches in the hidden
Markov model (HIVI1VI)
based PFAM database of conserved protein family domains. (See Table 3.) Data
from
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BLAST_PRODOM and BLAST DOMO analyses provide further corroborative evidence
that SEQ
ID NO:6 is a potassium chancel. In an alternative example, SEQ D7 N0:10 is 99%
identical, from
residue M1 to residue I418, 95% identical, from residue 5420 to residue 5680,
and 94% identical, from
residue P665 to residue H894, to human Eag-related gene member 2 (GenBank ID
g11878259) 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 117 N0:10 is localized to the plasma membrane, has
transporter and
channel activity and is a voltage-gated potassium channel, as determined by
BLAST analysis using the
PROTEOME database. SEQ ll~ N0:10 also contains a PAC domain, cyclic nucleotide
binding
domain and ion transport domain as determined by searching for statistically
significant matches in the
hidden Markov model (HNINI) based PFAM database of conserved protein family
domains. (See
Table 3.) SEQ ID N0:10 contains five transmembrane-spanning regions as
determined by
T'LR analysis. Data from further BLAST analyses of the PRODOM and DOMO
databases
provide additional corroborative evidence that SEQ lD N0:10 is a potassium
channel. In an
alternative example, SEQ ID N0:11 is 86% identical, from residue A94 to
residue S785, to rat
potassium channel (GenBank ID g2745729) 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. As determined
by BLAST
analysis using the PROTEOME database, SEQ ID N0:11 is localized to the plasma
membrane, is
homologous to rat ether-a-go-go related 2, which is a slowly activating
delayed rectifier potassium
channel, and may facilitate the differentiation of pre-vertebral neurons
(PROTEOME ll7
331276~Rn.10875); SEQ 177 N0:11 is also homologous to rat ether-a-go-go-
related gene 3 which is an
inward rectifier potassium channel that functions in potassium transport
specifically in the nervous
system (PROTEOME ll~ 331274~Rn.10874). SEQ ID N0:11 also contains a cyclic
nucleotide-binding domain and an ion transport protein domain as determined by
searching for
statistically significant matches in the hidden Markov model (HIVIM) based
PFAM database of
conserved protein family domains. (See Table 3.) Data from additional BLAST
analyses provide
further corroborative evidence that SEQ ID N0:11 is a potassium channel. In an
alternative example,
SEQ ID N0:14 is 38% identical, from residue Q13 to residue 51049, to
Schizosaccharomyces pombe
membrane ATPase (GenBank 1D g3451312) as determined by the Basic Local
Alignment Search
Tool (BLAST). (See Table 2.) The BLAST probability score is 1.5e-189, which
indicates the
probability of obtaining the observed polypeptide sequencealignment by chance.
SEQ ID N0:14 is
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localized to the membrane, and is a member of the P-type, Ca2+-type, ATPase
subfamily, as
determined by BLAST analysis using the PROTEOME database. SEQ ID N0:14 also
contains an
E1-E2 ATPase domain as determined by searching for statistically significant
matches in the hidden
Markov model (I~V1M)-based PFAM database of conserved protein family domains.
(See Table 3.)
Data from BLllVIPS, MOTIFS, and PROFILESCAN analyses provide further
corroborative evidence
that SEQ ID N0:14 is a membrane ATPase. In an alternative example, SEQ ID
N0:19 is 98%
identical, from residue M1 to residue L602, to human sodium-dependent high-
affinity dicarboxylate
transporter (GenBank ID 88132324) 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 D7 N0:19
also has homology
to proteins that have transporter gene function and are sodium-dependent
dicarboxylate transporters,
as determined by BLAST analysis using the PROTEOME database. SEQ ID N0:19 also
contains a
sodium-dependent dicarboxylate transporter domain as determined by searching
for statistically
significant matches in the hidden Markov model (I~VIM)-based PFAM database of
conserved protein
family domains. (See Table 3.) Data from BLllVIPS and BLAST analyses provide
further
corroborative evidence that SEQ ID N0:19 is a sodium-dependent dicarboxylate
transporter. SEQ D7
N0:2, SEQ ll7 N0:4,-5, SEQ ID N0:7-9, SEQ ll7 NO:12-13, SEQ ID NO:15-18, and
SEQ ID
NO:20-26 were analyzed and annotated in a sin-iilar manner. The algorithms and
parameters for the
analysis of SEQ JD N0:1-26 are described in Table 7.
As shown in Table 4, the full length polynucleotide embodiments 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 ID NO:), the corresponding Incyte polynucleotide consensus sequence number
(Incyte ll~) 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 embodiments, and of
fragments of the
polynucleotides which are useful, for example, in hybridization or
amplification technologies that
identify SEQ ID NO:B-14 or that distinguish between SEQ ID N0:8-14 and related
polynucleotides.
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
polynucleotides. In addition, the
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polynucleotide fragments described in column 2 may identify sequences derived
from the ENSEMBL
(The Sauger 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 axons brought
together by an "axon
stitching" algorithm. For example, a polynucleotide sequence identified as
FL I~:~~~LXXX NI N~ YI'YYI'_N3 N4 represents a "stitched" sequence in which
~'~~~XXX is the
identification number of the cluster of sequences to which the algorithm was
applied, and YYYYYis the
number of the prediction generated by the algorithm, and N1,~,3,.,, if
present, represent specific axons
that may have been manually edited during analysis (See Example V).
Alternatively, the
polynucleotide fragments in column 2 may refer to assemblages of axons brought
together by an
"axon-stretching" algorithm. For example, a polynucleotide sequence identified
as
FLX~:~_gA~9AAA~BBBBB_1 N is a "stretched" sequence, with 1~'~~~XXX being the
Incyte
project identification number, gAAAAA being the GenBank identification number
of the human
genomic sequence to which the "axon-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 axons (See Example V). In instances where a RefSeq
sequence was used
as a protein homolog for the "axon-stretching" algorithm, a RefSeq identifier
(denoted by "NM,"
"NP," or "NT") maybe 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
methods associated with the prefixes (see Example IV and Example V).
Prefix Type of analysis andlor examples of programs
GNN, GFG,Exon prediction from genomic sequences using,
ENST for example,
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).
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INCY ~ Full length transcript and exon prediction from mapping of EST
sequences to the genome. Genomic location and EST
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
polynucleotides 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 polynucleotides. The tissues and vectors
which were used to
construct the cDNA libraries shown in Table 5 are described in Table 6.
Table 8 shows single nucleotide polymorphisms (SNPs) found in polynucleotide
sequences of
the invention, along with,allele frequencies in different human populations.
Columns 1 and 2 show the
polynucleotide sequence identification number (SEQ ID NO:) and the
corresponding Incyte project
identification number (PDT) for polynucleotides of the invention. Column 3
shows the Iucyte
identification number for the EST in which the SNP was detected (EST ID), and
column 4 shows the
identification number for the SNP (SNP m). Column 5 shows the position within
the EST sequence
at which the SNP is located (EST SNP), and column 6 shows the position of the
SNP within the full-
length polynucleotide sequence (CB 1 SNP). Column 7 shows the allele found in
the EST sequence.
Columns 8 and 9 show the two alleles found at the SNP site. Column 10 shows
the amino acid
encoded by the codon including the SNP site, based upon the allele found in
the EST. Columns 11-14
show the frequency of allele 1 in four different human populations. An entry
of n/d (not detected)
indicates that the frequency of allele 1 in the population was too low to be
detected, while n/a (not
available) indicates that the allele frequency was not determined for the
population.
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.
Various embodiments also encompass polynucleotides which encode TRICH. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:27-52, which encodes TRICH. The
polynucleotide
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sequences of SEQ D7 N0:27-52, 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 variants of a polynucleotide encoding TRICH. In
particular,
such a variant polynucleotide will have at least about 70%, or alternatively
at least about 85%, or even
at least about 95% polynucleotide sequence identity to a polynucleotide
encoding TRICH. A
particular aspect of the invention encompasses a variant of a polynucleotide
comprising a sequence
selected from the group consisting of SEQ ll7 N0:27-52 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 IIJ N0:27-52. Any one of
the polynucleotide
variants described above can encode a polypeptide 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 encoding TRICH. A splice variant may have portions which have
significant sequence
identity to a polynucleotide 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 a
polynucleotide 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 encoding TRICH.
For example, a polynucleotide comprising a sequence of SEQ ID N0:34 and a
polynucleotide
comprising a sequence of SEQ D7 N0:43 are splice variants of each other; a
polynucleotide
comprising a sequence of SEQ ID N0:46 and a polynucleotide comprising a
sequence of SEQ ID
N0:52 are splice variants of each other; a polynucleotide comprising a
sequence of SEQ ID N0:39
and a polynucleotide comprising a sequence of SEQ ID NO:50 are splice variants
of each other; and a
polynucleotide comprising a sequence of SEQ ID NO:32, a polynucleotide
comprising a sequence of
SEQ ID NO:36, and a polynucleotide comprising a sequence of SEQ TD NO:37 are
splice variants of
each other. Any one of the splice variants described above can encode a
polypeptide 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
53
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
similarity to the polynucleotide sequences of any known and naturally
occurring gene, may be
produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
sequence that could be made by selecting combinations based on possible codon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring TRICH, and all such variations
are to be considered as
being specifically disclosed.
Although polynucleotides which encode TRICH and its variants are generally
capable of
hybridizing to polynucleotides encoding naturally occurring TRICH under
appropriately selected
conditions of stringency, it may be advantageous to produce polynucleotides
encoding TRICH or its
derivatives possessing a substantially different codon usage, e.g., inclusion
of non-naturally occurring
codons. Codons may be selected to increase the rate at which expression of the
peptide occurs in a
particular prokaryotic or eukaryotic host in accordance with the frequency
with which particular
codons are utilized by the host. Other reasons for substantially altering the
nucleotide sequence
encoding TRICH and its derivatives without altering the encoded amino acid
sequences include the
production of RNA transcripts having more desirable properties, such as a
greater half life, than
transcripts produced from the naturally occurring sequence.
The invention also encompasses production of polynucleotides which encode
TRICH and
TRICH derivatives, or fragments thereof, entirely by synthetic chemistry.
After production, the
synthetic polynucleotide 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 polynucleotide encoding TRICH or any fragment
thereof.
Embodiments of the invention can also include polynucleotides that are capable
of hybridizing
to the claimed polynucleotides, and, in particular, to those having the
sequences shown iu SEQ 177
NO:27-52 and fragments thereof, under various conditions of stringency (Wahl,
G.M. and S.L. Berger
(1987) Methods Enzymol. 152:399-407; IKimmel, 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 Biosciences, Piscataway NJ),
or combinations
of polymerases and proofreading exonucleases such as those found in the
ELONGASE amplification
system (Iuvitrogen, Carlsbad CA). Preferably, sequence preparation is
automated with machines
54
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
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 (Amersham Biosciences),
or other
systems known in the art. The resulting sequences are analyzed using a variety
of algorithms which
are well known in the art (Ausubel et al., supra, ch. 7; Meyers, R.A. (1995)
Molecular Biology and
Biotechnolo~y, Wiley VCH, New York NY, pp. 856-853).
The nucleic acids encoding TRICH may be extended utilizing a partial
nucleotide sequence
and employing various PCR-based methods known in the art to detect upstxeam
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 (Sarkar, G. (1993) PCR Methods Applic. 2:318-322). Another
method, inverse PCR,
uses primers that extend in divergent directions to amplify unknown sequence
from a circularized
template. The template is derived from restriction fragments comprising a
known genomic locus and
surrounding sequences (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 (Lagerstrom, M. et al. (1991) PCR Methods
Applic. 1:111-119). In
this method, multiple restriction enzyme digestions and ligations may be used
to insert an engineered
double-stranded sequence into a region of unknown sequence before performing
PCR. Other
methods which may be used to retrieve unknown sequences are known in the art
(Parker, J.D. et al.
(1991) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested
primers, and
PROMOTERFIhTDER libraries (Clontech, Palo Alto CA) to walk genomic DNA. This
procedure
avoids the need to screen libraries and is useful in fording 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.
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirni the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, 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, polynucleotides 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 polynucleotides which encode substantially the same or a
functionally equivalent
polypeptides may be produced and used to express TRICH.
The polynucleotides of the invention can be engineered using methods generally
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
56
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
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
iri 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, polynucleotides encoding TRICH may be synthesized, in
whole or in
part, using one or more chemical methods well known in the art (Caruthers,
M.H. et al. (1980)
Nucleic Acids Symp. Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp.
Ser. 7:225-232).
Alternatively, TRICH itself or a fragment thereof may be synthesized using
chemical methods known
in the art. For example, peptide synthesis can be performed using various
solution-phase or
solid-phase techniques (Creighton, T. (1984) Proteins, Structures and
Molecular Properties, WH
Freeman, New York NY, pp. 55-60; 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
occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography (Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-
421). The
composition of the synthetic peptides may be confirmed by amino acid analysis
or by sequencing
(Creighton, supra, pp. 28-53).
2o In order to express a biologically active TRICH, the polynucleotides
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
polynucleotides encoding
TRICH. Such elements may vary in their strength and specificity. Specific
initiation signals may also
be used to achieve more efficient translation of polynucleotides encoding
TRICH. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where a
polynucleotide sequence 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
57
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
origins, both natural and synthetic. The efficiency of expression may be
enhanced by the inclusion of
enhaucers appropriate for the particular host cell system used (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 polynucleotides encoding TRICH and appropriate
transcriptional and translational
control elements. These methods include in vitro recombinant DNA techniques,
synthetic techniques,
and itZ vivo genetic recombination (Sambrook, J. et al. (1989) Molecular
Cloning, A Laboratory
Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel
et al., supra, ch. 1, 3,
and 15).
A variety of expression vector/host systems may be utilized to contain and
express
polynucleotides 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 (Sambrook, supra;
Ausubel et al., 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 Technolo~y
(1992) McGraw Hill, New York NY, pp. 191-196; Logan, J. and T. Shenk (1984)
Proc. Natl. Acad.
Sci. USA 81:3655-3659; 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 polynucleotides to the targeted organ,
tissue, or cell population
(Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5:350-356; Yu, M. et al. (1993)
Proc. Natl. Acad. Sci.
USA 90:6340-6344; Buller, R.M. et al. (1985) Nature 317:813-815; McGregor,
D.P. et al. (1994) Mol.
T_mmunol. 31:219-226; 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 polynucleotides encoding TRICH. For example, routine
cloning, subcloning,
and propagation of polynucleotides encoding TRICH can be achieved using a
multifunctional E. eoli
vector such as PBLUESCRIPT (Stratagem, La Jolla CA) or PSPORT1 plasmid
(Invitrogen).
Ligation of polynucleotides encoding TRICH into the vector's multiple cloning
site disrupts the lacZ
58
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
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 iri the cloned
sequence (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 Saccharomyces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable integration
of foreign polynucleotide sequences into the host genome for stable
propagation (Ausubel et al.,
supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; Scorer, C.A.
et al. (1994)
Bio/Technology 12:181-184).
Plant systems may also be used for expression of TRICH. Transcription of
polynucleotides
encoding TRICH may be driven by viral promoters, e.g., the 35S and 19S
promoters of CaMV used
alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Brogue,
R. et al. (1984)
Science 224:838-843; Winter, J. et al. (1991) Results Probl. Cell Differ.
17:85-105). These constructs
can be introduced into plant cells by direct DNA transformation or pathogen-
mediated transfection
(The McGraw Hill Yearbook of Science and TechnoloQV (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, polynucleotides 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 (Logan, J. and T. Shenk
(1984) Proc. Natl. Acad.
Sci. USA 81:3655-3659). In addition, transcription enhancers, such as the Rous
sarcoma virus (RSV)
enhancer, may be used to increase expression in mammalian host cells. SV40 or
EBV based vectors
may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
59
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
DNA than can be contained in and expressed from a plasmid. HA.Cs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino polymers,
or vesicles) for therapeutic purposes (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, polynucleotides 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 rnay 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 apt cells, respectively
(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; rieo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively (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 (Hartman, S.C. and R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA
85:8047-8051). Visible markers, e.g., anthocyanins, green fluorescent proteins
(GFP; Clontech), ~3-
glucuronidase and its substrate (3-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 (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 polynucleotides 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
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
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 polynucleotide 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
(Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN, Sect.
1V; Coligan, J.E. et al. (1997) Current Protocols in Itnmunolo~y, Greene Pub.
Associates and Wiley-
Interscience, New York NY; Pound, J.D. (1998) Tmmunochemical 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, polynucleotides 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
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
Biosciences, 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 polynucleotides 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
61
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
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 polynucleotides or to process the expressed protein in the desired
fashion. Such modifications
of the polypeptide include, but are not limited to, acetylation,
carboxylation, glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro" or
"pro" form of the protein may also be used to specify protein targeting,
folding, and/or activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
l0 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
polynucleotides
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
2o 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 hemagglutinir (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 TR.ICH encoding sequence and the heterologous protein
sequence, so that
TRICH may be cleaved away from the heterologous moiety following purification.
Methods for
fusion protein expression and purification are discussed in Ausubel et al.
(supra, ch. 10 and 16). A
variety of commercially available kits may also be used to facilitate
expression and purification of
3o fusion proteins.
In another embodiment, synthesis of radiolabeled TRICH may be achieved in
vitro using the
TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These
systems couple
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CA 02458625 2004-02-16
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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-metluonine.
TRICH, fragments of TRICH, or variants of TRICH may be used to screen for
compounds
that specifically bind to TRICH.. One or more test compounds may be screened
for specific binding to
TRICH. In various embodiments, 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test
compounds can be screened
for specific binding to TRICH. Examples of test compounds can include
antibodies, anticalins,
oligonucleotides, proteins (e.g., ligands or receptors), or small molecules.
In related embodiments, variants of TRICH can be used to screen for binding of
test
compounds, such as antibodies, to TRICH, a variant of T1ZICH, or a combination
of TRICH and/or
one or more variants TRICH. In an embodiment, a variant of TRICH can be used
to screen for
compounds that bind to a variant of TRICH, but not to TRICH having the exact
sequence of a
sequence of SEQ LD N0:1-26. TRICH variants used to perform such screening can
have a range of
about 50% to about 99% sequence identity to T1ZICH, with various embodiments
having 60%, 70%,
75%o, 80%, 85%, 90%, and 95% sequence identity.
In an embodiment, a compound identified in a screen for specific binding to
TRICH can be
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 (Coligan, J.E.
et al. (1991) C~xrrent
Protocols in hnmunolo~y 1(2):Chapter 5). In another embodiment, the compound
thus identified can
be a natural ligand of a receptor TRICH (Howard, A.D. et al. (2001) Trends
Pharmacol. Sci.22:132-
140; Wise, A. et al. (2002) Drug Discovery Today 7:235-246).
In other embodiments, a compound identified in a screen for specific binding
to TRICH can be
closely related to the natural receptor to which TRICH binds, at least a
fragment of the receptor, or a
fragment of the receptor including all or a portion of the ligand binding site
or binding pocket. For
example, the compound may be a receptor for T1ZICH which is capable of
propagating a signal, or a
decoy receptor for TRICH which is not capable of propagating a signal
(Ashkenazi, A. and V.M.
Divit (1999) Curr. Opin. Cell Biol. 11:255-260; Mantovani, A. et al. (2001)
Trends Tmmunol. 22:328-
336). The compound can be rationally designed using known techniques. Examples
of such
techniques include those used to construct the compound etanercept (ENBREL;
Amgen Inc.,
Thousand Oaks CA), which is efficacious for treating rheumatoid arthritis in
humans. Etanercept is
an engineered p75 tumor necrosis factor (TNF) receptor dimer linked to the Fc
portion of human IgGI
(Taylor, P.C. et al. (2001) Curr. Opin. Tmmunol. 13:611-616).
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rr-mro rv.i
In one embodiment, two or more antibodies having similar or, alternatively,
different
specificities can be screened for specific binding to TRICH, fragments of
TRICH, or variants of
TRICH.~ The binding specificity of the antibodies thus screened can thereby be
selected to identify
particular fragments or variants of TRICH. In one embodiment, an antibody can
be selected such that
its binding specificity allows for preferential identification of specific
fragments or variants of TRICH.
In another embodiment, an antibody can be selected such that its binding
specificity allows for
preferential diagnosis of a specific disease or condition having increased,
decreased, or otherwise
abnormal production of TRICH.
In an embodiment, anticalins can be screened for specific binding to TRICH,
fragments of
TRICH, or variants of TRICH. Anticalins are ligand-binding proteins that have
been constructed
based on a lipocalin scaffold (Weiss, G.A. and H.B. Lowman (2000) Chem. Biol.
7:8177-8184;
Skerra, A. (2001) J. Biotechnol. 74:257-275). The protein architecture of
lipocalins can include a
beta-barrel having eight antiparallel beta-strands, which supports four loops
at its open end. These
loops form the natural ligand-binding site of the lipocalins, a site which can
be re-engineered in vitro
by amino acid substitutions to impart novel binding specificities. The amino
acid substitutions can be
made using methods known in the art or described herein, and can include
conservative substitutions
(e.g., substitutions that do not alter binding specificity) or substitutions
that modestly, moderately, or
significantly alter binding specificity.
In one embodiment, screening for compounds which specifically bind to,
stimulate, or inhibit
TRICH 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.
An assay canbe used to assess the ability of a compound to bind to its natural
ligand andlor to
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inhibit the binding of its natural ligand to its natural receptors. Examples
of such assays include radio-
labeling assays such as those described in U.S. Patent No. 5,914,236 and U.S.
Patent No. 6,372,724.
In a related embodiment, one or more amino acid substitutions can be
introduced into a polypeptide
compound (such as a receptor) to improve or alter its ability to bind to its
natural ligands (Matthews,
D.J. and J.A. Wells. (1994) Chem. Biol. 1:25-30). In another related
embodiment, one or more amino
acid substitutions can be introduced into a polypeptide compound (such as a
ligand) to improve or alter
its ability to bind to its natural receptors (Cunningham, B.C. and J.A. Wells
(1991) Proc. Natl. Acad.
Sci. USA 88:3407-3411; Lowman, H.B. et al. (1991) J. Biol. Chem. 266:10982-
10988).
TRICH, fragments of TRICH, or variants of TRICH 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
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). Fox 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) Clip. 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
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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 itt 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
convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu.
Rev. 4:55-74).
THERAPEUTTCS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists between
regions of TRICH and transporters and ion channels. The expression of TRICH is
closely associated
with normal heart tissue, liver tumor tissue and diseased corpus callosum
tissue. In addition, examples
of tissues expressing TRICH can be found in Table 6 and can also be found in
Example XI.
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 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,
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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,
atnyotrophic 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, priors 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, postherpetic
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
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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, seleroderma, 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 heltninthic 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
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a disorder associated with decreased expression or activity of TRICH
including, but not limited to,
those provided above.
In still auother 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. Iu. 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 au 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 protein, agonist, antagonist, antibody,
complementary sequence, or
vector embodiments may be administered in combination with other appropriate
therapeutic agents.
Selection of the appropriate agents for use in combination therapy may be made
by one of ordinary
skill in the art, according to conventional pharmaceutical principles. The
combination of therapeutic
agents may act synergistically to effect the treatment or prevention of the
various disorders described
above. Using this approach, one may be able to achieve therapeutic efficacy
with lower dosages of
each agent, thus reducing the potential for adverse side effects.
An antagonist of 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.
Biotechnol. 74:277-302).
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, camels,
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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 (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al.
(1985) J. Tmmunol. Methods
81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030;
Cole, S.P. et al. (1984)
Mol. Cell Biol. 62:109-120).
In addition, techniques developed fox 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 (Morrison, S.L. et
al. (1984) Proc. Natl. Acad.
Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608;
Takeda, S. et al. (1985)
Nature 314:4.52-454). 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 (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 (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-
3837; Winter, G. et al.
(1991) Nature 349:293-299).
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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~z 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
(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, Ka, 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 Ka 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 1012 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
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/mla preferably 5-10 mg
specific antibody/ml, is generally employed in procedures requiring
precipitation of TRICH-antibody
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complexes. Procedures for evaluating antibody specificity, titer, and avidity,
and guidelines for
antibody quality and usage in various applications, are generally available
(Catty, supra; Coligan et al.,
supra).
In another embodiment of the invention, 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 (Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press, 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 (Slater, J.E. et
al. (1998) J. Allergy Clip. Itnmunol. 102:469-475; Scanlon, K.J. et al. (1995)
9:1288-1296). Antisense
sequences can also be introduced intracellularly through the use of viral
vectors, such as retrovirus and
adeno-associated virus vectors (Miller, A.D. (1990) Blood 76:271; Ausubel et
al., supra; Uckert, W.
and W. Walther (1994) Pharmacol. Ther. 63:323-347). Other gene delivery
mechanisms include
liposome-derived systems, artificial viral envelopes, and other systems known
in the art (Rossi, J.J.
(1995) Br. Med. Bull. 51:217-225; Boado, R.J. et al. (1998) J. Pharm. Sci.
87:1308-1315; Morris,
M.C. et al. (1997) Nucleic Acids Res. 25:2730-2736).
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 (SC117)-X1 disease
characterized by X-
linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672),
severe combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-
242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
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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
Trypanosoma 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
(Iuvitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La
Jolla CA),
2o 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.
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 LIPll7
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
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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. Aced. 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 (Range, 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;
Range, U. et al. (1998)
Proc. Natl. Aced. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
In an embodiment, 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
(Csete, M.E. et al. (1995) Trausplantation 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.
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Rev. Nutr. 19:511-544) and Verma, LM. and N. Somia (1997; Nature 18:389:239-
242).
In another embodiment, 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. 5804,413 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). 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 embodiment, 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) C~rr. 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
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
CA 02458625 2004-02-16
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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 (Gee, J.E. et al. (1994) in Huber, B.E. and
B.I. Carr, Molecular and
T_m_m__unolog_lc Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-177). A
complementary
sequence or antisense molecule may also be designed to block trauslation 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 RNA molecules encbding 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 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 trauscription of DNA
molecules encoding
TRICH. Such DNA sequences may be incorporated into a wide variety of vectors
with suitable RNA
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polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs
that synthesize
complementary RNA, constitutively or inducibly, can be introduced into cell
lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3' ends
of the molecule, or the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages
within the backbone of the molecule. This concept is inherent in the
production of PNAs and can be
extended in all of these molecules by the inclusion of nontxaditional 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 TRICH is exposed to at least one test compound thus
obtained. The sample
3o 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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CA 02458625 2004-02-16
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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 carried out, for example, using a Schizosaccharomyces pombe gene
expression system
(Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000)
Nucleic Acids Res.
28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000)
Biochem. Biophys. Res.
1o 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 (Goldman, C.K. et al. (1997)
Nat. Biotechnol. 15:462-
466).
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
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~ton'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,
~s
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sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
case of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of fast-
s 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, liposo~ne
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
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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 xange depending upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
The exact dosage will be.determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and response
to therapy. Long-acting compositions may be administered every 3 to 4 days,
every week, or
biweekly depending on the half life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about 0.1 ,ug to 100,000 fig, up to a
total dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind 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 sarr~e 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
so
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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, polynucleotides encoding TRICH may be
used for
diagnostic purposes. The polynucleotides which may be used include
oligonucleotides, 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
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
polynucleotides,
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 S'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 m
N0:27-52 or from
genomic sequences including promoters, enhancers, and introns of the TRICH
gene.
Means for producing specific hybridization probes for polynucleotides encoding
TRICH
include the cloning of polynucleotides 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.
Polynucleotides 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
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neoplasms, prostate cancer, cardiac disorders associated with transport, e.g.,
angina, bradyarrythmia,
tachyatTyrhinia, 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,
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, priors 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, postherpetic
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
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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 (AIDS),
Addison's disease,
adult respiratory distress syndrome, allergies, ankylosing spondylitis,
amyloidosis, anemia, asthma,
atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis,
autoimmune
polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis,
cholecystitis, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema, episodic
lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum,
atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis,
hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's
syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial,
fungal, parasitic, protozoal, and hehninthic 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 vets,
psoriasis, primary thrombocythemia, and cancers including adenocarcinoma,
leukemia, lymphoma,
melanoma, myeloma, sarcoma, teratocarcinorna, 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. Polynucleotides 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, polynucleotides encoding TRICH may be used in assays
that detect the
presence of associated disorders, particularly those mentioned above.
Polynucleotides complementary
to sequences encoding TRICH may be labeled by standard methods and added to a
fluid or tissue
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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 iri comparison to a
control sample then the presence of altered levels of polynucleotides 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
1o 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
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
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condition. Oligomers may also be employed under less stringent conditions for
detection or
quantification of closely related DNA or RNA sequences.
Iu a particular aspect, oligonucleotide primers derived from polynucleotides
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 polynucleotides
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 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 5-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
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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 (Melby, P.C. et al. (1993) J. Tmmunol. Methods 159:235-244;
Duplaa, C. et al. (1993)
Anal. Biochem. 212:229- 236). The speed of quantitation of multiple samples
maybe 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
polynucleotides 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
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 (Seilhamer et al., "Comparative Gene Transcript Analysis," U.S.
Patent No. 5,840,484;
hereby 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
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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). If a test compound has a signature similar to
that of a compound
with known toxicity, it is likely to share those toxic properties. These
fingerprints or signatures are
most useful and refined when they contain expression information from a large
number of genes and
gene families. Ideally, a genome-wide measurement of expression provides the
highest quality
signature. Even genes whose expression is not altered by any tested compounds
are important as
well, as the levels of expression of these genes are used to normalize the
rest of the expression data.
The normalization procedure is useful for comparison of expression data after
treatment with different
compounds. While the assignment of gene function to elements of a toxicant
signature aids in
interpretation of toxicity mechanisms, knowledge of gene function is not
necessary for the statistical
matching of signatures which leads. to prediction of toxicity (see, for
example, Press Release 00-02
from the National Institute of Environmental Health Sciences, released
February 29, 2000, available at
http://www.niehs.nih.gov/oc/news/toxchip.htm). Therefore, it is important and
desirable in
toxicological screening using toxicant signatures to include all expressed
gene sequences.
In an embodiment, the toxicity of a test compound can be 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 embodiment relates to the use of the polypeptides disclosed herein to
analyze the
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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
S profile of a cell's proteome may thus.be generated by separating and
analyzing the polypeptides of a
particular tissue or cell type. In one embodiment, the separation is achieved
using two-dimensional gel
electrophoresis, in which proteins from a sample are separated by isoelectric
focusing in the first ,
dimension, and then according to molecular weight by sodium dodecyl sulfate
slab gel electrophoresis
in the second dimension (Steiner and Anderson, supra). The proteins are
visualized in the gel as
discrete and uniquely positioned spots, typically by staining the gel with an
agent such as Coomassie
Blue or silver or-fluorescent stains. The optical density of each protein spot
is generally proportional to
the level of the protein in the sample. The optical densities of equivalently
positioned protein spots
from different samples, for example, from biological samples either treated or
untreated with a test
compound or therapeutic, agent, are compared to identify any changes in
protein spot density related to
the treatment. The proteins in the spots are partially sequenced using, for
example, standard methods
employing chemical or enzymatic cleavage followed by mass spectrometry. The
identity of the protein
in a spot may be determined by comparing its partial sequence, preferably of
at least 5 contiguous
amino acid residues, to the polypeptide sequences of interest. 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
25' 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 maybe
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
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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
to 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
(Brennan,
T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc.
Natl. Acad. Sci. USA
93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalon, D. et al. (1995)
2o PCT application WO95/35505; Heller, R.A. et al. (1997) Proc. Natl. Acad.
Sci. USA 94:2150-2155;
Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662). Various types of
microarrays are well known
and thoroughly described in Schena, M., ed. (1999; DNA Microarrays: A
Practical Approach, Oxford
University Press, London).
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 (PACs), bacterial artificial chromosomes
(BACs), bacterial P1
constructions, or single chromosome cDNA libraries (Harrington, J.J. et al.
(1997) Nat. Genet. 15:345-
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355; Price, C.M. (1993) Blood Rev. 7:127-134; Trask, B.J. (1991) Trends Genet.
7:149-154). Once
mapped, the nucleic acid sequences 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) (Lander, E.S. and D. Botstein
(1986) Proc. Natl.
Acad. Sci. LTSA 83:7353-7357).
Fluorescent iti situ hybridization (FISH) may be correlated with other
physical and genetic
map data (Heinz-Ulrich, et al. (1995) in Meyers, supf-a, 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 11q22-23, any
sequences mapping to that area may represent associated or regulatory genes
for further investigation
(Gatti, R.A. et al. (1988) Nature 336:577-580). The nucleotide sequence of the
instant invention may
also be used to detect differences~in the chromosomal location due to
translocation, inversion, etc.,
among normal, carrier, or affected individuals.
In another embodiment of the invention, 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 (Geysen, et al.
(1984) PCT application
WO84/03564). 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
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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.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following preferred specific
embodiments are, therefore, to be construed as merely illustrative, and not
limitative of the remainder
of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below,
2o including U.S. Ser. No. 60/313,242, U.S. Ser. No. 60/324,782, U.S. Ser. No.
60/328,184, U.S. Ser.
No. 60/345,937, U.S. Ser. No. 60/335,698, U.S. Ser. No. 60/332,804, U.S. Ser.
No. 60/333,922, U.S.
Ser. No. 60/388,180, U.S. Ser. No. 60/375,637, and U.S. Ser. No. 60/377,444,
are hereby expressly
incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries
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 (Invitrogen), a monophasic solution of phenol and
guanidine
isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or
extracted with
chloroform. RNA was precipitated from the lysates with either isopropanol or
sodium acetate and
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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 (Invitrogen),
using the
recommended procedures or similar methods known in the art (Ausubel et al.,
supf-a, ch. 5). 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
S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham
Biosciences) or preparative agarose gel electrophoresis. cDNAs were ligated
into compatible
restriction enzyme sites of the polylinker of a suitable plasmid, e.g.,
PBLUESCRIPT plasmid
(Stratagene), PSPORT1 plasmid (Invitrogen), 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-BlueMRF, or SOLR from Stratagene or DHSa, DH10B, or ElectroMAX
DH10B
from Invitrogen.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo
excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega); an
AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid,
QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
R.E.A.L. PREP
96 plasmid purification kit from QIAGEN. Following precipitation, plasmids
were resuspended in 0.1
ml of distilled water and stored, with or without lyophilization, at
4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
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high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
3 84-well plates, and the concentration of amplified plasmid DNA was
quantified fluorometrically using
PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence
scanner
(Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation such
as the ABI CATALYST 800 (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 Biosciences 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 carried out using the MEGABACE 1000 DNA sequencing system (Amersham
Biosciences);
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 (Ausubel et al.,
supra, ch. 7). Some of the cDNA sequences were selected for extension using
the techniques
disclosed in Example V7JI.
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 nofvegicus, Mus rnusculus, Caeraor-ltabditis elegans,
Sacchar~omyces cer~evisiae,
Schizosacchar~omyces pombe, and Can.dida albicans (Incyte Genomics, Palo Alto
CA); hidden
Markov model (HMM)-based protein family databases such as PFAM, INCY, and
TIGRFAM (Haft,
D.H. et al. (2001) Nucleic Acids Res. 29:41-43); and FEVIM-based protein
domain databases such as
SMART (Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864;
Letunic, I. et al. (2002)
Nucleic Acids Res. 30:242-244). (HMM is a probabilistic approach which
analyzes consensus
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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
IllVIMER. 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 1V 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 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 (IITZM)-based protein family databases
such as PFAM,
INCY, and TIGRFAM; and I~~lM based protein domain databases such as SMART.
Full length
polynucleotide sequences are also analyzed using MACDNASIS PRO software
(MiraiBio, Alameda
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
Iucyte 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
N0:27-52. 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
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Putative transporters and ion channels were initially identified by running
the Genscan gene
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 (Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94;
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 chancels, the encoded polypeptides were
analyzed by querying
against PFAM models for transporters and ion channels. Potential transporters
and ion channels were
also identified by homology to Incyte cDNA sequences that had been annotated
as transporters and
ion chancels. 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
con~trm 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 llI. 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 1V. 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 progranuriing 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
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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
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 proteinhomolog.
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 TRICII Encoding Polynucleotides
The sequences which were used to assemble SEQ ID N0:27-52 were compared with
sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ ID N0:27-52 were assembled into clusters of contiguous and overlapping
sequences using
3o 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
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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 au interval, in centiMorgans, is measured relative to the terminus
of the chromosome's p-
arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb)
of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM distances
are based on genetic markers mapped by 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.govlgenemap~, 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 (Sambrook, supra, ch. 7;
Ausubel et al., supt-a,
ch. 4).
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in databases such as GenBank or L1FESEQ (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
5 x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. The product score is a normalized value between
0 and 100, and is
calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
product is divided by (5 times the length of the shorter of the two
sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by
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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.
Alternatively, polynucleotides 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 polynucleotides are 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
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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 Mga+, (NH4)aS04,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE
enzyme
(Invitrogen), and Pfu DNA polymerase (Stratagene), with the following
parameters for~primer pair
PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step
3: 60°C, 1 min; Step 4: 68 °C, 2
min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 °C, 5 min;
Step 7: storage at 4 °C. In the
alternative, the parameters for primer pair T7 and SK+ were as follows: Step
1: 94 °C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57 °C, 1 min; Step 4: 68 °C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 ~.1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 p1 of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ,u1 to 10 ,u1 aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 ~/o 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 religation into pUC 18 vector (Amersham
Biosciences). For shotgun
sequencing, the digested nucleotides were separated on low concentration (0.6
to 0.8/0) agarose gels,
fragments were excised, and agar digested with Agar ACE (Promega). Extended
clones were
religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector
(Amersham
Biosciences), treated with Pfu DNA polymerase (Stratagene) to fill-in
restriction site overhangs, and
transfected into competent E. coli cells. Transformed cells were selected on
antibiotic-containing
media, and individual colonies were picked and cultured overnight at 37
°C in 384-well plates in LB/2x
carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Biosciences) and Pfu DNA polymerase (Stxatagene) 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
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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 Biosciences) or the ABI PRISM BIGDYE
Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotides 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.
1o IX. Identification of Single Nucleotide Polymorphisms in TRICH Encoding
Polynucleotides
Common DNA sequence variants known as single nucleotide polymorphisms (SNPs)
were
identified in SEQ 117 NO:27-52 using the L1FESEQ database (Iucyte 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 aligmnent
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 were 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
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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 ID N0:27-52 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 /,tCi of
[~y-3aP] adenosine triphosphate (Amersham Biosciences), 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 Biosciences). An
aliquot containing 10'
counts per minute of the labeled probe is used in a typical membrane-based
hybridization analysis of
human genomic DNA digested with one of the following endonucleases: Ase I, Bgl
1I, 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 et al., 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, M., ed.
(1999) DNA Microarrays: A Practical Approach, Oxford University Press,
London). 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 (Schena, M. et al. (1995)
Science 270:467-470;
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Shalon, 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
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/~,l oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 unitslpl RNase inhibitor, 500 ACM dATP, 500 ~,M dGTP, 500
p,M dTTP, 40 p,M
dCTP, 40 ~,M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences). The reverse
transcription
2o reaction is performed in a 25 ml volume containing 200 ng poly(A)+ RNA with
GEMBRIGHT kits
(Incyte Genomics). Specific control poly(A)+ RNAs are synthesized by i~ 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 0.5M sodium
hydroxide and incubated for
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, 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 ~,15X
SSC/0.2% SDS.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses
primers complementary to the vector sequences flanking the cDNA insert. Array
elements are
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amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5 fig.
Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Biosciences).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water, and
coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are
cured in a 110°C
oven.
Array elements are applied to the coated glass substrate using a procedure
described in U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 ~.1 of the array
element DNA, at an average
concentration of 100 ng/~.1, is loaded into the open capillary printing
element by a lugh-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, lnc., Bedford MA) for 30 minutes at 60°
C followed by washes in 0.2%
SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 ~Cl of sample mixture consisting of 0.2 ~,g
each of Cy3 and
2o 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 cma 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
~.l 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 (1X SSC, 0.1%
SDS), three times for 10 minutes each at 45° C in a second wash buffer
(0.1X SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
lnnova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 llln 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-
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scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
'The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that location
to be correlated with a weight ratio of hybridizing species of 1:100,000. When
two samples 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 1BM-
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
Genomics). Array
elements that exhibited at least about a two-fold change in expression, a
signal-to background ratio of
at least 2.5, and an element spot size of at least 40% were identified as
differentially expressed.
Expression
For example, SEQ )D N0:30 showed differential expression in bone osteosarcoma
tissues
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versus normal osteocytes, as determined by microarray analysis. Messenger RNA
from normal
human osteoblasts was compared with mRNA from biopsy specimens, osteosarcoma
tissues, primary
cultures, or metastasized tissues. A normal osteoblast primary culture, NHOst
5488, was chosen as
the reference in the initial experiments. One basic set of experiments is
defined as the comparison of
mRNA from biopsy specimen with that of debnitive surgical specimen from the
same patient.
Extended study of this basic set includes mRNA from primary cell cultures of
the definitive surgical
specimen, muscle, or cartilage tissue from the same patient. Biopsy specimens,
definitive surgical
specimens, or lung metastatic tissues from different individuals were also
included to reveal individual
variability. °The expression of SEQ ID N0:30 was increased by at least
two-fold in bone
osteosarcoma tissues relative to normal osteocytes. Therefore, in various
embodiments, SEQ m
N0:30 can be used for one or more of the following: i) monitoring treatment of
bone cancer, ii)
diagnostic assays for bone cancer, and iii) developing therapeutics and/or
other treatments for bone
cancer.
In an alternative example, SEQ ID N0:33 showed differential expression in lung
squamous
carcinoma tissues versus normal lung tissues as determined by microarray
analysis. In matched tissue
experiments, the expression of SEQ 1D N0:33 was decreased by at least two-fold
in lung squamous
carcinoma tissues relative to grossly uninvolved normal lung tissues from the
same donors. Therefore,
in various embodiments, SEQ ID N0:33 can be used for one or more of the
following: i) monitoring
treatment of lung cancer, ii) diagnostic assays for lung cancer, and iii)
developing therapeutics and/or
other treatments for lung cancer.
SEQ ~ NO:33 also showed differential expression in ovarian adenocarcinoma
tissues versus
normal ovarian tissues as determined by microarray analysis. The expression of
SEQ )D N0:33 was
decreased by at least two-fold in ovarian adenocarcinoma tissues relative to
grossly uninvolved normal
ovarian tissues from the same donor. Therefore, in various embodiments, SEQ ID
N0:33 can be used
for one or more of the following: i) monitoring treatment of ovarian cancer,
ii) diagnostic assays for
ovarian cancer, and iii) developing therapeutics and/or other treatments for
ovarian cancer.
SEQ 117 N0:33 and SEQ ID NO:40 showed differential expression in association
with
immune and inflammatory responses as determined by microarray analysis. The
expression of SEQ
ll~ N0:33 was increased by at least two-fold in human umbilical vein cells
treated with PMA and
ionomycin relative to untreated human umbilical vein cells. Human umbilical
vein cells are derived
from the endothelium of the human umbilical vein, and have been used as an
experimental model for
investigating the functional biology of human endothelial cells in vitro. PMA
is a broad activator of
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protein kinase C-dependent pathways and ionomycin is a calcium ionophore that
permits the entry of
calcium in the cell, hence increasing the cytosofic calcium concentration. The
expression of SEQ m
N0:40 was increased by at least 2.5-fold in vascular endothelial tissue
treated with TNFa and IL-1(3
compared with untreated vascular endothelial tissue, as determined by
microarray analysis. Human
coronary artery endothelial cells and human coronary artery smooth muscle
cells (BioWhittaker, Inc.,
San Diego CA) obtained from the same donor were cultured in tissue culture
flasks in Endothelium
Growth Medium or Smooth Muscle Growth Medium, respectively (BioWhittaker).
Cultures at 85%
confluency were either treated with recombinant human TNFoc and IL-1(3 (R&D
Systems,
Minneapolis MN) at 10 ng/ml each for 24 hours at 37° C or were left
untreated. Therefore, in various
embodiments, SEQ ID NO:33 and SEQ 177 NO:40 can each be used for one or more
of the following:
i) monitoring treatment of immune/inflammatory responses, ii) diagnostic
assays for
immune/inflammatory responses, and iii) developing therapeutics and/or other
treatments for
immune/inflammatory responses.
In an alternative example, SEQ )D N0:3 8 showed differential expression in
breast carcinoma
cell lines versus primary mammary epithelial cells as determined by microarray
analysis. The breast
carcinoma cell lines include MCF7, a breast adenocarcinoma cell line derived
from the pleural effusion
of a 69-year-old female; T-47D, a breast carcinoma cell line derived from a
pleural effusion from a
54-year-old female with an infiltrating ductal carcinoma of the breast; Sk-BR-
3, a breast
adenocarcinoma cell line isolated from a malignant pleural effusion of a 43-
year-old female; MDA-
mb-231, a metastatic breast tumor cell line derived from the pleural effusion
of a 51-year-old female
with metastatic breast carcinoma; and MDA-mb-4355, a spindle shaped strain
that evolved from a cell
line isolated from the pleural effusion of a 31 year old female with
metastatic, ductal adenocarcinoma
of the breast. The primary mammary epithelial cell line HLV>EC was derived
from normal human
mammary tissue (Clonetics, San Diego, CA). All cell cultures were propagated
in a chemically-
defined medium, according to the supplier's recommendations and grown to 70-
80% confluence prior
to RNA isolation. The microarray experiments showed that the expression of SEQ
m N0:38 was
decreased by at least two-fold iu all five breast carcinoma lines (MCF7, T-
47D, Sk-BR-3, MDA-mb-
231, and MDA-mb-4355) relative to primary mammary epithelial cells. Therefore,
in various
embodiments, SEQ m N0:3 8 can be used for one or more of the following: i)
monitoring treatment of
breast cancer, ii) diagnostic assays for breast cancer, and iii) developing
therapeutics and/or other
treatments for breast cancer.
SEQ )D NO:38 also showed differential expression in certain prostate carcinoma
cell lines
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versus normal prostate epithelial cells as determined by microarray analysis.
The prostate carcinoma
cell lines include CA-HPV-10, DU 145, LNCaP, PC-3, and MDAPCa2b. CA-HPV-7 was
derived
from cells from a 63 year old male with prostate adenocarcinoma and was
transformed by
transfection with HPV18 DNA. DU 145 was isolated from a metastatic site in the
brain of a 69 year
old male with widespread metastatic prostate carcinoma. DU 145 has no
detectable sensitivity to
hormones; forms colonies in semi-solid medium; is only weakly positive for
acid phosphatase; and cells
are negative for prostate specific antigen (PSA). LNCaP is a prostate
carcinoma cell line isolated
from a lymph node biopsy of a 50 year old male with metastatic prostate
carcinoma. LNCaP
expresses PSA, produces prostate acid phosphatase, and expresses androgen
receptors. PC-3, a
prostate adenocarcinoma cell line, was isolated from a metastatic site in the
bone of a 62 year old
male with grade 1V prostate adenocarcinoma. MDAPCa2b is a prostate
adenocarcinoma cell line
isolated from a metastatic site in the bone of a 63 year old male. The
MDAPCa2b cell line expresses
PSA and androgen receptor and is androgen sensitive. The normal epithelial
cell line, PrEC, is a
primary prostate epithelial cell line isolated from a normal donor. The
expression of SEQ ll~ N0:38
was decreased by at least two-fold in three out of five prostate carcinoma
lines (DU 145, LNCaP, and
PC-3) relative to cells from the normal prostate epithelial cell line, PrEC.
Therefore, in various
embodiments, SEQ m NO:3 8 can be used for one or more of the following: i)
monitoring treatment of
prostate cancer, ii) diagnostic assays for prostate cancer, and iii)
developing therapeutics and/or other
treatments for prostate cancer.
2o In addition, SEQ ID NO:38 and SEQ ID N0:43 showed differential expression
in toxicology
studies as determined by microarray analysis. The expression of SEQ m N0:43
was increased by at
least two-fold in C3A hepatoblastoma cells treated with 1-100 p,M
beclomethazone as compared with
untreated C3A hepatoblastoma cells. The human C3A cell line is a clonal
derivative of HepG2/C3
(hepatoma cell line, isolated from a 15-year-old male with liver tumor), which
was selected for strong
contact inhibition of growth. The use of a clonal population enhances the
reproducibility of the cells.
C3A cells have many characteristics of primary human hepatocytes in culture:
i) expression of insulin
receptor and insulin-like growth factor 1I receptor; ii) secretion of a high
ratio of serum albumin
compared with a-fetoprotein iii) conversion of ammonia to urea and glutamine;
iv) metabolism of
aromatic amino acids; and v) proliferation in glucose-free and insulin-free
medium. The C3A cell line
3o is now well established as an in vitro model of the mature human liver
(Mickelson et al. (1995)
Hepatology 22:866-875; Nagendra et al. (1997) Am J Physiol 272:6408-6416). C3A
cells were
treated with 1-100 p,M beclomethazone for lhr, 3hr, 6hr and compared with
untreated cells. In
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addition, the expression of SEQ 117 N0:34 was increased by at least two-fold
in early confluent C3A
cells treated with progesterone, beclomethasone, medroxyprogesterone,
budesonide, prednisone,
dexamethasone, or betamethasone, for 1, 3, or 6 hours, as compared to
untreated C3A cells. In
addition, the expression of SEQ 117 N0:38 was decreased by at least two-fold
in early confluent C3A
cells treated with progesterone, beclomethasone, medroxyprogesterone,
budesonide, prednisone,
dexamethasone, or betamethasone, for 1, 3, or 6 hours, as compared to
untreated C3A cells. The
effects of steroids on liver metabolism are important to the understanding of
the pharmacodynamics of
drugs. Therefore, in various embodiments, SEQ D7 N0:34, SEQ ID N0:38 and SEQ
T17 N0:43 can
each be used for one or more of the following: i) monitoring treatment of
liver toxicity, diseases and
disorders, ii) diagnostic assays for liver toxicity, diseases and disorders,
and iii) developing therapeutics
and/or other treatments for liver toxicity, diseases and disorders.
In yet another example, the expression of SEQ ID N0:48 was differentially
expressed in a
specific region of human brain tissue as compared to pooled brain tissue
control. Characterization of
region-specific gene expression in the human brain provides a context and
background for molecular
neurobiology research in general. This knowledge may provide insight into the
genetic basis of brain
structure and function. The expression of SEQ 117 N0:48 was decreased by at
least two-fold in
normal human amygdala, entorhinal cortex, brain tissue as compared to the
normal human pooled brain
tissue used as a control. These experiments indicate that, SEQ 117 N0:48
exhibited significant
differential expression patterns using microarray techniques, and further
establishes its utility as a
diagnostic marker or therapeutic agent which maybe useful in neurological
disorders. Therefore, in
various embodiments, SEQ 117 NO:48 can be used for one or more of the
following: i) monitoring
treatment of neurological disorders, ii) diagnostic assays for neurological
disorders, and iii) developing
therapeutics and/or other treatments for neurological disorders.
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
anal 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.
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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
tfp-lac (tac) hybrid
promoter and the TS or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21(DE3).
Antibiotic resistant bacteria express TR1CH 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 Autogr~aplaica califo~nica
nuclear polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding TRTCH 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 (Engelhard, E.K. et a1.
(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum.
Gene Ther. 7:1937-
1945).
In most expression systems, TR1CH 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 Sclaistosoma japonicum, enables the purification of fusion
proteins on immobilized
glutathione under conditions that maintain protein activity and antigenicity
(Amersham Biosciences).
Following purification, the GST moiety can be proteolytically cleaved from
TRICH at specifically
engineered sites. FLAG, an 8-amino acid peptide, enables immunoaf~nity
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 (QIA.GEN).
Methods for protein expression and purification are discussed in Ausubel et
al. (supt~a, ch. 10 and 16).
Purified TRICH obtained by these methods can be used directly in the assays
shown in Examples
XVII, XV)II, and XIX, where applicable.
XIV. Functional Assays
TRICH function is assessed by expressing the sequences encoding TRICH at
physiologically
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elevated levels in mammalian cell culture systems. cDNA is subcloned into a
mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice
include PCMV SPORT plasmid (Invitrogen, Carlsbad CA) and PCR3.1 plasmid
(Invitrogen), 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 ~g of an additional plasmid
containing sequences
encoding a marker proteinare co-transfected. Expression of a marker protein
provides a means to
distinguish transfected cells from nontransfected cells and is a reliable
predictor of cDNA expression
from the recombinant vector. Marker proteins of choice include, e.g., Green
Fluorescent Protein
(GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser
optics based technique, is used to identify transfected cells expressing GFP
or CD64-GFP and to
evaluate the apoptotic state of the cells and other cellular properties. FCM
detects and quantifies the
uptake of fluorescent molecules that diagnose events preceding or coincident
with cell death. These
events include changes in nuclear DNA content as measured by staining of DNA
with propidium
iodide; changes in cell size and granularity as measured by forward light
scatter and 90 degree side
light scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine
uptake; alterations in 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.
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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 (Ausubel et al., supf-a, 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
KLH (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) to
increase immunogenicity (Ausubel et al., supra). Rabbits are immunized with
the oligopeptide-I~LH
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,
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 Biosciences). 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 lzsl Bolton-
Hunter reagent
(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
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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(3~y 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 1~I Bolton-Hunter reagent. (See, e.g.,
Bolton A.E. and W.M.
Hunter (1973) Biochem. J. 133:529-539.) Candidate molecules previously arrayed
in the wells of a
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, 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 lexA, 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
are discussed in Niethammer, M. and M. Sheng (1998, Meth. Enzymol. 293:104-
122).
TRICH may also be used in the PATHCALL1NG process (C~raGen 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, I~.
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
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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 !3-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
13-galactosidase sequences alone, are used as controls and tested in parallel.
Cells expressing TRICH
will have higher anion or canon 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,
and the associated conductance.
Alternatively, ion channel activity of TRICH is measured as current flow
across a TRICH-
containing Xeuopus laevis oocyte membrane using the two-electrode voltage-
clamp technique (Ishi et
al., supf~a; 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 I~-
gluconate, 4 mM KCl,
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+2 (in the form of CaCl2), where
appropriate.
Electrode resistance is set at 2-5 MSZ 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 (82.5mM NaCl,
2.5 mM KCl, 1mM CaCl2,
1mM MgCl2, 1mM NaaHP04, 5 mM Hepes, 3.8 mM NaOH , 50p,g/ml gentamycin, pH 7.8)
to allow
expression of TRICH. Oocytes are then transferred to standard uptake medium
(100mM NaCl, 2
113
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
mM KCl, 1mM CaCl2, 1mM MgCl2, lO~mM HepeslTris pH 7.5). TJptake 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
30 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.
ATPase activity associated with TRICH can be measured by hydrolysis of
radiolabeled ATP-
[y-32P~, separation of the hydrolysis products by chromatographic methods, and
quantitation of the
recovered 3aP using a scintillation counter. The reaction mixture contains ATP-
[~y-32P] 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
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-paltmitic 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 p1 aliquot of
1 p.M TRICH in 10 mM Tris (pH 7.5), 2 mM EDTA, and 500 mM NaCl is placed in a
1 cm path
length quartz cuvette and 1 p1 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.
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
114
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
Ca2+ indicator Fluo-4 AM, sodium-sensitive dyes such as SBFI and sodium green,
or the Cl- 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.
Various modifications and variations of the described compositions, 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. It will be appreciated that the invention provides novel and
useful proteins, and their
encoding polynucleotides, which can be used in the drug discovery process, as
well as methods for
using these compositions for the detection, diagnosis, and treatment of
diseases and conditions.
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.
Nor should the description of such embodiments be considered exhaustive or
limit the invention to the
precise forms disclosed. Furthermore, elements from one embodiment can be
readily recombined with
elements from one or more other embodiments. Such combinations can form a
number of
embodiments within the scope of the invention. It is intended that the scope
of the invention be
defined by the following claims and their equivalents.
115
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
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CA 02458625 2004-02-16
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Table 5
PolynucleotideIncyte ProjectRepresentative Library
SEQ ID:
ID NO:
27 1853191CB1 PROSNOT15
28 7497369CB I~IDEUNE02
1
29 1700438CB1 BLADTUT05
30 535939CB IiEARFET01
1
31 55118067CB1LIVRTUT13
33 7500819CB CORPNOT02
1
34 7503413CB LPARNOT02
1
35 7500007CB BRAITUT02
1
36 7500025CB COLCTUT02
1
37 7502736CB BRANDINOl
1
38 7503570CB1 CARGDITOl
39 7504008CB1 THP1TXT03
40 7503559CB MCLDTXN03
1
41 6243872CB1 TESTNOT17
44 90113658CB PROSTUT04
1
45 3942766CB1 FIBRTXS07
46 7501987CB BRAENOT04
1
47 7503223CB1 PROSTUT10
48 7503566CB1 BSTMNON02
49 7505122CB SINTNOT 13
1
50 7511620CB COLNNOT23
1
51 7506995CB BRAENOT04
1
52 ~ 7506996CB BRAENOT04
1 ~
166
CA 02458625 2004-02-16
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<110> INCYTE GENOMICS, INC.
TANG, Y. Tom
LAL, Preeti G.
YUE, Henry
BAUGHN, Mariah R.
NGUYEN, Danniel B.
YAO, Monique G.
GREENE, Barrie D.
BOROWSKY, Mark L.
LEE, Sally
EMERLING, Brooke M.
XU, Yuming
BECHA, Shanya D.
GORVAD, Ann E.
AZIMZAI, Yalda
YUE, Huibin
ELLIOTT, Vicki S.
LEE, Ernestine A.
YANG, Junming
LEHR-MASON, Patricia M.
RAMKUMAR, Jayalaacmi
LEE, Soo Yeun
FARIS, Mary
TURNER, Christopher
FURNESS, Michael
BUCHBINDER, Jenny L.
WALIA, Narinder K.
LI, Joana X
FORSYTHE, Ian J.
GRIFFIN, Jennifer A.
GIETZEN, Kimberly J.
SWARNAKAR, Anita
HAFALIA, April J.A.
LINDQUIST, Erika A.
JIANG, Xin
JACKSON, Alan A.
WILSON, Amy D.
JIN, Pei
KHARE, Reena
MARQUIS, Joseph P.
<120> TRANSPORTERS AND ION CHANNELS
<130> PF-1148 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/313,242
<151> 2001-08-17
<150> 60/324,782
<151> 2001-09-21
<150> 60/328,184
<151> 2001-10-02
1/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
<150> 60/345,937
<151> 2001-10-26
<150> 60/335,698
<151> 2001-11-01
<150> 60/332,804
<151> 2001-11-13
<150> 60/333,922
<151> 2001-11-27
<150> 60/375,637
<151> 2002-04-26
<150> 60/377,444
<151> 2002-05-03
<150> 60/388,180
<151> 2002-06-11
<160> 52
<170> PERL Program
<210> 1
<211> 652
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 1853191CD1
<400> 1
Met His Cys Leu Gly Ala Glu Tyr Leu Val Ser Ala Glu Gly Ala
1 5 10 15
Pro Arg Gln Arg Glu Trp Arg Pro Gln Ile Tyr Arg Lys Cys Thr
20 25 30
Asp Thr Ala Trp Leu Phe Leu Phe Phe Leu Phe Trp Thr Gly Leu
35 40 45
Val Phe Ile Met Gly Tyr Ser Val Val Ala Gly Ala Ala Gly Arg
50 55 60
Leu Leu Phe Gly Tyr Asp Ser Phe Gly Asn Met Cys Gly Lys Lys
65 70 75
Asn Ser Pro Val Glu Gly Ala Pro Leu Ser Gly Gln Asp Met Thr
80 85 90
Leu Lys Lys His Val Phe Phe Met Asn Ser Cys Asn Leu Glu Val
95 100 105
Lys Gly Thr Gln Leu Asn Arg Met Ala Leu Cys Val Ser Asn Cys
110 115 120
Pro Glu Glu Gln Leu Asp Ser Leu Glu Glu Val Gln Phe Phe Ala
125 130 135
Asn Thr Ser Gly Ser Phe Leu Cys Val Tyr Ser Leu Asn Ser Phe
140 145 150
Asn Tyr Thr His Ser Pro Lys Ala Asp Ser Leu Cys Pro Arg Leu
155 160 165
2/65
CA 02458625 2004-02-16
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Pro Val Pro Pro Ser Lys Ser Phe Pro Leu Phe Asn Arg Cys Val
170 175 180
Pro Gln Thr Pro Glu Cys Tyr Ser Leu Phe Ala Ser Val Leu Ile
185 190 195
Asn Asp Val Asp Thr Leu His Arg Ile Leu Ser Gly Ile Met Ser
200 205 210
Gly Arg Asp Thr Ile Leu Gly Leu Cys Ile Leu Ala Leu Ala Leu
215 220 225
Ser Leu Ala Met Met Phe Thr Phe Arg Phe Ile Thr Thr Leu Leu
230 235 240
Val His Ile Phe Ile Ser Leu Val Ile Leu Gly Leu Leu Phe Val
245 250 255
Cys Gly Val Leu Trp Trp Leu Tyr Tyr Asp Tyr Thr Asn Asp Leu
260 265 270
Ser Ile Glu Leu Asp Thr Glu Arg Glu Asn Met Lys Cys Val Leu
275 280 285
Gly Phe Ala Ile Val Ser Thr Gly Ile Thr Ala Val Leu Leu Val
290 . 295 300
Leu Ile Phe Val Leu Arg Lys Arg Ile Lys Leu Thr Val Glu Leu
305 310 315
Phe Gln Ile Thr Asn Lys Ala Ile Ser Ser Ala Pro Phe Leu Leu
320 325 330
Phe Gln Pro Leu Trp Thr Phe Ala Ile Leu Ile Phe Phe Trp Val
335 340 345
Leu Trp Val Ala Val Leu Leu Ser Leu Gly Thr Ala Gly Ala Ala
350 355 360
Gln Val Met Glu Gly Gly Gln Val Glu Tyr Lys Pro Leu Ser Gly
365 370 375
Ile Arg Tyr Met Trp Ser Tyr His Leu Ile Gly Leu Ile Trp Thr
380 385 390
Ser Glu Phe Ile Leu Ala Cys Gln Gln Met Thr Ile Ala Gly Ala
395 400 405
Val Val Thr Cys Tyr Phe Asn Arg Ser Lys Asn Asp Pro Pro Asp
410 415 420
His Pro Ile Leu Ser Ser Leu Ser Ile Leu Phe Phe Tyr His Gln
425 430 435
Gly Thr Ile Val Lys Gly Ser Phe Leu Ile Ser Val Val Arg Ile
440 445 450
Pro Arg Ile Ile Val Met Tyr Met Gln Asn Ala Leu Lys Glu Gln
455 460 465
His Gly Ala Leu Ser Arg Tyr Leu Phe Arg Cys Cys Tyr Cys Cys
470 475 480
Phe Trp Cys Leu Asp Lys Tyr Leu Leu His Leu Asn Gln Asn Ala
485 490 495
Tyr Thr Thr Thr Ala Ile Asn Gly Thr Asp Phe Cys Thr Ser Ala
500 505 510
Lys Asp Ala Phe Lys Ile Leu Ser Lys Asn Ser Ser His Phe Thr
515 520 525
Ser Ile Asn Cys Phe Gly Asp Phe Ile Ile Phe Leu Gly Lys Val
530 535 540
Leu Val Val Cys Phe Thr Val Phe Gly Gly Leu Met Ala Phe Asn
545 550 555
Tyr Asn Arg Ala Phe Gln Val Trp Ala Val Pro Leu Leu Leu Val
560 565 570
Ala Phe Phe Ala Tyr Leu Val Ala His Ser Phe Leu Ser Val Phe
575 580 585
3/65
CA 02458625 2004-02-16
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Glu Thr Val Leu Asp Ala Leu Phe Leu Cys Phe Ala Val Asp Leu
590 595 600
Glu Thr Asn Asp Gly Ser Ser Glu Lys Pro Tyr Phe Met Asp G1n
605 610 615
Glu Phe Leu Ser Phe Val Lys Arg Ser Asn Lys Leu Asn Asn Ala
620 625 630
Arg Ala Gln Gln Asp Lys His Ser Leu Arg Asn Glu Glu Gly Thr
635 640 645
Glu Leu Gln Ala Ile Val Arg
650
<210> 2
<211> 345
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7497369CD1
<400> 2
Met Pro Gly Lys Arg Met Gly Tyr Arg Val Thr Glu Asn Tyr Ser
1 5 10 15
Asp Val Leu Pro Leu Ser Leu Gly Val Gly Trp Gly Pro Trp Pro
20 25 30
Leu Pro Leu Leu Pro Gly Gly Gly Gly Gly Ser Arg Gly His Pro
35 40 45
His Gln Arg Cys Arg Phe Pro Ala Leu Phe Pro Glu Ser Pro Cys
50 55 60
Trp Leu Leu Ala Thr Gly Gln Val Ala Arg Ala Arg Lys Ile Leu
65 70 75
Trp Arg Phe Ala Glu Ala Ser Gly Val Gly Pro Gly Asp Ser Pro
80 85 90
Leu Glu Glu Asn Ser Leu Ala Thr Glu Leu Thr Met Leu Ser Ala
95 100 105
Arg Ser Pro Gln Pro Arg Tyr His Ser Pro Leu Gly Leu Leu Arg
110 115 120
Thr Arg Val Thr Trp Arg Asn Gly Leu Ile Leu Gly Phe Ser Ser
125 130 135
Leu Val Gly Gly Gly Ile Arg Ala Ser Phe Arg Arg Ser Leu Ala
140 145 150
Pro Gln Val Pro Thr Phe Tyr Leu Pro Tyr Phe Leu Glu Ala Gly
155 160 165
Leu Glu Ala Ala Ala Leu Val Phe Leu Leu Leu Thr Ala Asp Cys
170 175 180
Cys Gly Arg Arg Pro Val Leu Leu Leu Gly Thr Met Val Thr Gly
185 190 195
Leu Ala Ser Leu Leu Leu Leu Ala Gly Ala Gln Tyr Leu Pro Gly
200 205 210
Trp Thr Val Leu Phe Leu Ser Val Leu Gly Leu Leu Ala Ser Arg
215 220 225
Ala Val Ser Ala Leu Ser Ser Leu Phe Ala Ala Glu Val Phe Pro
230 235 240
Thr Val Ile Arg Gly Ala Gly Leu Gly Leu Val Leu Gly Ala Gly
245 250 255
Phe Leu Gly Gln Ala Ala Gly Pro Leu Asp Thr Leu His Gly Arg
4/65
CA 02458625 2004-02-16
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260 265 270
Gln Gly Phe Phe Leu Gln Gln Val Val Phe Ala Ser Leu Ala Val
275 280 285
Leu Ala Leu Leu Cys Val Leu Leu Leu Pro Glu Ser Arg Ser Arg
290 295 300
Gly Leu Pro Gln Ser Leu Gln Asp Ala Asp Arg Leu Arg Arg Ser
305 310 315
Pro Leu Leu Arg Gly Arg Pro Arg Gln Asp His Leu Pro Leu Leu
320 325 330
Pro Pro Ser Asn Ser Tyr Trp Ala Gly His Thr Pro Glu Gln His
335 340 345
<210> 3
<211> 150
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1700438CD1
<400> 3
Met Gln Arg Leu Leu Cys Met Cys His Glu Gly Trp Glu Ala Tyr
1 5 10 15
Cys Arg Gln Met Val Phe Leu Ala Gly Leu Cys Leu Val Phe Leu
20 25 30
Tyr Met Thr Val Leu Gly Ser Gly Gly Ile Ile Thr Gly Tyr Ala
35 40 45
Cys Thr Gln Gly Val Gly Asp Ser Leu Leu Ser Ile Leu Thr Ala
50 55 60
Leu Ser Ala Leu Ser Gly Leu Met Gly Thr Val Leu Phe Thr Gln
65 70 75
Leu Arg Gly His Tyr Gly Leu Val Thr Thr Gly Val Ile Ser Ser
80 85 90
Gln Leu His Leu Gly Cys Leu Met Leu Cys Met Phe Ser Val Leu
95 100 105
Ala Pro Gly Asn Ser Phe Asp Leu Ala Val Phe Ser Leu Pro Leu
110 115 ~ 120
Ser Lys Asn Pro Ser Asn Tyr Glu Leu Leu Val Gln Trp Met Glu
125 130 135
Glu Gln Ser Arg Gly Met Ala Trp Phe Arg Phe Leu Ser Lys Gly
140 145 150
<210> 4
<211> 537
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 535939CD1
<400> 4
Met Gly Asp Glu Asp Lys Arg Ile Thr Tyr Glu Asp Ser Glu Pro
5/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
1 5 10 15
Ser Thr Gly Met Asn Tyr Thr Pro Ser Met His Gln Glu Ala Gln
20 25 30
Glu Glu Thr Val Met Lys Leu Lys Gly Ile Asp Ala Asn Glu Pro
35 40 45
Thr Glu Gly Ser Ile Leu Leu Lys Ser Ser Glu Lys Lys Leu Gln
50 55 60
Glu Thr Pro Thr Glu Ala Asn His Val Gln Arg Leu Arg Gln Met
65 70 75
Leu Ala Cys Pro Pro His Gly Leu Leu Asp Arg Val Ile Thr Asn
80 85 90
Val Thr Ile Ile Val Leu Leu Trp Ala Val Val Trp Ser Ile Thr
95 100 105
Gly Ser Glu Cys Leu Pro Gly Gly Asn Leu Phe Gly Ile Ile Ile
110 115 120
Leu Phe Tyr Cys Ala Ile Ile Gly Gly Lys Leu Leu Gly Leu Ile
125 130 135
Lys Leu Pro Thr Leu Pro Pro Leu Pro Ser Leu Leu Gly Met Leu
140 145 150
Leu Ala Gly Phe Leu Ile Arg Asn Ile Pro Val Ile Asn Asp Asn
155 160 165
Val Gln Ile Lys His Lys Trp Ser Ser Ser Leu Arg Ser Ile Ala
170 175 180
Leu Ser Ile Ile Leu Val Arg Ala Gly Leu Gly Leu Asp Ser Lys
185 190 195
Ala Leu Lys Lys Leu Lys Gly Val Cys Val Arg Leu Ser Met Gly
200 205 210
Pro Cys Ile Val Glu Ala Cys Thr Ser Ala Leu Leu Ala His Tyr
215 220 225
Leu Leu Gly Leu Pro Trp Gln Trp Gly Phe Ile Leu Gly Phe Val
230 235 240
Leu Gly Ala Val Ser Pro Ala Val Val Val Pro Ser Met Leu Leu
245 250 255
Leu Gln Gly Gly Gly Tyr Gly Val Glu Lys Gly Val Pro Thr Leu
260 265 270
Leu Met Ala Ala Gly Ser Phe Asp Asp Ile Leu Ala Ile Thr Gly
275 280 285
Phe Asn Thr Cys Leu Gly Ile Ala Phe Ser Thr Gly Ser Thr Val
290 295 300
Phe Asn Val Leu Arg Gly Val Leu Glu Val Val Ile Gly Val Ala
305 310 315
Thr Gly Ser Val Leu Gly Phe Phe Ile Gln Tyr Phe Pro Ser Arg
320 325 330
Asp Gln Asp Lys Leu Val Cys Lys Arg Thr Phe Leu Val Leu Gly
335 340 345
Leu Ser Val Leu Ala Val Phe Ser Ser Val His Phe Gly Phe Pro
350 355 360
Gly Ser Gly Gly Leu Cys Thr Leu Val Met Ala Phe Leu Ala Gly
365 370 375
Met Gly Trp Thr Ser Glu Lys Ala Glu Val Glu Lys Ile Ile Ala
380 385 390
Val Ala Trp Asp Ile Phe Gln Pro Leu Leu Phe Gly Leu Ile Gly
395 400 405
Ala Glu Val Ser Ile Ala Ser Leu Arg Pro Glu Thr Val Gly Leu
410 415 420
Cys Val Ala Thr Val Gly Ile Ala Val Leu Ile Arg Ile Leu Thr
6/65
CA 02458625 2004-02-16
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425 430 435
Thr Phe Leu Met Val Cys Phe Ala Gly Phe Asn Leu Lys Glu Lys
440 445 450
Ile Phe Ile Ser Phe Ala Trp Leu Pro Lys Ala Thr Val Gln Ala
455 460 465
Ala Ile Gly Ser Val Ala Leu Asp Thr Ala Arg Ser His Gly Glu
470 475 480
Lys Gln Leu Glu Asp Tyr Gly Met Asp Val Leu Thr Val Ala Phe
485 490 495
Leu Ser Ile Leu Ile Thr Ala Pro Ile Gly Ser Leu Leu Ile Gly
500 505 510
Leu Leu Gly Pro Arg Leu Leu Gln Lys Val Glu His Gln Asn Lys
515 520 525
Asp Glu Glu Val Gln Gly Glu Thr Ser Val Gln Val
530 535
<210> 5
<211> 1119
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 55118067CD1
<400> 5
Met Thr Ala Ala Ala Ala Ser Asn Trp' Gly Leu Ile Thr Asn Ile
1 5 10 15
Val Asn Ser Ile Val Gly Val Ser Val Leu Thr Met Pro Phe Cys
20 25 30
Phe Lys Gln Cys Gly Ile Val Leu Gly Ala Leu Leu Leu Val Phe
35 40 45
Cys Ser Trp Met Thr His Gln Ser Cys Met Phe Leu Val Lys Ser
50 55 60
Ala Ser Leu Ser Lys Arg Arg Thr Tyr Ala Gly Leu Ala Phe His
65 70 75
Ala Tyr Gly Lys Ala Gly Lys Met Leu Val Glu Thr Ser Met Ile
80 85 90
Gly Leu Met Leu Gly Thr Cys Ile Ala Phe Tyr Val Val Ile Gly
95 100 105
Asp Leu Gly Ser Asn Phe Phe Ala Arg Leu Phe Gly Phe Gln Val
110 115 120
Gly Gly Thr Phe Arg Met Phe Leu Leu Phe Ala Val Ser Leu Cys
125 130 135
Ile Val Leu Pro Leu Ser Leu Gln Arg Asn Met Met Ala Ser Ile
140 145 150
Gln Ser Phe Ser Ala Met Ala Leu Leu Phe Tyr Thr Val Phe Met
155 160 165
Phe Val Ile Val Leu Ser Ser Leu Lys His Gly Leu Phe Ser Gly
170 175 180
Gln Trp Leu Arg Arg Val Ser Tyr Val Arg Trp Glu Gly Val Phe
185 190 195
Arg Cys Ile Pro Ile Phe Gly Met Ser Phe Ala Cys Gln Ser Gln
200 205 210
Val Leu Pro Thr Tyr Asp Ser Leu Asp Glu Pro Ser Val Lys Thr
215 220 225
7/65
CA 02458625 2004-02-16
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Met Ser Ser Ile Phe Ala Ser Ser Leu Asn Val Val Thr Thr Phe
230 235 240
Tyr Val Met Val Gly Phe Phe Gly Tyr Val Ser Phe Thr Glu Ala
245 250 255
Thr Ala Gly Asn Val Leu Met His Phe Pro Ser Asn Leu Val Thr
260 265 270
Glu Met Leu Arg Val Gly Phe Met Met Ser Val Ala Val Gly Phe
275 280 285
Pro Met Met Ile Leu Pro Cys Arg Gln Ala Leu Ser Thr Leu Leu
290 295 300
Cys Glu Gln Gln Gln Lys Asp Gly Thr Phe Ala Ala Gly Gly Tyr
305 310 315
Met Pro Pro Leu Arg Phe Lys Ala Leu Thr Leu Ser Val Val Phe
320 325 330
Gly Thr Met Val Gly Gly Ile Leu Ile Pro Asn Val Glu Thr Ile
335 340 345
Leu Gly Leu Thr Gly Ala Thr Met Gly Ser Leu Ile Cys Phe Ile
350 355 360
Cys Pro Ala Leu Ile Tyr Lys Lys Ile His Lys Asn Ala Leu Ser
365 370 375
Ser Gln Val Val Leu Trp Val Gly Leu Gly Val Leu Val Val Ser
380 385 390
Thr Val Thr Thr Leu Ser Val Ser Glu Glu Val Pro Glu Asp Leu
395 400 405
Ala Glu Glu Ala Pro Gly Gly Arg Leu Gly Glu Ala Glu Gly Leu
410 415 420
Met Lys Val Glu Ala Ala Arg Leu Ser Ala Gln Asp Pro Val Val
425 430 435
Ala Val Ala Glu Asp Gly Arg Glu Lys Pro Lys Leu Pro Lys Glu
440 445 450
Arg Glu Glu Leu Glu Gln Ala Gln Ile Lys Gly Pro Val Asp Val
455 460 465
Pro Gly Arg Glu Asp Gly Lys Glu Ala Pro Glu Glu Ala Gln Leu
470 475 480
Asp Arg Pro Gly Gln Gly Ile Ala Val Pro Val Gly Glu Ala His
485 490 495
Arg His Glu Pro Pro Val Pro His Asp Lys Val Val Val Asp Glu
500 505 510
Gly Gln Asp Arg Glu Val Pro Glu Glu Asn Lys Pro Pro Ser Arg
515 520 525
His Ala Gly Gly Lys Ala Pro Gly Val Gln Gly Gln Met Ala Pro
530 535 540
Pro Leu Pro Asp Ser Glu Arg Glu Lys Gln Glu Pro Glu Gln Gly
545 550 555
Glu Val Gly Lys Arg Pro Gly Gln Ala Gln Ala Leu Glu Glu Ala
560 565 570
Gly Asp Leu Pro Glu Asp Pro Gln Lys Val Pro Glu Ala Asp Gly
575 580 585
Gln Pro Ala Val Gln Pro Ala Lys Glu Asp Leu Gly Pro Gly Asp
590 595 600
Arg Gly Leu His Pro Arg Pro Gln Ala Val Leu Ser Glu Gln Gln
605 610 615
Asn Gly Leu Ala Val Gly Gly Gly Glu Lys Ala Lys Gly Gly Pro
620 625 630
Pro Pro Gly Asn Ala Ala Gly Asp Thr Gly Gln Pro Ala Glu Asp
635 640 645
8/65
CA 02458625 2004-02-16
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Ser Asp His Gly Gly Lys Pro Pro Leu Pro Ala Glu Lys Pro Ala
650 655 660
Pro Gly Pro Gly Leu Pro Pro Glu Pro Arg Glu Gln Arg Asp Val
665 670 675
Glu Arg Ala Gly Gly Asn Gln Ala Ala Ser Gln Leu Glu Glu Ala
680 685 690
Gly Arg Ala Glu Met Leu Asp His Ala Val Leu Leu Gln Val Ile
695 700 705
Lys Glu Gln Gln Val Gln Gln Lys Arg Leu Leu Asp Gln Gln Glu
710 715 720
Lys Leu Leu Ala Val Ile Glu Glu Gln His Lys Glu Ile His Gln
725 730 735
Gln Arg Gln Glu Asp Glu Glu Asp Lys Pro Arg Gln Val Glu Val
740 745 750
His Gln Glu Pro Gly Ala Ala Val Pro Arg Gly Gln Glu Ala Pro
755 760 765
Glu Gly Lys Ala Arg Glu Thr Val Glu Asn Leu Pro Pro Leu Pro
770 775 780
Leu Asp Pro Val Leu Arg Ala Pro Gly Gly Arg Pro Ala Pro Ser
785 790 795
Gln Asp Leu Asn Gln Arg Ser Leu Glu His Ser Glu Gly Pro Val
800 805 810
Gly Arg Asp Pro Ala Gly Pro Pro Asp Gly Gly Pro Asp Thr Glu
815 820 825
Pro Arg Ala Ala Gln Ala Lys Leu Arg Asp Gly Gln Lys Asp Ala
830 835 840
Ala Pro Arg Ala Ala Gly Thr Val Lys Glu Leu Pro Lys Gly Pro
845 850 855
Glu Gln Val Pro Val Pro Asp Pro Ala Arg Glu Ala Gly Gly Pro
860 865 870
Glu Glu Arg Leu Ala Glu Glu Phe Pro Gly Gln Ser Gln Asp Val
875 880 885
Thr Gly Gly Ser Gln Asp Arg Lys Lys Pro Gly Lys Glu Val Ala
890 895 900
Ala Thr Gly Thr Ser Ile Leu Lys Glu Ala Asn Trp Leu Val Ala
905 910 915
Gly Pro Gly Ala Glu Thr Gly Asp Pro Arg Met Lys Pro Lys Gln
920 925 930
Val Ser Arg Asp Leu Gly Leu Ala Ala Asp Leu Pro Gly Gly Ala
935 940 945
Glu Gly Ala Ala Ala Gln Pro Gln Ala Val Leu Arg Gln Pro Glu
950 955 960
Leu Arg Val Ile Ser Asp Gly Glu Gln Gly Gly Gln Gln Gly His
965 970 975
Arg Leu Asp His Gly Gly His Leu Glu Met Arg Lys Ala Arg Gly
980 985 990
Gly Asp His Val Pro Val Ser His Glu Gln Pro Arg Gly Gly Glu
995 1000 1005
Asp Ala Ala Val Gln Glu Pro Arg Gln Arg Pro Glu Pro Glu Leu
1010 1015 1020
Gly Leu Lys Arg Ala Val Pro Gly Gly Gln Arg Pro Asp Asn Ala
1025 1030 1035
Lys Pro Asn Arg Asp Leu Lys Leu Gln Ala Gly Ser Asp Leu Arg
1040 1045 1050
Arg Arg Arg Arg Asp Leu Gly Pro His Ala Glu Gly Gln Leu Ala
1055 1060 1065
9/65
CA 02458625 2004-02-16
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Pro Arg Asp Gly Val Ile Ile Gly Leu Asn Pro Leu Pro Asp Val
1070 1075 1080
Gln Val Asn Asp Leu Arg Gly Ala Leu Asp Ala Gln Leu Arg Gln
1085 1090 1095
Ala Ala Gly Gly Ala Leu Gln Val Val His Ser Arg Gln Leu Arg
1100 1105 1110
Gln Ala Pro Gly Pro Pro Glu Glu Ser
1115
<210> 6
<211> 947
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7502087CD1
<400> 6
Met Pro Val Arg Arg Gly His Val Ala Pro Gln Asn Thr Tyr Leu
1 5 10 15
Asp Thr Ile Ile Arg Lys Phe Glu Gly Gln Ser Arg Lys Phe Leu
20 25 30
Ile Ala Asn Ala Gln Met Glu Asn Cys Ala Ile Ile Tyr Cys Asn
35 40 45
Asp Gly Phe Cys Glu Leu Phe Gly Tyr Ser Arg Val Glu Val Met
50 55 60
Gln Gln Pro Cys Thr Cys Asp Phe Leu Thr Gly Pro Asn Thr Pro
65 70 75
Ser Ser Ala Val Ser Arg Leu Ala Gln Ala Leu Leu Gly Ala Glu
80 85 90
Glu Cys Lys Val Asp Ile Leu Tyr Tyr Arg Lys Asp Ala Ser Ser
95 100 105
Phe Arg Cys Leu Val Asp Val Val Pro Val Lys Asn Glu Asp Gly
110 115 120
Ala Val Ile Met Phe Ile Leu Asn Phe Glu Asp Leu Ala Gln Leu
125 130 135
Leu Ala Lys Cys Ser Ser Arg Ser Leu Ser Gln Arg Leu Leu Ser
140 145 150
Gln Ser Phe Leu Gly Ser Glu Gly Ser His Gly Arg Pro Gly Gly
155 160 165
Pro Gly Pro Gly Thr Gly Arg Gly Lys Tyr Arg Thr Ile Ser Gln
170 175 180
Ile Pro Gln Phe Thr Leu Asn Phe Val Glu Phe Asn Leu Glu Lys
185 190 195
His Arg Ser Ser Ser Thr Thr Glu Ile Glu Ile Ile Ala Pro His
200 205 210
Lys Val Val Glu Arg Thr Gln Asn Val Thr Glu Lys Val Thr Gln
215 220 225
Val Leu Ser Leu Gly Ala Asp Val Leu Pro Glu Tyr Lys Leu Gln
230 235 240
Ala Pro Arg Ile His Arg Trp Thr Ile Leu His Tyr Ser Pro Phe
245 250 255
Lys Ala Val Trp Asp Trp Leu Ile Leu Leu Leu Val Ile Tyr Thr
260 265 270
Ala Val Phe Thr Pro Tyr Ser Ala Ala Phe Leu Leu Ser Asp Gln
10/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
275 280 285
Asp Glu Ser Arg Arg Gly Ala Cys Ser Tyr Thr Cys Ser Pro Leu
290 295 300
Thr Val Val Asp Leu Ile Val Asp Ile Met Phe Val Val Asp Ile
305 310 315
Val Ile Asn Phe Arg Thr Thr Tyr Val Asn Thr Asn Asp Glu Val
320 325 , 330
Val Ser His Pro Arg Arg Ile Ala Val His Tyr Phe Lys Gly Trp
335 340 345
Phe Leu Ile Asp Met Val Ala Ala Ile Pro Phe Asp Leu Leu Ile
350 355 360
Phe Arg Thr Gly Ser Asp Glu Thr Thr Thr Leu Ile Gly Leu Leu
365 370 375
Lys Thr Ala Arg Leu Leu Arg Leu Val Arg Val Ala Arg Lys Leu
380 385 390
Asp Cys Tyr Ser Glu Tyr Gly Ala Ala Val Leu Phe Leu Leu Met
395 400 405
Cys Thr Phe Ala Leu Ile Ala His Trp Leu Ala Cys Ile Trp Tyr
410 415 420
Ala Ile Gly Asn Val Glu Arg Pro Tyr Leu Glu His Lys Ile Gly
425 430 435
Trp Leu Asp Ser Leu Gly Val Gln Leu Gly Lys Arg Tyr Asn Gly
440 445 450
Ser Asp Pro Ala Ser Gly Pro Ser Val Gln Asp Lys Tyr Val Thr
455 460 465
Ala Leu Tyr Phe Thr Phe Ser Ser Leu Thr Ser Val Gly Phe Gly
470 475 480
Asn Val Ser Pro Asn Thr Asn Ser Glu Lys Val Phe Ser Ile Cys
485 490 495
Val Met Leu Ile Gly Ser Leu Met Tyr Ala Ser Ile Phe Gly Asn
500 505 510
Val Ser Ala Ile Ile Gln Arg Leu Tyr Ser Gly Thr Ala Arg Tyr
515 520 525
His Thr Gln Met Leu Arg Val Lys Glu Phe Ile Arg Phe His Gln
530 535- 540
Ile Pro Asn Pro Leu Arg Gln Arg Leu Glu Glu Tyr Phe Gln His
545 550 555
Ala Trp Ser Tyr Thr Asn Gly Ile Asp Met Asn Ala Val Leu Lys
560 565 570
Gly Phe Pro Glu Cys Leu Gln Ala Asp Ile Cys Leu His Leu His
575 580 585
Arg Ala Leu Leu Gln His Cys Pro Ala Phe Ser Gly Ala Gly Lys
590 595 600
Gly Cys Leu Arg Ala Leu Ala Val Lys Phe Lys Thr Thr His Ala
605 610 615
Pro Pro Gly Asp Thr Leu Val His Leu Gly Asp Val Leu Ser Thr
620 625 630
Leu Tyr Phe Ile Ser Arg Gly Ser Ile Glu Ile Leu Arg Asp~Asp
635 640 645
Val Val Val Ala Ile Leu Gly Lys Asn Asp Ile Phe Gly Glu Pro
650 655 660
Val Ser Leu His Ala Gln Pro Gly Lys Ser Ser Ala Asp Val Arg
665 670 675
Ala Leu Thr Tyr Cys Asp Leu His Lys Ile Gln Arg Ala Asp Leu
680 685 690
Leu Glu Val Leu Asp Met Tyr Pro Ala Phe Ala Glu Ser Phe Trp
11/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
695 700 705
Ser Lys Leu Glu Val Thr Phe Asn Leu Arg Asp Ala Pro Gly Ser
710 715 720
Gln Asp His Gln Gly Phe Phe Leu Ser Asp Asn Gln Ser Asp Ala
725 730 735
Ala Pro Pro Leu Ser Ile Ser Asp Ala Ser Gly Leu Trp Pro Glu
740 745 750
Leu Leu Gln Glu Met Pro Pro Arg His Ser Pro Gln Ser Pro Gln
755 760 765
Glu Asp Pro Asp Cys Trp Pro Leu Lys Leu Gly Ser Arg Leu Glu
770 775 780
Gln Leu Gln Ala Gln Met Asn Arg Leu Glu Ser Arg Val Ser Ser
785 790 795
Asp Leu Ser Arg Ile Leu Gln Leu Leu Gln Lys Pro Met Pro Gln
800 805 810
Gly His Ala Ser Tyr Ile Leu Glu Ala Pro Ala Ser Asn Asp Leu
815 820 825
Ala Leu Val Pro Ile Ala Ser Glu Thr Thr Ser Pro Gly Pro Arg
830 835 840
Leu Pro Gln Gly Phe Leu Pro Pro Ala Gln Thr Pro Ser Tyr Gly
845 850 855
Asp Leu Asp Asp Cys Ser Pro Lys His Arg Asn Ser Ser Pro Arg
860 865 870
Met Pro His Leu Ala Val Ala Met Asp Lys Thr Leu Ala Pro Ser
875 880 885
Ser Glu Gln Glu Gln Pro Glu Gly Leu Trp Pro Pro Leu Ala Ser
890 895 900
Pro Leu His Pro Leu Glu Val Gln Gly Leu Ile Cys Gly Pro Cys
905 910 915
Phe Ser Ser Leu Pro Glu His Leu Gly Ser Val Pro Lys Gln Leu
920 925 930
Asp Phe Gln Arg His Gly Ser Asp Pro Gly Phe Ala Gly Ser Trp
935 940 945
Gly His
<210> 7
<211> 80
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7500819CD1
<400> 7
Met Glu Leu Val Leu Val Phe Leu Cys Ser Leu Leu Ala Pro Met
1 5 10 15
Val Leu Ala Ser Ala Ala Glu Lys Glu Lys Glu Met Asp Pro Phe
20 25 30
His Tyr Asp Tyr Gln Thr Leu Arg Ile Gly Gly Leu Val Phe Ala
35 40 45
Val Val Leu Phe Ser Val Gly Ile Leu Leu Ile Leu Ser Arg Arg
50 55 60
Cys Lys Cys Ser Phe Tyr Ser Ala Pro Gly Glu Cys Val Pro Cys
65 70 75
12/65
CA 02458625 2004-02-16
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Ile Ser Ser Gln Gln
<210> 8
<211> 531
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_~eature
<223> Incyte ID No: 7503413CD1
<400> 8
Met Ala Ser Ala Leu Ser Tyr Val Ser Lys Phe Lys Ser Phe Val
1 5 10 15
Ile Leu Phe Val Thr Pro Leu Leu Leu Leu Pro Leu Val Ile Leu
20 25 30
Met Pro Ala Lys Phe Val Arg Cys Ala Tyr Val Ile Ile Leu Met
35 40 45
Ala Ile Tyr Trp Cys Thr Glu Val Ile Pro Leu Ala Val Thr Ser
50 55 60
Leu Met Pro Val Leu Leu Phe Pro Leu Phe Gln Ile Leu Asp Ser
65. 70 75
Arg Gln Val Cys Val Gln Tyr Met Lys Asp Thr Asn Met Leu Phe
80 85 90
Leu Gly Gly Leu Ile Val Ala Val Ala Val Glu Arg Trp Asn Leu
95 100 105
His Lys Arg Ile Ala Leu Arg Thr Leu Leu Trp Val Gly Ala Lys
110 115 120
Pro Ala Arg Leu Met Leu Gly Phe Met Gly Val Thr Ala Leu Leu
125 130 ~ 135
Ser Met Trp Ile Ser Asn Thr Ala Thr Thr Ala Met Met Val Pro
140 145 150
Ile Val Glu Ala Ile Leu Gln Gln Met Glu Ala Thr Ser Ala Ala
155 160 165
Thr Glu Ala Gly Leu Glu Leu Val Asp Lys Gly Lys Ala Lys Glu
170 175 180
Leu Pro Gly Ser Gln Val Ile Phe Glu Gly Pro Thr Leu Gly Gln
185 190 195
Gln Glu Asp Gln Glu Arg Lys Arg Leu Cys Lys Ala Met Thr Leu
200 205 210
Cys Ile Cys Tyr Ala Ala Ser Ile Gly Gly Thr Ala Thr Leu Thr
215 220 225
Gly Thr Gly Pro Asn Val Val Leu Leu Gly Gln Met Asn Glu Leu
230 235 240
Phe Pro Asp Ser Lys Asp Leu Val Asn Phe Ala Ser Trp Phe Ala
245 250 255
Phe Ala Phe Pro Asn Met Leu Val Met Leu Leu Phe Ala Trp Leu
260 265 270
Trp Leu Gln Phe Val Tyr Met Arg Phe Asn Phe Lys Lys Ser Trp
275 280 285
Gly Cys Gly Leu Glu Ser Lys Lys Asn Glu Lys Ala Ala Leu Lys
290 295 300
Val Leu Gln Glu Glu Tyr Arg Lys Leu Gly Pro Leu Ser Phe Ala
305 310 315
Glu Ile Asn Val Leu Ile Cys Phe Phe Leu Leu Val Ile Leu Trp
13/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
320 325 330
Phe Ser Arg Asp Pro Gly Phe Met Pro Gly Trp Leu Thr Val Ala
335 340 345
Trp Val Glu Glu Arg Lys Thr Pro Phe Tyr Pro Pro Pro Leu Leu
350 355 360
Asp Trp Lys Val Thr Gln Glu Lys Val Pro Trp Gly Ile Val Leu
365 370 ~ 375
Leu Leu Gly Gly Gly Phe Ala Leu Ala Lys Gly Ser Glu Ala Ser
380 385 390
Gly Leu Ser Val Trp Met Gly Lys Gln Met Glu Pro Leu His Ala
395 400 405
Val Pro Pro Ala Ala Ile Thr Leu Ile Leu Ser Leu Leu Val Ala
410 415 420
Val Phe Thr Glu Cys Thr Ser Asn Val Ala Thr Thr Thr Leu Phe
425 430 435
Leu Pro Ile Phe Ala Ser Met Ser Arg Ser Ile Gly Leu Asn Pro
440 445 450
Leu Tyr Ile Met Leu Pro Cys Thr Leu Ser Ala Ser Phe Ala Phe
455 460 465
Met Leu Pro Val Ala Thr Pro Pro Asn Ala Ile Val Phe Thr Tyr
470 475 480
Gly His Leu Lys Val Ala Asp Met Val Lys Thr Gly Val Ile Met
485 490 495
Asn Ile Ile Gly Val Phe Cys Val Phe Leu Ala Val Asn Thr Trp
500 505 510
Gly Arg Ala Ile Phe Asp Leu Asp His Phe Pro Asp Trp Ala Asn
515 520 525
Val Thr His Ile Glu Thr
530
<210> 9
<211> 510
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7500007CD1
<400> 9
Met Asp Ser Arg Val Ser Gly Thr Thr Ser Asn Gly Glu Thr Lys
1 5 10 15
Pro Val Tyr Pro Val Met Glu Lys Lys Glu Glu Asp Gly Thr Leu
20 25 30
Glu Arg Gly His Trp Asn Asn Lys Met Glu Phe Val Leu Ser Val
35 40 45
Ala Gly Glu Ile Ile Gly Leu Gly Asn Val Trp Arg Phe Pro Tyr
50 55 60
Leu Cys Tyr Lys Asn Gly Gly Glu His Cys Met Glu Phe Gln Lys
65 70 75
Thr Asn Gly Ser Leu Asn Gly Thr Ser Glu Asn Ala Thr Ser Pro
80 85 90
Val Ile Glu Phe Trp Glu Arg Arg Val Leu Lys Ile Ser Asp Gly
95 100 105
Ile Gln His Leu Gly Ala Leu Arg Trp Glu Leu Ala Leu Cys Leu
110 115 120
14/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
Leu Leu Ala Trp Val Ile Cys Tyr Phe Cys Ile Trp Lys Gly Val
125 130 135
Lys Ser Thr Gly Lys Val Val Tyr Phe Thr Ala Thr Phe Pro Tyr
140 145 150
Leu Met Leu Val Val Leu Leu Ile Arg Gly Val Thr Leu Pro Gly
155 160 165
Ala Ala Gln Gly Ile Gln Phe Tyr Leu Tyr Pro Asn Leu Thr Arg
170 175 180
Leu Trp Asp Pro Gln Val Trp Met Asp Ala Gly Thr Gln Ile Phe
185 190 195
Phe Ser Phe Ala Ile Cys Leu Gly Cys Leu Thr Ala Leu Gly Ser
200 205 210
Tyr Asn Lys Tyr His Asn Asn Cys Tyr Arg Asp Cys Ile Ala Leu
215 220 225
Cys Phe Leu Asn Ser Gly Thr Ser Phe Val Ala Gly Phe Ala Ile
230 235 240
Phe Ser Ile Leu Gly Phe Met Ser Gln Glu Gln Gly Val Pro Ile
245 250 255
Ser Glu Val Ala Glu Ser Gly Pro Gly Leu Ala Phe Ile Ala Tyr
260 265 270
Pro Arg Ala Val Val Met Leu Pro Phe Ser Pro Leu Trp Ala Cys
275 280 285
Cys Phe Phe Phe Met Val Val Leu Leu Gly Leu Asp Ser Gln Phe
290 295 300
Val Cys Val Glu Ser Leu Val Thr Ala Leu Val Asp Met Tyr Pro
305 310 315
His Val Phe Arg Lys Lys Asn Arg Arg Glu Val Leu Ile Leu Gly
320 325 330
Val Ser Val Val Ser Phe Leu Val Gly Leu Ile Met Leu Thr Glu
335 340 345
Gly Gly Met Tyr Val Phe Gln Leu Phe Asp Tyr Tyr Ala Ala Ser
350 355 360
Gly Met Cys Leu Leu Phe Val Ala Ile Phe Glu Ser Leu Cys Val
365 370 375
Ala Trp Val Tyr Gly Ala Lys Arg Phe Tyr Asp Asn Ile Glu Asp
380 385 390
Met Ile Gly Tyr Arg Pro Trp Pro Leu Ile Lys Tyr Cys Trp Leu
395 400 405
Phe Leu Thr Pro Ala Val Cys Thr Ala Thr Phe Leu Phe Ser Leu
410 415 420
Ile Lys Tyr Thr Pro Leu Thr Tyr Asn Lys Lys Tyr Thr Tyr Pro
425 430 435
Trp Trp Gly Asp Ala Leu Gly Trp Leu Leu Ala Leu Ser Ser Met
440 445 450
Val Cys Ile Pro Ala Trp Ser Leu Tyr Arg Leu Gly Thr Leu Lys
455 460 4&5
Gly Pro Phe Arg Glu Arg Ile Arg Gln Leu Met Cys Pro Ala Glu
470 475 480
Asp Leu Pro Gln Arg Asn Pro Ala Gly Pro Ser Ala Pro Ala Thr
485 490 495
Pro Arg Thr Ser Leu Leu Arg Leu Thr Glu Leu Glu Ser His Cys
500 505 510
<210> 10
<211> 894
15/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7500025CD1
<400> 10
Met Ser Val Arg Arg Gly His Val Ala Pro Gln Asn Thr Tyr Leu
1 5 10 15
Asp Thr Ile Ile Arg Lys Phe Glu Gly Gln Ser Arg Lys Phe Leu
20 25 30
Ile Ala Asn Ala Gln Met Glu Asn Cys Ala Ile Ile Tyr Cys Asn
35 40 45
Asp Gly Phe Cys Glu Leu Phe Gly Tyr Ser Arg Val Glu Val Met
50 55 60
Gln Gln Pro Cys Thr Cys Asp Phe Leu Thr Gly Pro Asn Thr Pro
65 70 75
Ser Ser Ala Val Ser Arg Leu Ala Gln Ala Leu Leu Gly Ala Glu
80 85 90
Glu Cys Lys Val Asp Ile Leu Tyr Tyr Arg Lys Asp Ala Ser Ser
95 100 105
Phe Arg Cys Leu Val Asp Val Val Pro Val Lys Asn Glu Asp Gly
110 115 120
Ala Val Ile Met Ser Ile Leu Asn Phe Glu Asp Leu Ala Gln Leu
125 130 135
Leu Ala Lys Cys Ser Ser Arg Ser Leu Ser Gln Arg Leu Leu Ser
140 145 150
Gln Ser Phe Leu Gly Ser Glu Gly Ser His Gly Arg Pro Gly Gly
155 160 165
Pro Gly Pro Gly Thr Gly Arg Gly Lys Tyr Arg Thr Ile Ser Gln
170 ~ 175 180
Ile Pro Gln Phe Thr Leu Asn Phe Val Glu Phe Asn Leu Glu Lys
185 190 195
His Arg Ser Ser Ser Thr Thr Glu Ile Glu Ile Ile Ala Pro His
200 205 210
Lys Val Val Glu Arg Thr Gln Asn Val Thr Glu Lys Val Thr Gln
215 220 225
Val Leu Ser Leu Gly Ala Asp Val Leu Pro Glu Tyr Lys Leu Gln
230 235 240
Ala Pro Arg Ile His Arg Trp Thr Ile Leu His Tyr Ser Pro Phe
245 250 255
Lys Ala Val Trp Asp Trp Leu Ile Pro Leu Leu Val Ile Tyr Thr
260 265 270
Ala Val Phe Thr Pro Tyr Ser Ala Ala Phe Leu Leu Ser Asp Gln
275 280 285
Asp Glu Ser Arg Arg Gly Ala Cys Ser Tyr Thr Cys Ser Pro Leu
290 295 300
Thr Val Val Asp Leu Ile Val Asp Ile Met Phe Val Val Asp Ile
305 310 315
Val Ile Asn Phe Arg Thr Thr Tyr Val Asn Thr Asn Asp Glu Val
320 325 330
Val Ser His Pro Arg Arg Ile Ala Val His Tyr Phe Lys Gly Trp
335 340 345
Phe Leu Ile Asp Met Val Ala Ala Ile Pro Phe Asp Leu Leu Ile
350 355 360
16/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
Phe Arg Thr Gly Ser Asp Glu Thr Thr Thr Leu Ile Gly Leu Leu
365 370 375
Lys Thr Ala Arg Leu Leu Arg Leu Val Arg Val Ala Arg Lys Leu
380 385 390
Asp Arg Tyr Ser Glu Tyr Gly Ala Ala Val Leu Phe Leu Leu Met
395 400 405
Cys Thr Phe Ala Leu Ile Ala His Trp Leu Ala Cys Ile Cys Ser
410 415 420
Leu Thr Ser Val Gly Phe Gly Asn Val Ser Pro Asn Thr Asn Ser
425 430 435
Glu Lys Val Phe Ser Ile Cys Val Met Leu Ile Gly Ser Leu Met
440 445 450
Tyr Ala Ser Ile Phe Gly Asn Val Ser Ala Ile Ile Gln Arg Leu
455 460 465
Tyr Ser Gly Thr Ala Arg Tyr His Thr Gln Met Leu Arg Val Lys
470 475 480
Glu Phe Ile Arg Phe His Gln Ile Pro~Asn Pro Leu Arg Gln Arg
485 490 495
Leu Glu Glu Tyr Phe Gln His Ala Trp Ser Tyr Thr Asn Gly Ile
500 505 510
Asp Met Asn Ala Val Leu Lys Gly Phe Pro Glu Cys Leu Gln Ala
515 520 525
Asp Ile Cys Leu His Leu His Arg Ala Leu Leu Gln His Cys Pro
530 535 540
Ala Phe Ser Gly Ala Gly Lys Gly Cys Leu Arg Ala Leu Ala Val
545 550 555
Lys Phe Lys Thr Thr His Ala Pro Pro Gly Asp Thr Leu Val His
560 565 570
Leu Gly Asp Val Leu Ser Thr Leu Tyr Phe Ile Ser Arg Gly Ser
575 580 585
Ile Glu Ile Leu Arg Asp Asp Val Val Val Ala Ile Leu Gly Lys
590 595 600
Asn Asp Ile Phe Gly Glu Pro Val Ser Leu His Ala Gln Pro Gly
605 610 615
Lys Ser Ser Ala Asp Val Arg Ala Leu Thr Tyr Cys Asp Leu His
620 625 630
Lys Ile Gln Arg Ala Asp Leu Leu Glu Val Leu Asp Met Tyr Pro
635 640 645
Ala Phe Ala Glu Ser Phe Trp Ser Lys Leu Glu Val Thr Phe Asn
650 655 660
Leu Arg Asp Ala Pro Gly Ser Gln Asp His Gln Gly Phe Phe Leu
665 670 675
Ser Asp Asn Gln Ser Asp Ala Ala Pro Pro Leu Ser Ile Ser Asp
680 685 690
Ala Ser Gly Leu Trp Pro Glu Leu Leu Gln Glu Met Pro Pro Arg
695 700 705
His Ser Pro Gln Ser Pro Gln Glu Asp Pro Asp Cys Trp Pro Leu
710 715 720
Lys Leu Gly Ser Arg Leu Glu Gln Leu Gln Ala Gln Met Asn Arg
725 730 735
Leu Glu Ser Arg Val Ser Ser Asp Leu Ser Arg Ile Leu Gln Leu
740 745 750
Leu Gln Lys Pro Met Pro Gln Gly His Ala Ser Tyr Ile Leu Glu
755 760 765
Ala Pro Ala Ser Asn Asp Leu Ala Leu Val Pro Ile Ala Ser Glu
770 775 780
17/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
Thr Thr Ser Pro Gly Pro Arg Leu Pro Gln Gly Phe Leu Pro Pro
785 790 795
Ala Gln Thr Pro Ser Tyr Gly Asp Leu Asp Asp Cys Ser Pro Lys
800 805 810
His Arg Asn Ser Ser Pro Arg Met Pro His Leu Ala Val Ala Met
815 820 825
Asp Lys Thr Leu Ala Pro Ser Ser Glu Gln Glu Gln Pro Glu Gly
830 835 840
Leu Trp Pro Pro Leu Ala Ser Pro Leu His Pro Leu Glu Val Gln
845 850 855
Gly Leu Ile Cys Gly Pro Cys Phe Ser Ser Leu Pro Glu His Leu
860 865 870
Gly Ser Val Pro Lys Gln Leu Asp Phe Gln Arg His Gly Ser Asp
875 880 885
Pro Gly Phe Ala Gly Ser Trp Gly His
890
<210> 11
<211> 788
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7502736CD1
<400> 11
Met Ala Gly Gln Ala Asp Gln Gly Gln Ala Gln Ala Gly Ala Ser
1 5 10 15
Thr Gly Pro Ser Ala Arg Ser His Ser Ser Arg Ser Thr Ser Trp
20 25 30
Ser Ser Thr Trp Arg Ser Thr Ala Pro Ala Pro Pro Arg Arg Leu
35 40 45
Arg Ser Ser Arg Pro Ile Arg Trp Trp Ser Gly His Arg Thr Ser
50 55 60
Leu Arg Arg Ser Pro Arg Ser Cys Pro Trp Ala Arg Met Cys Cys
65 70 75
Arg Ser Thr Ser Cys Arg Arg Arg Ala Ser Thr Ala Gly Pro Ser
80 85 90
Cys Thr Thr Ala Pro Phe Lys Ala Val Trp Asp Trp Leu Ile Leu
95 100 105
Leu Leu Val Ile Tyr Thr Ala Val Phe Thr Pro Tyr Ser Ala Ala
110 115 120
Phe Leu Leu Ser Asp Gln Asp Glu Ser Arg Arg Gly Ala Cys Ser
125 130 135
Tyr Thr Cys Ser Pro Leu Thr Val Val Asp Leu Ile Val Asp Ile
140 145 150
Met Phe Val Val Asp Ile Val Ile Asn Phe Arg Thr Thr Tyr Val
155 160 165
Asn Thr Asn Asp Glu Val Val Ser His Pro Arg Arg Ile Ala Val
170 175 180
His Tyr Phe Lys Gly Trp Phe Leu Ile Asp Met Val Ala Ala Ile
185 190 195
Pro Phe Asp Leu Leu Ile Phe Arg Thr Gly Ser Asp Glu Thr Thr
200 205 210
Thr Leu Ile Gly Leu Leu Lys Thr Ala Arg Leu Leu Arg Leu Val
18/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
215 220 225
Arg Val Ala Arg Lys Leu Asp Arg Tyr Ser Glu Tyr Gly Ala Ala
230 235 240
Val Leu Phe Leu Leu Met Cys Thr Phe Ala Leu Ile Ala His Trp
245 250 255
Leu Ala Cys Ile Trp Tyr Ala Ile Gly Asn Val Glu Arg Pro Tyr
260 265 270
Leu Glu His Lys Ile Gly Trp Leu Asp Ser Leu Gly Val Gln Leu
275 280 285
Gly Lys Arg Tyr Asn Gly Ser Asp Pro Ala Ser Gly Pro Ser Val
290 295 300
Gln Asp Lys Tyr Val Thr Ala Leu Tyr Phe Thr Phe Ser Ser Leu
305 310 315
Thr Ser Val Gly Phe Gly Asn Val Ser Pro Asn Thr Asn Ser Glu
320 325 330
Lys Val Phe Ser Ile Cys Val Met Leu Ile Gly Ser Leu Met Tyr
335 340 345
Ala Ser Ile Phe Gly Asn Val Ser Ala Ile Ile Gln Arg Leu Tyr
350 355 360
Ser Gly Thr Ala Arg Tyr His Thr Gln Met Leu Arg Val Lys Glu
365 370 375
Phe Ile Arg Phe His Gln Ile Pro Asn Pro Leu Arg Gln Arg Leu
380 385 390
Glu Glu Tyr Phe Gln His Ala Trp Ser Tyr Thr Asn Gly Ile Asp
395 400 405
Met Asn Ala Val Leu Lys Gly Phe Pro Glu Cys Leu Gln Ala Asp
410 415 420
Ile Cys Leu His Leu His Arg Ala Leu Leu Gln His Cys Pro Ala
425 430 435
Phe Ser Gly Ala Gly Lys Gly Cys Leu Arg Ala Leu Ala Val Lys
440 445 450
Phe Lys Thr Thr His Ala Pro Pro Gly Asp Thr Leu Val His Leu
455 460 465
Gly Asp Val Leu Ser Thr Leu Tyr Phe Ile Ser Arg Gly Ser Ile
470 475 480
Glu Ile Leu Arg Asp Asp Val Val Val Ala Ile Leu Gly Lys Asn
485 490 495
Asp Ile Phe Gly Glu Pro Val Ser Leu His Ala Gln Pro Gly Lys
500 505 510
Ser Ser Ala Asp Val Arg Ala Leu Thr Tyr Cys Asp Leu His Lys
515 520 525
Ile Gln Arg Ala Asp Leu Leu Glu Val Leu Asp Met Tyr Pro Ala
530 535 540
Phe Ala Glu Ser Phe Trp Ser Lys Leu Glu Val Thr Phe Asn Leu
545 550 555
Arg Asp Ala Pro Gly Ser Gln Asp His Gln Gly Phe Phe Leu Ser
560 565 570
Asp Asn Gln Ser Asp Ala Ala Pro Pro Leu Ser Ile Ser Asp Ala
575 580 585
Ser Gly Leu Trp Pro Glu Leu Leu Gln Glu Met Pro Pro Arg His
590 595 600
Ser Pro Gln Ser Pro Gln Glu Asp Pro Asp Cys Trp Pro Leu Lys
605 610 615
Leu Gly Ser Arg Leu Glu Gln Leu Gln Ala Gln Met Asn Arg Leu
620 625 630
Glu Ser Arg Val Ser Ser Asp Leu Ser Arg Ile Leu Gln Leu Leu
19/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
635 640 645
Gln Lys Pro Met Pro Gln Gly His Ala Ser Tyr Ile Leu Glu Ala
650 655 660
Pro Ala Ser Asn Asp Leu Ala Leu Val Pro Ile Ala Ser Glu Thr
665 670 675
Thr Ser Pro Gly Pro Arg Leu Pro Gln Gly Phe Leu Pro Pro Ala
680 685 690
Gln Thr Pro Ser Tyr Gly Asp Leu Asp Asp Cys Ser Pro Lys His
695 700 705
Arg Asn Ser Ser Pro Arg Met Pro His Leu Ala Val Ala Met Asp
710 715 720
Lys Thr Leu Ala Pro Ser Ser Glu Gln Glu Gln Pro Glu Gly Leu
725 730 735
Trp Pro Pro Leu Ala Ser Pro Leu His Pro Leu Glu Val Gln Gly
740 745 750
Leu Ile Cys Gly Pro Cys Phe Ser Ser Leu Pro Glu His Leu Gly
755 760 765
Ser Val Pro Lys Gln Leu Asp Phe Gln Arg His Gly Ser Asp Pro
770 T75 780
Gly Phe Ala Gly Ser Trp Gly His
785
<210> 12
<211> 501
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503570CD1
<400> 12
Met Ser Gln Asp Thr Glu Val Asp Met Lys Glu Val Glu Leu Asn
1 5 10 15
Glu Leu Glu Pro Glu Lys Gln Pro Met Asn Ala Ala Ser Gly Ala
20 25 30
Ala Met Ser Leu Ala Gly Ala Glu Lys Asn Gly Leu Val Lys Ile
35 40 45
Lys Val Ala Glu Asp Glu Ala Glu Ala Ala Ala Ala Ala Lys Phe
50 55 60
Thr Gly Leu Ser Lys Glu Glu Leu Leu Lys Val Ala Gly Ser Pro
65 70 75
Gly Trp'Val Arg Thr Arg Trp Ala Leu Leu Leu Leu Phe Trp Leu
80 85 90
Gly Trp Leu Gly Met Leu Ala Gly Ala Val Val Ile Ile Val Arg
95 100 105
Ala Pro Arg Cys Arg Glu Leu Pro Ala Gln Lys Trp Trp His Thr
110 115 120
Gly Ala Leu Tyr Arg Ile Gly Asp Leu Gln Ala Phe Gln Gly His
125 130 135
Gly Ala Gly Asn Leu Ala Gly Leu Lys Gly Arg Leu Asp Tyr Leu
140 14.5 150
Ser Ser Leu Lys Val Lys Gly Leu Val Leu Gly Pro Ile His Lys
155 160 165
Asn Gln Lys Asp Asp Val Ala Gln Thr Asp Leu Leu Gln Ile Asp
170 175 180
20/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
Pro Asn Phe Gly Ser Lys Glu Asp Phe Asp Ser Leu Leu Gln Ser
185 190 195
Ala Lys Lys Lys Ser Ile Arg Val Ile Leu Asp Leu Thr Pro Asn
200 205 210
Tyr Arg Gly Glu Asn Ser Trp Phe Ser Thr Gln Val Asp Thr Val
215 220 225
Ala Thr Lys Val Lys Asp Ala Leu Glu Phe Trp Leu Gln Ala Gly
230 235 240
Val Asp Gly Phe Gln Val Arg Asp Ile Glu Asn Leu Lys Asp Ala
245 250 255
Ser Ser Phe Leu Ala Glu Trp Gln Asn Ile Thr Lys Gly Phe Ser
260 265 270
Glu Asp Arg Leu Leu Ile Ala Gly Thr Asn Ser Ser Asp Leu Gln
275 280 285
Gln Ile Leu Ser Leu Leu Glu Ser Asn Lys Asp Leu Leu Leu Thr
290 295 300
Ser Ser Tyr Leu Ser Asp Ser Gly Ser Thr Gly Glu His Thr Lys
305 310 315
Ser Leu Val Thr Gln Tyr Leu Asn Ala Thr Gly Asn Arg Trp Cys
320 325 330
Ser Trp Ser Leu Ser Gln Ala Arg Leu Leu Thr Ser Phe Leu Pro
335 340 345
Ala Gln Leu Leu Arg Leu Tyr Gln Leu Met Leu Phe Thr Leu Pro
350 355 360
Gly Thr Pro Val Phe Ser Tyr Gly Asp Glu Ile Gly Leu Asp Ala
365 370 375
Ala Ala Leu Pro Gly Gln Gly Gln Ser Glu Asp Pro Gly Ser Leu
380 385 390
Leu Ser Leu Phe Arg Arg Leu Ser Asp Gln Arg Ser Lys Glu Arg
395 400 405
Ser Leu Leu His Gly Asp Phe His Ala Phe Ser Ala Gly Pro Gly
410 415 420
Leu Phe Ser Tyr Ile Arg His Trp Asp Gln Asn Glu Arg Phe Leu
425 430 435
Val Val Leu Asn Phe Gly Asp Val Gly Leu Ser Ala Gly Leu Gln
440 445 450
Ala Ser Asp Leu Pro Ala Ser Ala Ser Leu Pro Ala Lys Ala Asp
455 460 465
Leu Leu Leu Ser Thr Gln Pro Gly Arg Glu Glu Gly Ser Pro Leu
470 475 480
Glu Leu Glu Arg Leu Lys Leu Glu Pro His Glu Gly Leu Leu Leu
485 490 495
Arg Phe Pro Tyr Ala Ala
500
<210> 13
<211> 721
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7504008CD1
<400> 13
Met Gly Leu Ala Asp Ala Ser Gly Pro Arg Asp Thr Gln Ala Leu
21/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
1 5 10 15
Leu Ser Ala Thr Gln Ala Met Asp Leu Arg Arg Arg Asp Tyr His
20 25 30
Met Glu Arg Pro Leu Leu Asn Gln Glu His Leu Glu Glu Leu Gly
35 40 45
Arg Trp Gly Ser Ala Pro Arg Thr His Gln Trp Arg Thr Trp Leu
50 55 60
Gln Cys Ser Arg Ala Arg Ala Tyr Ala Leu Leu Leu Gln His Leu
65 70 75
Pro Val Leu Val Trp Leu Pro Arg Tyr Pro Val Arg Asp Trp Leu
80 85 90
Leu Gly Asp Leu Leu Ser Gly Leu Ser Val Ala Ile Met Gln Leu
95 100 105
Pro Gln Gly Leu Ala Tyr Ala Leu Leu Ala Gly Leu Pro Pro Val
110 115 120
Phe Gly Leu Tyr Ser Ser Phe Tyr Pro Val Phe Ile Tyr Phe Leu
125 130 135
Phe Gly Thr Ser Arg His Ile Ser Val Gly Thr Phe Ala Val Met
140 145 150
Ser Val Met Val Gly Ser Val Thr Glu Ser Leu Ala Pro Gln Ala
155 160 165
Leu Asn Asp Ser Met Ile Asn Glu Thr Ala Arg Asp Ala Ala Arg
170 175 180
Val Gln Val Ala Ser Thr Leu Ser Val Leu Val Gly Leu Phe Gln
185 190 195
Val Gly Leu Gly Leu Ile His Phe Gly Phe Val Val Thr Tyr Leu
200 205 210
Ser Glu Pro Leu Val Arg Gly Tyr Thr Thr Ala Ala Ala Val Gln
215 220 225
Val Phe Val Ser Gln Leu Lys Tyr Val Phe Gly Leu His Leu Ser
230 235 240
Ser His Ser Gly Pro Leu Ser Leu Ile Tyr Thr Val Leu Glu Val
245 250 255
Cys Trp Lys Leu Pro Gln Ser Lys Leu Ile Gly Ala Thr Gly Ile
260 265 270
Ser Tyr Gly Met Gly Leu Lys His Arg Phe Glu Val Asp Val Val
275 280 285
Gly Asn Ile Pro Ala Gly Leu Val Pro Pro Val Ala Pro Asn Thr
290 295 300
Gln Leu Phe Ser Lys Leu Val Gly Ser Ala Phe Thr Ile Ala Val
305 310 315
Val Gly Phe Ala Ile Ala Ile Ser Leu Gly Lys Ile Phe Ala Leu
320 325 330
Arg His Gly Tyr Arg Val Asp Ser Asn Gln Glu Leu Val Ala Leu
335 340 345
Gly Leu Ser Asn Leu Ile Gly Gly Ile Phe Gln Cys Phe Pro Val
350 355 360
Ser Cys Ser Met Ser Arg Ser Leu Val Gln Glu Ser Thr Gly Gly
365 370 375
Asn Ser Gln Val Ala Gly Ala Ile Ser Ser Leu Phe Ile Leu Leu
380 385 390
Ile Ile Val Lys Leu Gly Glu Leu Phe His Asp Leu Pro Lys Ala
395 400 405
Val Leu Ala Ala Ile Ile Ile Val Asn Leu Lys Gly Met Leu Arg
410 415 420
Gln Leu Ser Asp Met Arg Ser Leu Trp Lys Ala Asn Arg Ala Asp
22/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
425 430 435
Leu Leu Ile Trp Leu Val Thr Phe Thr Ala Thr Ile Leu Leu Asn
440 445 450
Leu Asp Leu Gly Leu Val Val Ala Val Ile Phe Ser Leu Leu Leu
455 460 465
Val Val Val Arg Thr Gln Met Pro His Tyr Ser Val Leu Gly Gln
470 475 480
Val Pro Asp Thr Asp Ile Tyr Arg Asp Val Ala Glu Tyr Ser Glu
485 490 495
Ala Lys Glu Val Arg Gly Val Lys Val Phe Arg Ser Ser Ala Thr
500 505 510
Val Tyr Phe Ala Asn Ala Glu Phe Tyr Ser Asp Ala Leu Lys Gln
515 520 525
Arg Cys Gly Val Asp Val Asp Phe Leu Ile Ser Gln Lys Lys Lys
530 535 540
Leu Leu Lys Lys Gln Glu Gln Leu Lys Leu Lys Gln Leu Gln Lys
545 550 555
Glu Glu Lys Leu Arg Lys Gln Ala Ala Ser Pro Lys Gly Ala Ser
560 565 570
Val Ser Ile Asn Val Asn Thr Ser Leu Glu Asp Met Arg Ser Asn
575 580 585
Asn Val Glu Asp Cys Lys Met Met Gln Val Ser Ser Gly Asp Lys
590 595 600
Met Glu Asp Ala Thr Ala Asn Gly Gln Glu Asp Ser Lys Ala Pro
605 610 615
Asp Gly Ser Thr Leu Lys Ala Leu Gly Leu Pro Gln Pro Asp Phe
620 625 630
His Ser Leu Ile Leu Asp Leu Gly Ala Leu Ser Phe Val Asp Thr
635 640 645
Val Cys Leu Lys Ser Leu Lys Asn Ile Phe His Asp Phe Arg Glu
650 655 660
Ile Glu Val Glu Val Tyr Met Ala Ala Cys His Ser Pro Val Val
665 670 675
Ser Gln Leu Glu Ala Gly His Phe Phe Asp Ala Ser Ile Thr Lys
680 685 690
Lys His Leu Phe Ala Ser Val His Asp Ala Val Thr Phe Ala Leu
695 700 705
Gln His Pro Arg Pro Val Pro Asp Ser Pro Val Ser Val Thr Arg
710 715 720
Leu
<210> 14
<211> 1226
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503559CD1
<400> 14
Met Asp Arg Glu Glu Arg Lys Thr Ile Asn Gln Gly Gln Glu Asp
1 5 10 15
Glu Met Glu Ile Tyr Gly Tyr Asn Leu Ser Arg Trp Lys Leu Ala
20 25 30
23/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
Ile Val Ser Leu Gly Val Ile Cys Ser Gly Gly Phe Leu Leu Leu
35 40 45
Leu Leu Tyr Trp Met Pro Glu Trp Arg Val Lys Ala Thr Cys Val
50 55 60
Arg Ala Ala Ile Lys Asp Cys Glu Val Val Leu Leu Arg Thr Thr
65 70 75
Asp Glu Phe Lys Met Trp Phe Cys Ala Lys Ile Arg Val Leu Ser
80 85 90
Leu Glu Thr Tyr Pro Val Ser Ser Pro Lys Ser Met Ser Asn Lys
95 100 105
Leu Ser Asn Gly His Ala Val Cys Leu Ile Glu Asn Pro Thr Glu
110 115 ~ 120
Glu Asn Arg His Arg Ile Ser Lys Tyr Ser Gln Thr Glu Ser Gln
125 130 135
Gln Ile Arg Tyr Phe Thr His His Ser Val Lys Tyr Phe Trp Asn
140 145 150
Asp Thr Ile His Asn Phe Asp Phe Leu Lys Gly Leu Asp Glu Gly
155 160 165
Val Ser Cys Thr Ser Ile Tyr Glu Lys His Ser Ala Gly Leu Thr
170 175 180
Lys Gly Met His Ala Tyr Arg Lys Leu Leu Tyr Gly Val Asn Glu
185 190 195
Ile Ala Val Lys Val Pro Ser Val Phe Lys Leu Leu Ile Lys Glu
200 205 210
Val Leu Asn Pro Phe Tyr Ile Phe Gln Leu Phe Ser Val Ile Leu
215 220 225
Trp Ser Thr Asp Glu Tyr Tyr Tyr Tyr Ala Leu Ala Ile Val Val
230 235 240
Met Ser Ile Val Ser Ile Val Ser Ser Leu Tyr Ser Ile Arg Lys
245 , 250 255
Gln Tyr Val Met Leu His Asp Met Val Ala Thr His Ser Thr Val
260 265 270
Arg Val Ser Val Cys Arg Val Asn Glu Glu Ile Glu Glu Ile Phe
275 280 285
Ser Thr Asp Leu Val Pro Gly Asp Val Met Val Ile Pro Leu Asn
290 295 300
Gly Thr Ile Met Pro Cys Asp Ala Val Leu Ile Asn Gly Thr Cys
305 310 315
Ile Val Asn Glu Ser Met Leu Thr Gly Glu Ser Val Pro Val Thr
320 325 330
Lys Thr Asn Leu Pro Asn Pro Ser Val Asp Val Lys Gly Ile Gly
335 340 345
Asp Glu Leu Tyr Asn Pro Glu Thr His Lys Arg His Thr Leu Phe
350 355 360
Cys Gly Thr Thr Val Ile Gln Thr Arg Phe Tyr Thr Gly Glu Leu
365 370 375
Val Lys Ala Ile Val Val Arg Thr Gly Phe Ser Thr Ser Lys Gly
380 385 390
Gln Leu Val Arg Ser Ile Leu Tyr Pro Lys Pro Thr Asp Phe Lys
395 400 405
Leu Tyr Arg Asp Ala Tyr Leu Phe Leu Leu Cys Leu Val Ala Val
410 415 420
Ala Gly Ile Gly Phe Ile Tyr Thr Ile Ile Asn Ser Ile Leu Asn
425 430 435
Glu Val Gln Val Gly Val Ile Ile Ile Glu Ser Leu Asp Ile Ile
440 445 450
24/65
Val Val Val Arg Thr Gln Met Pro His Tyr Ser Val Leu G
CA 02458625 2004-02-16
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Thr Ile Thr Val Pro Pro Ala Leu Pro Ala Ala Met Thr Ala Gly
455 460 465
Ile Val Tyr Ala Gln Arg Arg Leu Lys Lys Ile Gly Ile Phe Cys
470 475 480
Ile Ser Pro Gln Arg Ile Asn Ile Cys Gly Gln Leu Asn Leu Val
485 490 495
Cys Phe Asp Lys Thr Gly Thr Leu Thr Glu Asp Gly Leu Asp Leu
500 505 510
Trp Gly Ile Gln Arg Val Glu Asn Ala Arg Phe Leu Ser Pro Glu
515 520 525
Glu Asn Val Cys Asn Glu Met Leu Val Lys Ser Gln Phe Val Ala
530 535 540
Cys Met Ala Thr Cys His Ser Leu Thr Lys Ile Glu Gly Val Leu
545 550 555
Ser Gly Asp Pro Leu Asp Leu Lys Met Phe Glu Ala Ile Gly Trp
560 565 570
Ile Leu Glu Glu Ala Thr Glu Glu Glu Thr Ala Leu His Asn Arg
575 580 585
Ile Met Pro Thr Val Val Arg Pro Pro Lys Gln Leu Leu Pro Glu
590 595 600
Ser Thr Pro Ala Gly Asn Gln Glu Met Glu Leu Phe Glu Leu Pro
605 610 615
Ala Thr Tyr Glu Ile Gly Ile Val Arg Gln Phe Pro Phe Ser Ser
620 625 630
Ala Leu Gln Arg Met Ser Val Val Ala Arg Val Leu Gly Asp Arg
635 640 645
Lys Met Asp Ala Tyr Met Lys Gly Ala Pro Glu Ala Ile Ala Gly
650 655 660
Leu Cys Lys Pro Glu Thr Val Pro Val Asp Phe Gln Asn Val Leu
665 670 675
Glu Asp Phe Thr Lys Gln Gly Phe Arg Val Ile Ala Leu Ala His
680 685 690
Arg Lys Leu Glu Ser Lys Leu Thr Trp His Lys Val Gln Asn Ile
695 700 705
Ser Arg Asp Ala Ile Glu Asn Asn Met Asp Phe Met Gly Leu Ile
710 715 720
Ile Met Gln Asn Lys Leu Lys Gln Glu Thr Pro Ala Val Leu Glu
725 730 735
Asp Leu His Lys Ala Asn Ile Arg Thr Val Met Val Thr Gly Asp
740 745 750
Ser Met Leu Thr Ala Val Ser Val Ala Arg Asp Cys Gly Met Ile
755 760 765
Leu Pro Gln Asp Lys Val Ile Ile Ala Glu Ala Leu Pro Pro Lys
770 775 780
Asp Gly Lys Val Ala Lys Ile Asn Trp His Tyr Ala Asp Ser Leu
785 790 795
Thr Gln Cys Ser His Pro Ser Ala Ile Asp Pro Glu Ala Ile Pro
800 805 810
Val Lys Leu Val His Asp Ser Leu Glu Asp Leu Gln Met Thr Arg
815 820 825
Tyr His Phe Ala Met Asn Gly Lys Ser Phe Ser Val Ile Leu Glu
830 835 840
His Phe Gln Asp Leu Val Pro Lys Leu Met Leu His Gly Thr Val
845 850 855
Phe Ala Arg Met Ala Pro Asp Gln Lys Thr Gln Leu Ile Glu Ala
860 865 870
25/65
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Leu Gln Asn Val Asp Tyr Phe Val Gly Met Cys Gly Asp Gly Ala
875 880 885
Asn Asp Cys Gly Ala Leu Lys Arg Ala His Gly Gly Ile Ser Leu
890 895 900
Ser Glu Leu Glu Ala Ser Val Ala Ser Pro Phe Thr Ser Lys Thr
905 910 ~ 915
Pro Ser Ile Ser Cys Val Pro Asn Leu Ile Arg Glu Gly Arg Ala
920 925 930
Ala Leu Ile Thr Ser Phe Cys Val Phe Lys Phe Met Ala Leu Tyr
935 940 945
Ser Ile Ile Gln Tyr Phe Ser Val Thr Leu Leu Tyr Ser Ile Leu
950 955 960
Ser Asn Leu Gly Asp Phe Gln Phe Leu Phe Ile Asp Leu Ala Ile
965 970 975
Ile Leu Val Val Va1 Phe Thr Met Ser Leu Asn Pro Ala Trp Lys
980 985 990
Glu Leu Val Ala Gln Arg Pro Pro Ser Gly Leu Ile Ser Gly Ala
995 1000 1005
Leu Leu Phe Ser Val Leu Ser Gln Ile Ile Ile Cys Ile Gly Phe
1010 1015 1020
Gln Ser Leu Gly Phe Phe Trp Val Lys Gln Gln Pro Trp Tyr Glu
1025 1030 1035
Val Trp His Pro Lys Ser Asp Ala Cys Asn Thr Thr Gly Ser Gly
1040 1045 1050
Phe Trp Asn Ser Ser His Val Asp Asn Glu Thr Glu Leu Asp Glu
1055 1060 1065
His Asn Ile Gln Asn Tyr Glu Asn Thr Thr Val Phe Phe Ile Ser
1070 1075 1080
Ser Phe Gln Tyr Leu Ile Val Ala Ile Ala Phe Ser Lys Gly Lys
1085 1090 1095
Pro Phe Arg Gln Pro Cys Tyr Lys Asn Tyr Phe Phe Val Phe Ser
1100 1105 1110
Val Ile Phe Leu Tyr Ile Phe Ile Leu Phe Ile Met Leu Tyr Pro
1115 1120 1125
Val Ala Ser Va1 Asp Gln Val Leu Gln Ile Val Cys Val Pro Tyr
1130 1135 1140
Gln Trp Arg Val Thr Met Leu Ile Ile Val Leu Val Asn Ala Phe
1145 1150 1155
Val Ser Ile Thr Val Glu Glu Ser Val Asp Arg Trp Gly Lys Cys
1160 1165 1170
Cys Leu Pro Trp Ala Leu Gly Cys Arg Lys Lys Thr Pro Lys Ala
1175 1180 1185
Lys Tyr Met Tyr Leu Ala Gln Glu Leu Leu Val Asp Pro Glu Trp
1190 1195 1200
Pro Pro Lys Pro Gln Thr Thr Thr Glu Ala Lys Ala Leu Val Lys
1205 1210 ' 1215
Glu Asn Gly Ser Cys Gln Ile Ile Thr Ile Thr
1220 1225
<210> 15
<211> 638
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
26/65
CA 02458625 2004-02-16
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<223> Incyte ID No: 6243872CD1
<400> 15
Met Phe Val Gly Val Ala Arg His Ser Gly Ser Gln Asp Glu Val
1 5 10 15
Ser Arg Gly Val Glu Pro Leu Glu Ala Ala Arg Ala Gln Pro Ala
20 25 30
Lys Asp Arg Arg Ala Lys Gly Thr Pro Lys Ser Ser Lys Pro Gly
35 40 45
Lys Lys His Arg Tyr Leu Arg Leu Leu Pro Glu Ala Leu Ile Arg
50 55 60
Phe Gly Gly Phe Arg Lys Arg Lys Lys Ala Lys Ser Ser Val Ser
65 70 75
Lys Lys Pro Gly Glu Val Asp Asp Ser Leu Glu Gln Pro Cys Gly
80 85 90
Leu Gly Cys Leu Val Ser Thr Cys Cys Glu Cys Cys Asn Asn Ile
95 100 105
Arg Cys Phe Met Ile Phe Tyr Cys Ile Leu Leu Ile Cys Gln Gly
110 115 120
Val Val Phe Gly Leu Ile Asp Val Ser Ile Gly Asp Phe Gln Lys
125 130 135
Glu Tyr Gln Leu Lys Thr Ile Glu Lys Leu Ala Leu Glu Lys Ser
140 145 150
Tyr Asp Ile Ser Ser Gly Leu Thr Val Gln Gly Ile Ala Gly Met
155 160 165
Pro Leu Tyr Ile Leu Gly Ile Thr Phe Ile Asp Glu Asn Val Ala
170 175 180
Thr His Ser Ala Gly Ile Tyr Leu Gly Ile Ala Glu Cys Thr Ser
185 190 195
Met Ile Gly Tyr Ala Leu Gly Tyr Val Leu Gly Ala Pro Leu Val
200 205 210
Lys Val Pro Glu Asn Thr Thr Ser Ala Thr Asn Thr Thr Val Asn
215 220 225
Asn Gly Ser Pro Glu Trp Leu Trp Thr Trp Trp Ile Asn Phe Leu
230 235 240
Phe Ala Ala Val Val Ala Trp Cys Thr Leu Ile Pro Leu Ser Cys
245 250 255
Phe Pro Asn Asn Met Pro Gly Ser Thr Arg Ile Lys Ala Arg Lys
260 265 270
Arg Lys Gln Leu His Phe Phe Asp Ser Arg Leu Lys Asp Leu Lys
275 280 285
Leu Gly Ile Asn Ile Lys Asp Leu Cys Ala Ala Leu Trp Ile Leu
290 295 300
Met Lys Asn Pro Val Leu Ile Cys Leu Ala Leu Ser Lys Ala Thr
305 310 315
Glu Tyr Leu Val Ile Ile Gly Ala Ser Glu Phe Leu Pro Ile Tyr
320 325 330
Leu Glu Asn Gln Phe Ile Leu Thr Pro Thr Val Ala Thr Thr Leu
335 340 345
Ala Gly Leu Val Leu Ile Pro Gly Gly Ala Leu Gly Gln Leu Leu
350 355 360
Gly Gly Val Ile Val Ser Thr Leu Glu Met Ser Cys Lys Ala Leu
365 370 375
Met Arg Phe Ile Met Val Thr Ser Val Ile Ser Leu Ile Leu Leu
380 385 390
Val Phe Ile Ile Phe Val Arg Cys Asn Pro Val Gln Phe Ala Gly
27/65
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395 400 405
Ile Asn Glu Asp Tyr Asp Gly Thr Gly Lys Leu Gly Asn Leu Thr
410 415 420
Ala Pro Cys Asn Glu Lys Cys Arg Cys Ser Ser Ser Ile Tyr Ser
425 430 435
Ser Ile Cys Gly Arg Asp Asp Ile Glu Tyr Phe Ser Pro Cys Phe
440 445 450
Ala Gly Ile Val Ser Cys Leu Gln Tyr Ser Gln Met Tyr Tyr Asn
455 460 465
Cys Ser Cys Ile Lys Glu Gly Leu Ile Thr Ala Asp Ala Glu Gly
470 475 480
Asp Phe Ile Asp Ala Arg Pro Gly Lys Cys Asp Ala Lys Cys Tyr
485 490 495
Lys Leu Pro Leu Phe Ile Ala Phe Ile Phe Ser Thr Leu Ile Phe
500 505 510
Ser Gly Phe Ser Gly Val Pro Ile Val Leu Ala Met Thr Arg Val
515 520 525
Val Pro Asp Lys Leu Arg Ser Leu Ala Leu Gly Val Ser Tyr Val
530 535 540
Ile Leu Arg Ile Phe Gly Thr Ile Pro Gly Pro Ser Ile Phe Lys
545 550 555
Met Ser Gly Glu Thr Ser Cys Ile Leu Arg Asp Val Asn Lys Cys
560 565 570
Gly His Thr Gly Arg Cys Trp Ile Tyr Asn Lys Thr Lys Met Ala
575 580 585
Phe Leu Leu Val Gly Ile Cys Phe Leu Cys Lys Leu Cys Thr Ile
590 595 600
Ile Phe Thr Thr Ile Ala Phe Phe Ile Tyr Lys Arg Arg Leu Asn
605 610 615
Glu Asn Thr Asp Phe Pro Asp Val Thr Val Lys Asn Pro Lys Val
620 625 630
Lys Lys Lys Glu Glu Thr Asp Leu
635
<210> 16
<211> 507
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 90011608CD1
<400> 16
Met Thr Ala Ser Thr Pro Glu Ala Thr Pro Asn Met Glu Leu Lys
1 5 10 15
Ala Pro Ala Ala Gly Gly Leu Asn Ala Gly Pro Val Pro Pro Ala
20 25 30
Ala Leu Ser Thr Gln Arg Leu Arg Asn Glu Asp Tyr His Asp Tyr
35 40 45
Ser Ser Thr Asp Val Ser Pro Glu Glu Ser Pro Ser Glu Gly Leu
50 55 60
Asn Asn Leu Ser Ser Pro Gly Ser Tyr Gln Arg Phe Gly Gln Ser
65 70 75
Asn Ser Thr Thr Trp Phe Gln Thr Leu Ile His Leu Leu Lys Gly
80 85 90
28/65
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Asn Ile Gly Thr Gly Leu Leu Gly Leu Pro Leu Ala Val Lys Asn
95 100 105
Ala Gly Ile Val Met Gly Pro Ile Ser Leu Leu Ile Ile Gly Ile
110 115 120
Val Ala Val His Cys Met Gly Ile Leu Val Lys Cys Ala His His
125 130 135
Phe Cys Arg Arg Leu Asn Lys Ser Phe Val Asp Tyr Gly Asp Thr
140 145 ' 150
Val Met Tyr Gly Leu Glu Ser Ser Pro Cys Ser Trp Leu Arg Asn
155 160 165
His Ala His Trp Gly Arg Arg Val Val Asp Phe Phe Leu Ile Val
170 175 180
Thr Gln Leu Gly Phe Cys Cys Val Tyr Phe Val Phe Leu Ala Asp
185 190 195
Asn Phe Lys Gln Val Ile Glu Ala Ala Asn Gly Thr Thr Asn Asn
200 205 210
Cys His Asn Asn Glu Thr Val Ile Leu Thr Pro Thr Met Asp Ser
215 220 225
Arg Leu Tyr Met Leu Ser Phe Leu Pro Phe Leu Val Leu Leu Val
230 235 240
Phe Ile Arg Asn Leu Arg Ala Leu Ser Ile Phe Ser Leu Leu Ala
245 250 255
Asn Ile Thr Met Leu Val Ser Leu Val Met Ile Tyr Gln Phe Ile
260 265 270
Val Gln Arg Ile Pro Asp Pro Ser His Leu Pro Leu Val Ala Pro
275 280 285
Trp Lys Thr Tyr Pro Leu Phe Phe Gly Thr Ala Ile Phe Ser Phe
290 295 300
Glu Gly Ile Gly Met Val Leu Pro Leu Glu Asn Lys Met Lys Asp
305 310 315
Pro Arg Lys Phe Pro Leu Ile Leu Tyr Leu Gly Met Val Ile Val
320 325 330
Thr Ile Leu Tyr Ile Ser Leu Gly Cys Leu Gly Tyr Leu Gln Phe
335 340 345
Gly Ala Asn Ile Gln Gly Ser Ile Thr Leu Asn Leu Pro Asn Cys
350 355 360
Trp Leu Tyr Gln Ser Val Lys Leu Leu Tyr Ser Ile Gly Ile Phe
365 370 375
Phe Thr Tyr Ala Leu Gln Phe Tyr Val Pro Ala Glu Ile Ile Ile
380 385 390
Pro Phe Phe Val Ser Arg Ala Pro Glu His Cys Glu Leu Val Val
395 400 405
Asp Leu Phe Val Arg Thr Val Leu Val Cys Leu Thr Cys Ile Leu
410 415 420
Ala Ile Leu Ile Pro Arg Leu Asp Leu Val Ile Ser Leu Val Gly
425 430 435
Ser Val Ser Ser Ser Ala Leu Ala Leu Ile Ile Pro Pro Leu Leu
440 445 450
Glu Val Thr Thr Phe Tyr Ser Glu Gly Met Ser Pro Leu Thr Ile
455 460 465
Phe Lys Asp Ala Leu Ile Ser Ile Leu Gly Phe Val Gly Phe Val
470 475 480
Val Gly Thr Tyr Glu Ala Leu Tyr Glu Leu Ile Gln Pro Ser Asn
485 490 495
Ala Pro Ile Phe Ile Asn Ser Thr Cys Ala Phe Ile
500 505
29/65
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<210> 17
<211> 568
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 90024583CD1
<400> 17
Met Ala Ser Ala Leu Ser Tyr Val Ser Lys Phe Lys Ser Phe Val
1 5 ~ 10 15
Ile Leu Phe Val Thr Pro Leu Leu Leu Leu Pro Leu Val Ile Leu
20 25 30
Met Pro Ala Lys Phe Val Arg Cys Ala Tyr Val Ile Ile Leu Met
35 40 45
Ala Ile Tyr Trp Cys Thr Glu Val Ile Pro Leu Ala Val Thr Ser
50 55 60
Leu Met Pro Val Leu Leu Phe Pro Leu Phe Gln Ile Leu Asp Ser
65 70 75
Arg Gln Val Cys Val Gln Tyr Met Lys Asp Thr Asn Met Leu Phe
80 85 90
Leu Gly Gly Leu Ile Val Ala Val Ala Val Glu Arg Trp Asn Leu
95 100 105
His Lys Arg Ile Ala Leu Arg Thr Leu Leu Trp Val Gly Ala Lys
110 115 120
Pro Ala Arg Leu Met Leu Gly Phe Met Gly Val Thr Ala Pro Leu
125 130 135
Ser Met Trp Ile Ser Asn Thr Ala Thr Thr Ala Met Met Val Pro
140 145 150
Ile Val Glu Ala Ile Leu Gln Gln Met Glu Ala Thr Ser Ala Ala
155 160 165
Thr Glu Ala Gly Leu Glu Leu Val Asp Lys Gly Lys Ala Lys Glu
170 175 180
Leu Pro Gly Ser Gln Val Ile Phe Glu Gly Pro Thr Leu Gly Gln
185 190 195
Gln Glu Asp Gln Glu Arg Lys Arg Leu Cys Lys Ala Met Thr Leu
200 205 210
Cys Ile Cys Tyr Ala Ala Ser Ile Gly Gly Thr Ala Thr Leu Thr
215 220 225
Gly Thr Gly Pro Asn Val Val Leu Leu Gly Gln Met Asn Glu Leu
230 235 240
Phe Pro Asp Ser Lys Asp Leu Val Asn Phe Ala Ser Trp Phe Ala
245 250 255
Phe Ala Phe Pro Asn Met Leu Val Met Leu Leu Phe Ala Trp Leu
260 265 270
Trp Leu Gln Phe Val Tyr Met Arg Phe Asn Phe Lys Lys Ser Trp
275 280 285
Gly Cys Gly Leu Glu Ser Lys Lys Asn Glu Lys Ala Ala Leu Lys
290 295 300
Val Leu Gln Glu Glu Tyr Arg Lys Leu Gly Pro Leu Ser Phe Ala
305 310 315
Glu Ile Asn Val Leu Ile Cys Phe Phe Leu Leu Val Ile Leu Trp
320 325 330
Phe Ser Arg Asp Pro Gly Phe Met Pro Gly Trp Leu Thr Val Ala
335 340 345
30/65
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Trp Val Glu Gly Glu Thr Lys Tyr Val Ser Asp Ala Thr Val Ala
350 355 360
Ile Phe Val Ala Thr Leu Leu Phe Ile Val Pro Ser Gln Lys Pro
365 370 375
Lys Phe Asn Phe Arg Ser Gln Thr Glu Glu Glu Arg Lys Thr Pro
380 385 390
Phe Tyr Pro Pro Pro Leu Leu Asp Trp Lys Val Thr Gln Glu Lys
395 400 405
Val Pro Trp Gly Ile Val Leu Leu Leu Gly Gly Gly Phe Ala Leu
410 415 420
Ala Lys Gly Ser Glu Ala Ser Gly Leu Ser Val Trp Met Gly Lys
425 430 435
Gln Met Glu Pro Leu His Ala Val Pro Pro Ala Ala Ile Thr Leu
440 445 450
Ile Leu Ser Leu Leu Val Ala Val Phe Thr Glu Cys Thr Ser Asn
455 460 465
Val Ala Thr Thr Thr Leu Phe Leu Pro Ile Phe Ala Ser Met Ser
470 475 480
Arg Ser Asn Gly Leu Asn Pro Leu Tyr Ile Met Leu Pro Cys Thr
485 490 495
Leu Ser Ala Ser Phe Ala Phe Met Leu Pro Val Ala Thr Pro Pro
500 505 510
Asn Ala Ile Val Phe Thr Tyr Gly His Leu Lys Val Ala Asp Met
515 520 525
Val Lys Thr Gly Val Ile Met Asn Ile Ile Gly Val Phe Cys Val
530 535 540
Phe Leu Ala Val Asn Thr Trp Gly Arg Ala Ile Phe Asp Leu Asp
545 550 555
His Phe Pro Asp Trp Ala Asn Val Thr His Ile Glu Thr
560 565
<210> 18
<211> 595
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 90113658CD1
<400> 18
Met Ile Leu Ile Lys Gln Lys Arg Leu Phe Pro Cys Trp Ile Pro
1 5 10 15
Ala Leu Phe Ile Gly Phe Ser Gln Phe Ser Asp Ser Phe Leu Leu
20 25 30
Asp Gln Pro Asn Phe Trp Cys Arg Gly Ala Gly Lys Gly Thr Glu
35 40 45
Leu Ala Gly Val Thr Thr Thr Gly Arg Gly Gly Asp Met Gly Asn
50 55 60
Trp Thr Ser Leu Pro Thr Thr Pro Phe Ala Thr Ala Pro Trp Glu
65 70 75
Ala Ala Gly Asn Arg Ser Asn Ser Ser Gly Ala Asp Gly Gly Asp
80 85 90
Thr Pro Pro Leu Pro Ser Pro Pro Asp Lys Gly Asp Asn Ala Ser
95 100 105
Asn Cys Asp Cys Arg Ala Trp Asp Tyr Gly Ile Arg Ala Gly Leu
31/65
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110 115 120
Val Gln Asn Val Val Ser Lys Trp Asp Leu Val Cys Asp Asn Ala
125 130 135
Trp Lys Val His Ile Ala Lys Phe Ser Leu Leu Val Gly Leu Ile
140 145 150
Phe Gly Tyr Leu Ile Thr Gly Cys Ile Ala Asp Trp Val Gly Arg
155 160 165
Arg Pro Val Leu Leu Phe Ser Ile Ile Phe Ile Leu Ile Phe Gly
170 175 180
Leu Thr Val Ala Leu Ser Val Asn Val Thr Met Phe Ser Thr Leu
185 190 195
Arg Phe Phe Glu Gly Phe Cys Leu Ala Gly Ile Ile Leu Thr Leu
200 205 210
Tyr Ala Leu Arg Ile Glu Leu Cys Pro Pro Gly Lys Arg Phe Met
215 220 225
Ile Thr Met Val Ala Ser Phe Val Ala Met Ala Gly Gln Phe Leu
230 235 240
Met Pro Gly Leu Ala Ala Leu Cys Arg Asp Trp Gln Val Leu Gln
245 250 255
Ala Leu Ile Ile Cys Pro Phe Leu Leu Met Leu Leu Tyr Trp Ser
260 265 270
Ile Phe Pro Glu Ser Leu Arg Trp Leu Met Ala Thr Gln Gln Phe
275 280 285
Glu Ser Ala Lys Arg Leu Ile Leu His Phe Thr Gln Lys Asn Arg
290 295 300
Met Asn Pro Glu Gly Asp Ile Lys Gly Val Ile Pro Glu Leu Glu
305 310 315
Lys Glu Leu Ser Arg Arg Pro Lys Lys Val Cys Ile Val Lys Val
320 325 330
Val Gly Thr Arg Asn Leu Trp Lys Asn Ile Val Val Leu Cys Val
335 340 345
Asn Ser Leu Thr Gly Tyr Gly Ile His His Cys Phe Ala Arg Ser
350 355 360
Met Met Gly His Glu Val Lys Val Pro Leu Leu Glu Asn Phe Tyr
365 370 375
Ala Asp Tyr Tyr Thr Thr Ala Ser Ile Ala Leu Val Ser Cys Leu
380 385 390
Ala Met Cys Val Val Val Arg Phe Leu Gly Arg Arg Gly Gly Leu
395 400 405
Leu Leu Phe Met Ile.Leu Thr Ala Leu Ala Ser Leu Leu Gln Leu
410 415 420
Gly Leu Leu Asn Leu Ile Gly Lys Tyr Ser Gln His Pro Asp Ser
425 430 435
Gly Met Ser Asp Ser Val Lys Asp Lys Phe Ser Ile Ala Phe Ser
440 445 450
Ile Val Gly Met Phe Ala Ser His Ala Val Gly Ser Leu Ser Val
455 460 465
Phe Phe Cys Ala.Glu Ile Thr Pro Thr Val Ile Arg Cys Gly Gly
470 475 480
Leu Gly Leu Val Leu Ala Ser Ala Gly Phe Gly Met Leu Thr Ala
485 490 495
Pro Ile Ile Glu Leu His Asn Gln Lys Gly Tyr Phe Leu His His
500 505 510
Ile Ile Phe Ala Cys Cys Thr Leu Ile Cys Ile Ile Cys Ile Leu
515 520 525
Leu Leu Pro Glu Ser Arg Asp Gln Asn Leu Pro Glu Asn Ile Ser
32/65
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530 535 540
Asn Gly Glu His Tyr Thr Arg Gln Pro Leu Leu Pro His Lys Lys
545 550 555
Gly Glu Gln Pro Leu Leu Leu Thr Asn Ala Glu Leu Lys Asp Tyr
560 565 570
Ser Gly Leu His Asp Ala Ala Ala Ala Gly Asp Thr Leu Pro Glu
575 580 585
Gly Ala Thr Ala Asn Gly Met Lys Ala Met
590 595
<210> 19
<211> 602
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3942766CD1
<400> 19
Met Ala Ala Leu Ala Ala Ala Ala Lys Lys Val Trp Ser Ala Arg
1 5 10 15
Arg Leu Leu Val Leu Leu Phe Thr Pro Leu Ala Leu Leu Pro Val
20 25 30
Val Phe Ala Leu Pro Pro Lys Glu Gly Arg Cys Leu Phe Val Ile
35 40 45
Leu Leu Met Ala Val Tyr Trp Cys Thr Glu Ala Leu Pro Leu Ser
50 55 60
Val Thr Ala Leu Leu Pro Ile Val Leu Phe Pro Phe Met Gly Ile
65 70 75
Leu Pro Ser Asn Lys Val Cys Pro Gln Tyr Phe Leu Asp Thr Asn
80 85 90
Phe Leu Phe Leu Ser Gly Leu Ile Met Ala Ser Ala Ile Glu Glu
95 100 105
Trp Asn Leu His Arg Arg Ile Ala Leu Lys Ile Leu Met Leu Val
110 115 120
Gly Val Gln Pro Ala Arg Leu Ile Leu Gly Met Met Val Thr Thr
125 130 135
Ser Phe Leu Ser Met Trp Leu Ser Asn Thr Ala Ser Thr Ala Met
140 145 150
Met Leu Pro Ile Ala Asn Ala Ile Leu Lys Ser Leu Phe Gly Gln
155 160 165
Lys Glu Val Arg Lys Asp Pro Ser Gln Glu Ser Glu Glu Asn Thr
170 175 180
Ala Ala Val Arg Arg Asn Gly Leu His Thr Val Pro Thr Glu Met
185 190 195
Gln Phe Leu Ala Ser Thr Glu Ala Lys Asp His Pro Gly Glu Thr
200 205 210
Glu Val Pro Leu Asp Leu Pro Ala Asp Ser Arg Lys Glu Asp Glu
215 220 225
Tyr Arg Arg Asn Ile Trp Lys Gly Phe Leu I1e Ser Ile Pro Tyr
230 235 240
Ser Ala Ser Ile Gly Gly Thr Ala Thr Leu Thr Gly Thr Ala Pro
245 250 255
Asn Leu Ile Leu Leu Gly Gln Leu Lys Ser Phe Phe Pro Gln Cys
260 265 270
33/65
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Asp Val Val Asn Phe Gly Ser Trp Phe Ile Phe Ala Phe Pro Leu
275 280 285
Met Leu Leu Phe Leu Leu Ala Gly Trp Leu Trp Ile Ser Phe Leu
290 295 300
Tyr Gly Gly Leu Ser Phe Arg Gly Trp Arg Lys Asn Lys Ser Glu
305 310 315
Ile Arg Thr Asn Ala Glu Asp Arg Ala Arg Ala Val Ile Arg Glu
320 325 330
Glu Tyr Gln Asn Leu Gly Pro Ile Lys Phe Ala Glu Gln Ala Val
335 340 345
Phe Ile Leu Phe Cys Met Phe Ala Ile Leu Leu Phe Thr Arg Asp
350 355 360
Pro Lys Phe Ile Pro Gly Trp Ala Ser Leu Phe Asn Pro Gly Phe
365 370 375
Leu Ser Asp Ala Val Thr Gly Val Ala Ile Val Thr Ile Leu Phe
380 385 390
Phe Phe Pro Ser Gln Arg Pro Ser Leu Lys Trp Trp Phe Asp Phe
395 400 405
Lys Ala Pro Asn Thr Glu Thr Glu Pro Leu Leu Thr Trp Lys Lys
410 415 420
Ala Gln Glu Thr Val Pro Trp Asn Ile Ile Leu Leu Leu Gly Gly
425 430 435
Gly Phe Ala Met Ala Lys Gly Cys Glu Glu Ser Gly Leu Ser Val
440 445 450
Trp Ile Gly Gly Gln Leu His Pro Leu Glu Asn Val Pro Pro Ala
455 460 465
Leu Ala Val Leu Leu Ile Thr Val Val Ile Ala Phe Phe Thr Glu
470 475 480
Phe Ala Ser Asn Thr Ala Thr Ile Ile Ile Phe Leu Pro Val Leu
485 490 495
Ala Glu Leu Ala Ile Arg Leu Arg Val His Pro Leu Tyr Leu Met
500 505 510
Ile Pro Gly Thr Val Gly Cys Ser Phe Ala Phe Met Leu Pro Val
515 520 525
Ser Thr Pro Pro Asn Ser Ile Ala Phe Ala Ser Gly His Leu Leu
530 535 540
Val Lys Asp Met Val Arg Thr Gly Leu Leu Met Asn Leu Met Gly
545 550 555
Val Leu Leu Leu Ser Leu Ala Met Asn Thr Trp Ala Gln Thr Ile
560 565 570
Phe Gln Leu Gly Thr Phe Pro Asp Trp Ala Asp Met Tyr Ser Val
575 580 585
Asn Val Thr Ala Leu Pro Pro Thr~Leu Ala Asn Asp Thr Phe Arg
590 595 600
Thr Leu
<210> 20
<211> 372
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7501987CD1
34/65
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<400> 20
Met Val Pro Ala Gly Trp Val Arg Gly Leu Glu Leu Ser Leu Trp
1 5 10 15
Gly Gly Asp Pro Val Val Pro Trp Ser Cys Arg Phe Cys Ser Gln
20 25 30
Gln Asp Asp Gly Gln Asp Arg Glu Arg Leu Thr Tyr Phe Gln Asn
35 40 45
Leu Pro Glu Ser Leu Thr Ser Leu Leu Val Leu Leu Thr Thr Ala
50 55 60
Asn Asn Pro Asp Val Met Ile Pro Ala Tyr Ser Lys Asn Arg Ala
65 70 75
Tyr Ala Ile Phe Phe Ile Val Phe Thr Val Ile Gly Ser Leu Phe
80 85 90
Leu Met Asn Leu Leu Thr Ala Ile Ile Tyr Ser Gln Phe Arg Gly
95 100 . 105
Tyr Leu Met Lys Ser Leu Gln Thr Ser Leu Phe Arg Arg Arg Leu
110 115 120
Gly Thr Arg Ala Ala Phe Glu Val Leu Ser Ser Met Val Gly Glu
125 130 135
Gly Gly Ala Phe Pro Gln Ala Val Gly Val Lys Pro Gln Asn Leu
140 145 150
Leu Gln Val Leu Gln Lys Val Gln Leu Asp Ser Ser His Lys Gln
155 160 165
Ala Met Met Glu Lys Val Arg Ser Tyr Gly Ser Val Leu Leu Ser
170 175 180
Ala Glu Glu Phe Gln Lys Leu Phe Asn Glu Leu Asp Arg Ser Val
185 190 195
Val Lys Glu His Pro Pro Arg Pro Glu Tyr Gln Ser Pro Phe Leu
200 205 210
Gln Ser Ala Gln Phe Leu Phe Gly His Tyr Tyr Phe Asp Tyr Leu
215 220 225
Gly Asn Leu Ile Ala Leu Ala Asn Leu Val Ser Ile Cys Val Phe
230 235 240
Leu Val Leu Asp Ala Asp Val Leu Pro Ala Glu Arg Asp Asp Phe
245 250 255
Ile Leu Gly Ile Leu Asn Cys Val Phe Ile Val Tyr Tyr Leu Leu
260 265 270
Glu Leu Leu Leu Lys Val Phe Ala Leu Gly Leu Arg Gly Tyr Leu
275 280 285
Ser Tyr Pro Ser Asn Val Phe Asp Gly Leu Leu Thr Val Val Leu
290 295 300
Leu Glu Ala Gly Asp Gly Gly Pro Ala Val Ala Val Gly His Asp
305 310 315
Pro His Ala Glu His Ala His Arg Val Pro Leu Pro Ala Tyr His
320 325 330
Pro Gln His Glu Ala Asp Gly Arg Gly Gly Gln Tyr Arg Pro Gly
335 340 345
Pro Gly Ala Glu His Ala Cys Val Trp Arg Asp Pro Gly Gly Gly
350 355 360
Leu Leu Arg Ile Cys His His Trp Asp Gln Leu Val
365 370
<210> 21
<211> 165
<212> PRT
<213> Homo Sapiens
35/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
<220>
<221> misc_feature
<223> Incyte ID No: 7503223CD1
<400> 21
Met Thr Leu Leu Pro Gly Asp Asn Ser Asp Tyr Asp Tyr Ser Ala
1 5 10 15
Leu Ser Cys Thr Ser Asp Ala Ser Phe His Pro Ala Phe Leu Pro
20 25 30
Gln Arg Gln Ala Ile Lys Gly Ala Phe Tyr Arg Arg Ala Gln Arg
35 40 45
Leu Arg Pro Gln Asp Glu Pro Arg Gln Gly Cys Gln Pro Glu Asp
50 55 60
Arg Arg Arg Arg Ile Ile Ile Asn Val Gly Gly Ile Lys Tyr Ser
65 70 75
Leu Pro Trp Thr Thr Leu Asp Glu Phe Pro Leu Thr Arg Leu Gly
80 85 90
Gln Leu Lys Ala Cys Thr Asn Phe Asp Asp Ile Leu Asn Val Cys
95 100 105
Asp Asp Tyr Asp Val Thr Cys Asn Glu Phe Phe Phe Asp Arg Asn
110 115 120
Pro Gly Ala Phe Gly Thr Ile Leu Thr Phe Leu Arg Ala Gly Lys
125 130 135
Leu Arg Leu Leu Arg Glu Met Cys Ala Leu Ser Phe Gln Asp Ser
140 145 150
Asp Ile Leu Phe Gly Ser Ala Ser Ser Asp Thr Arg Asp Asn Asn
155 160 165
<210> 22
<211> 497
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503566CD1
<400> 22
Met Leu Arg Thr Ile Leu Asp Ala Pro Gln Arg Leu Leu Lys Glu
1 5 10 15
Gly Arg Ala Ser Arg Gln Leu Val Leu Val Val Val Phe Val Ala
20 25 30
Leu Leu Leu Asp Asn Met Leu Phe Thr Val Val Val Pro Ile Val
35 40 45
Pro Thr Phe Leu Tyr Asp Met Glu Phe Lys Glu Val Asn Ser Ser
50 55 60
Leu His Leu Gly His Ala Gly Ser Ser Pro His Ala Leu Ala Ser
65 70 75
Pro Ala Phe Ser Thr Ile Phe Ser Phe Phe Asn Asn Asn Thr Val
80 85 90
Ala Val Glu Glu Ser Val Pro Ser Gly Ile Ala Trp Met Asn Asp
95 100 105
Thr Ala Ser Thr Ile Pro Pro Pro Ala Thr G1u Ala Ile Ser Ala
110 115 120
His Lys Asn Asn Cys Leu Gln Gly Thr Gly Phe Leu Glu Glu Glu
36/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
125 130 135
Thr Thr Arg Val Gly Val Leu Phe Ala Ser Lys Ala Val Met Gln
140 145 150
Leu Leu Val Asn Pro Phe Val Gly Pro Leu Thr Asn Arg Ile Gly
155 160 165
Tyr His Ile Pro Met Phe Ala Gly Phe Val Ile Met Phe Leu Ser
170 175 180
Thr Val Ser Leu Gly Met Leu Ala Ser Val Tyr Thr Asp Asp His
185 190 195
Glu Arg Gly Arg Ala Met Gly Thr Ala Leu Gly Gly Leu Ala Leu
200 205 210
Gly Leu Leu Val Gly Ala Pro Phe Gly Ser Val Met Tyr Glu Phe
215 220 225
Val Gly Lys Ser Ala Pro Phe Leu Ile Leu Ala Phe Leu Ala Leu
230 235 240
Leu Asp Gly Ala Leu Gln Leu Cys Ile Leu Gln Pro Ser Lys Val
245 250 255
Ser Pro Glu Ser Ala Lys Gly Thr Pro Leu Phe Met Leu Leu Lys
260 265 270
Asp Pro Tyr Ile Leu Val Ala Ala Gly Ser Ile Cys Phe Ala Asn
275 280 285
Met Gly Val Ala Ile Leu Glu Pro Thr Leu Pro Ile Trp Met Met
290 295 300
Gln Thr Met Cys Ser Pro Lys Trp Gln Leu Gly Leu Ala Phe Leu
305 310 315
Pro Ala Ser Val Ser Tyr Leu Ile Gly Thr Asn Leu Phe Gly Val
320 325 330
Leu Ala Asn Lys Met Gly Arg Trp Leu Cys Ser. Leu Ile Gly Met
335 340 345
Leu Val Val Gly Thr Ser Leu Leu Cys Val Pro Leu Ala His Asn
350 355 360
Ile Phe Gly Leu Ile Gly Pro Asn Ala Gly Leu Gly Leu Ala Ile
365 370 375
Gly Met Val Asp Ser Ser Met Met Pro Ile Met Gly His Leu Val
380 385 390
Asp Leu Arg His Thr Ser Val Tyr Gly Ser Val Tyr Ala Ile Ala
395 400 405
Asp Val Ala Phe Cys Met Gly Phe Ala Ile Gly Pro Ser Thr Gly
410 415 420
Gly Ala Ile Val Lys Ala Ile Gly Phe Pro Trp Leu Met Val Ile
425 430 435
Thr Gly Val Ile Asn Ile Val Tyr Ala Pro Leu Cys Tyr Tyr Leu
440 445 450
Arg Ser Pro Pro Ala Lys Glu Glu Lys Leu Ala Ile Leu Ser Gln
455 460 465
Asp Cys Pro Met Glu Thr Arg Met Tyr Ala Thr Gln Lys Pro Thr
470 475 480
Lys Glu Phe Pro Leu Gly Glu Asp Ser Asp Glu Glu Pro Asp His
485 490 495
Glu Glu
<210> 23
<211> 67
<212> PRT
<213> Homo Sapiens
37/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
<220>
<221> misc_feature
<223> Incyte ID No: 7505122CD1
<400> 23
Met Gln Lys Val Thr Leu Gly Leu Leu Val Phe Leu Ala Gly Phe
1 5 10 15
Pro Val Leu Asp Ala Asn Asp Leu Glu Asp Lys Asn Ser Pro Phe
20 25 30
Tyr Tyr Asp Trp His Ser Leu Gln Val Gly Gly Leu Ile Cys Ala
35 40 45
Gly Val Leu Cys Met Ala Gly Pro His Leu Thr Ser Arg Lys Arg
50 55 60
Val Ser Leu Phe Asn Phe Phe
<210> 24
<211> 152
<212> PRT
~213> Homo sapiens
~220>
<221> misc_feature
<223> Incyte ID No: 7511620CD1
<400> 24
Met Gly Leu Ala Asp Ala Ser Gly Pro Arg Asp Thr Gln Ala Leu
1 5 10 15
Leu Ser Ala Thr Gln Ala Met Asp Leu Arg Arg Arg Asp Tyr His
20 25 30
Met Glu Arg Pro Leu Leu Asn Gln Glu His Leu Glu Glu Leu Gly
35 40 45
Arg Trp Gly Ser Ala Pro Arg Thr His Gln Trp Arg Thr Trp Leu
50 55 60
Gln Cys Ser Arg Ala Arg Ala Tyr Ala Leu Leu Leu Gln His Leu
65 70 75
Pro Val Leu Val Trp Leu Pro Arg Tyr Pro Val Arg Asp Trp Leu
80 85 90
Leu Gly Asp Leu Leu~Ser Gly Leu Ser Val Ala Ile Met Gln Leu
95 100 105
Pro Gln Gly Leu Ala Tyr Ala Leu Leu Ala Gly Leu Pro Pro Val
110 115 120
Phe Gly Leu Tyr Ser Ser Phe Tyr Pro Val Phe Ile Tyr Phe Leu
125 130 135
Phe Gly Thr Ser Arg His Ile Ser Val Gly Leu Glu Arg Leu His
140 145 150
Asp Gln
<210> 25
<211> 467
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
38/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
~223> Incyte ID No: 7506995CD1
<400> 25
Met Val Pro Ala Gly Trp Val Arg Gly Leu Glu Leu Ser Leu Trp
1 5 10 15
Gly Gly Asp Pro Val Val Pro Trp Ser Cys Arg Phe Cys Ser Gln
20 25 30
Gln Asp Asp Gly Gln Asp Arg Glu Arg Leu Thr Tyr Phe Gln Asn
35 40 45
Leu Pro Glu Ser Leu Thr Ser Leu Leu Val Leu Leu Thr Thr Ala
50 55 60
Asn Asn Pro Asp Val Met Ile Pro Ala Tyr Ser Lys Asn Arg Ala
65 70 75
Tyr Ala Ile Phe Phe Ile Val Phe Thr Val Ile Gly Ser Leu Phe
80 85 90
Leu Met Asn Leu Leu Thr Ala Ile Ile Tyr Ser Gln Phe Arg Gly
95 100 105
Tyr Leu Met Lys Ser Leu Gln Thr Ser Leu Phe Arg Arg Arg Leu
110 115 120
Gly Thr Arg Ala Ala Phe Glu Val Leu Ser Ser Met Val Gly Glu
125 130 135
Gly Gly Ala Phe Pro Gln Ala Thr Arg Arg Gly Pro Ser Thr Ser
140 145 150
Leu Arg Phe Cys Arg Ala Pro Ser Ser Ser Ser Ala Thr Thr Thr
155 160 165
Leu Thr Thr Trp Gly Thr Ser Ser Pro Trp Gln Thr Trp Cys Pro
170 175 180
Phe Ala Cys Ser Trp Cys Trp Met Gln Met Cys Cys Leu Leu Ser
185 190 195
Val Met Thr Ser Ser Trp Gly Phe Ser Thr Ala Ser Ser Leu Cys
200 205 210
Thr Thr Cys Trp Arg Cys Cys Ser Arg Ser Leu Pro Trp Ala Cys
215 220 225
Glu Gly Thr Cys Pro Thr Pro Ala Thr Cys Leu Thr Gly Ser Ser
230 235 240
Pro Leu Ser Cys Trp Arg Pro Glu Met Val Gly Leu Leu Ser Leu
245 250 255
Trp Asp Met Thr Arg Met Leu Asn Met Leu Ile Val Phe Arg Phe
260 265 270
Leu Arg Ile Ile Pro Ser Met Lys Pro Met Ala Val Val Ala Ser
275 280 285
Thr Val Leu Gly Leu Val Gln Asn Met Arg Ala Phe Gly Gly Ile
290 295 300
Leu Val Val Val Tyr Tyr Val Phe Ala Ile Ile Gly Ile Asn Leu
305 310 315
Phe Arg Gly Val Ile Val Ala Leu Pro Gly Asn Ser Ser Leu Ala
320 325 330
Pro Ala Asn Gly Ser Ala Pro Cys Gly Ser Phe Glu Gln Leu Glu
335 340 345
Tyr Trp Ala Asn Asn Phe Asp Asp Phe Ala Ala Ala Leu Val Thr
350 355 360
Leu Trp Asn Leu Met Val Val Asn Asn Trp Gln Val Phe Leu Asp
365 370 375
Ala Tyr Arg Arg Tyr Ser Gly Pro Trp Ser Lys Ile Tyr Phe Val
380 385 390
Leu Trp Trp Leu Val Ser Ser Val Ile Trp Val Asn Leu Phe Leu
39/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
395 400 405
Ala Leu Ile Leu Glu Asn Phe Leu His Lys Trp Asp Pro Arg Ser
410 415 420
His Leu Gln Pro Leu Ala Gly Thr Pro Glu Ala Thr Tyr Gln Met
425 430 435
Thr Val Glu Leu Leu Phe Arg Asp Ile Leu Glu Glu Pro Gly Glu
440 445 450
Asp Glu Leu Thr Glu Arg Leu Ser Gln His Pro His Leu Trp Leu
455 460 465
Cys Arg
<210> 26
<211> 490
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7506996CD1
<400> 26
Met Val Pro Ala Gly Trp Val Arg Gly Leu Glu Leu Ser Leu Trp
1 5 . 10 15
Gly Gly Asp Pro Val Val Pro Trp Ser Cys Arg Phe Cys Ser Gln
20 25 30
Gln Asp Asp Gly Gln Asp Arg Glu Arg Leu Thr Tyr Phe Gln Asn
35 40 45
Leu Pro Glu Ser Leu Thr Ser Leu Leu Val Leu Leu Thr Thr Ala
50 55 60
Asn Asn Pro Asp Val Met Ile Pro Ala Tyr Ser Lys Asn Arg Ala
65 70 75
Tyr Ala Ile Phe Phe Ile Val Phe Thr Val Ile Gly Ser Leu Phe
80 85 90
Leu Met Asn Leu Leu Thr Ala Ile Ile Tyr Ser Gln Phe Arg Gly
95 100 105
Tyr Leu Met Lys Ser Leu Gln Thr Ser Leu Phe Arg Arg Arg Leu
110 115 120
Gly Thr Arg Ala Ala Phe Glu Val Leu Ser Ser Met Val Gly Glu
125 130 135
Gly Gly Ala Phe Pro Gln Ala Val Gly Val Lys Pro Gln Asn Leu
140 145 150
Leu Gln Val Leu Gln Lys Val Gln Leu Asp Ser Ser His Lys Gln
155 160 165
Ala Met Met Glu Lys Val Arg Ser Tyr Gly Ser Val Leu Leu Ser
170 175 180
Ala Glu Glu Phe Gln Lys Leu Phe Asn Glu Leu Asp Arg Ser Val
185 190 195
Val Lys Glu His Pro Pro Arg Pro Glu Tyr Gln Ser Pro Phe Leu
200 205 210
Gln Ser Ala Gln Phe Leu Phe Gly His Tyr Tyr Phe Asp Tyr Leu
215 220 225
Gly Asn Leu Ile Ala Leu Ala Asn Leu Val Ser Ile Cys Val Phe
230 235 240
Leu Val Leu Asp Ala Asp Val Leu Pro Ala Glu Arg Asp Asp Phe
245 250 255
40/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
Ile Leu Gly Ile Leu Asn Cys Val Phe Ile Val Tyr Tyr Leu Leu
260 265 270
Glu Leu Leu Leu Lys Val Phe Ala Leu Gly Leu Arg Gly Tyr Leu
275 280 285
Ser Tyr Pro Ser Asn Val Phe Asp Gly Leu Leu Thr Val Val Leu
290 295 300
Leu Pro Met Ala Val Val Ala Ser Thr Val Leu Gly Leu Val Gln
305 310 315
Asn Met Arg Ala Phe Gly Gly Ile Leu Val Val Val Tyr Tyr Val
320 325 330
Phe Ala Ile Ile Gly Ile Asn Leu Phe Arg Gly Val Ile Val Ala
335 340 345
Leu Pro Gly Asn Ser Ser Leu Ala Pro Ala Asn Gly Ser Ala Pro
350 355 360
Cys Gly Ser Phe Glu Gln Leu Glu Tyr Trp Ala Asn Asn Phe Asp
365 370 375
Asp Phe Ala Ala Ala Leu Val Thr Leu Trp Asn Leu Met Val Val
380 385 390
Asn Asn Trp Gln Val Phe Leu Asp Ala Tyr Arg Arg Tyr Ser Gly
395 400 405
Pro Trp Ser Lys Ile Tyr Phe Val Leu Trp Trp Leu Val Ser Ser
410 415 420
Val Ile Trp Val Asn Leu Phe Leu Ala Leu Ile Leu Glu Asn Phe
425 430 435
Leu His Lys Trp Asp Pro Arg Ser His Leu Gln Pro Leu Ala Gly
440 445 450
Thr Pro Glu Ala Thr Tyr Gln Met Thr Val Glu Leu Leu Phe Arg
455 460 465
Asp Ile Leu Glu Glu Pro Gly Glu Asp Glu Leu Thr Glu Arg Leu
470 475 480
Ser Gln His Pro His Leu Trp Leu Cys Arg
485 490
<210> 27
<211> 2343
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1853191CB1
<400> 27
cgcgtacagg aggcggcggc ggctcccagt caccggcccc cgccggcgag cgcacgatgc 60
actgcctggg cgccgagtac ctggtttctg cagaaggagc ccctaggcaa agggagtggc 120
gaccccagat ttataggaaa tgcacagata cggcatggtt attcctgttc tttctctttt 180
ggactggttt ggtgtttatc atgggctact cggtggtggc tggagccgcg ggaagactcc 240
tctttggcta tgacagcttt ggcaacatgt gtggcaagaa gaactccccc gtggaagggg 300
cccctctttc agggcaggac atgaccctaa aaaaacacgt gttctttatg aattcctgca 360
acctggaagt caaaggtacg cagctcaacc gcatggccct ctgtgtatcc aactgccctg 420
aagagcagct tgactccctg gaagaggtcc agttctttgc aaacaccagt gggtccttcc 480
tgtgtgttta tagtttgaat tccttcaact atacccacag tccaaaagca gactcactgt 540
gtcccaggct accagttcct ccaagcaagt catttccctt atttaaccga tgtgtccctc 600
aaacacctga gtgctactcc ctatttgcat ctgttttgat aaatgatgtt gacaccctcc 660
accgaattct aagtggaatc atgtcgggaa gagatacaat ccttggcctg tgtatcctcg 720
cattagcctt gtctttggcc atgatgttta ccttcagatt catcaccacc cttctggttc 780
41/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
acattttcat ttcattggtt attttgggat tgttgtttgt ctgcggtgtt ttatggtggc 840
tgtattatga ctataccaac gacctcagca tagaattgga cacagaaagg gaaaatatga 900
agtgcgtgct ggggtttgct atcgtatcca caggcatcac ggcagtgctg ctcgtcttga 960
tttttgttct cagaaagaga ataaaattga cagttgagct tttccaaatc acaaataaag 1020
ccatcagcag tgctcccttc ctgctgttcc agccactgtg gacatttgcc atcctcattt 1080
tcttctgggt cctctgggtg gctgtgctgc tgagcctggg aactgcagga gctgcccagg 1140
ttatggaagg cggccaagtg gaatataagc ccctttcggg cattcggtac atgtggtcgt 1200
accatttaat tggcctcatc tggactagtg aattcatcct tgcgtgccag caaatgacta 1260
tagctggggc agtggttact tgttatttca acagaagtaa aaatgatcct cctgatcatc 1320
ccatcctttc gtctctctcc attctcttct tctaccatca aggaaccatt gtgaaagggt 1380
catttttaat ctctgtggtg aggattccga gaatcattgt catgtacatg caaaacgcac 1440
tgaaagaaca gcatggtgca ttgtccaggt acctgttccg atgctgctac tgctgtttct 1500
ggtgtcttga caaatacctg ctccatctca accagaatgc atatactaca actgctatta 1560
atgggacaga tttctgtaca tcagcaaaag atgcattcaa aatcttgtcc aagaactcaa 1620
gtcactttac atctattaac tgctttggag acttcataat ttttctagga aaggtgttag 1680
tggtgtgttt cactgttttt ggaggactca tggcttttaa ctacaatcgg gcattccagg 1740
tgtgggcagt ccctctgtta ttggtagctt tttttgccta cttagtagcc catagttttt 1800
tatctgtgtt tgaaactgtg ctggatgcac ttttcctgtg ttttgctgtt gatctggaaa 1860
caaatgatgg atcgtcagaa aagccctact ttatggatca agaatttctg agtttcgtaa 1920
aaaggagcaa caaattaaac aatgcaaggg cacagcagga caagcactca ttaaggaatg 1980
aggagggaac agaactccag gccattgtga gatagatacc catttaggta tctgtacctg 2040
gaaaacattt ccttctaaga gccatttaca gaatagaaga tgagaccact agagaaaagt 2100
tagtgaattt ttttttaaaa gacctaataa accctattct tcctcattgt ctttgtcatt 2160
attgtttgac caggtaacaa tactggaact atattagttt accttttttt gtacaaatta 2220
ggacagaaaa actcttctaa aaccatgttt atatgcatca acttacaaag tacactatgt 2280
aagaactgag gtaagtttgt aagtgcacaa ctaataaata aaccttttta agataaggaa 2340
aaa 2343
<210> 28
<211> 3145
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7497369CB1
<400> 28
ctggtgtccc cggggtccct cggatagagg tggaaacagg tcggacaaga gctggccgtg 60
ggggaagagc aatggtgtgc ggaagtgggg agccctatct agggacaagc agaaaactta 120
gggtggcaga tccccccgac cctgtgcagg ggctgccgga gaccagagag gctgctgtgt 180
gacctggacc aagccccttg ccctctcatg cctcagtttc cccttctctc cacggaggga 240
ttgagcagga catggagggc cccttccagt agccattctg ggactccctg cacctgtccc 300
caggggaggg tcctgagctg gcctccacgt ggctgctgtt ccccatagtg gaaccttgtg 360
tgtggagacg gctggaaggt cccgctggag caggtgagcc acctcctggg ctggctgctg 420
ggctgtgtca tcctgggagc aggctgtgac cggtgaggcc ccttcccttt tcctgctgcc 480
cagcagccct cccctccgcc cactctcagg gttcagctag acctggtgct gccaggatga 540
aggttggacc ctgggttatc ctgaccctgt ccaagcatgt cccccgctgc ccaccaggtt 600
tggacgccgg gcagtttttg tggcctccct ggtgctgacc acaggcctgg gggccagtga 660
ggccctggct gccagcttcc ctaccctgct ggtcctgcgc ctactccacg ggggcacatt 720
ggcaggggcc ctcctcgccc tgtatctggc tcgtgagtac cctgggggct gctgtcactg 780
ggggaggggc agtgggtggc ccgcaggcct ctgaggctcc cttgccgagg gccccgagct 840
gcagggacag tgagcagtga gtcccttggg catcccgctc ctgggcaggt caccaatagg 900
tccccgcagt tcccaatgga actgttccag tcctccccga ggcctccact tcaacctgtc 960
tgtgtctgcc caggcctgga gttgtgtgac cctccccacc gcctggcctt ctccatgggg 1020
gctggccttt tctcggtggt gggcaccctg ctgctgcccg gcctggctgc gcttgtgcag 1080
42/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
gactggcgtc ttctgcaggg gctgggcgcc ctgatgagtg gactcttgct gctcttttgg 1140
gggtaagtgt ggtgggagct gggccagcag ccctcatcgg ggctgactat gtagcttccc 1200
tctgcaccga cgtatcccat gccaggtaaa cgcatgggtt acagggtcac agagaactac 1260
agtgatgtcc tgcccctgag cctgggggtg gggtgggggc cctggccttt gcctctcctt 1320
ccaggaggag gtggagggag ccgtgggcat cctcaccaac gctgcaggtt cccggccctg 1380
ttccccgagt ctccctgctg gctgctggcc acaggtcagg tagctcgagc caggaagatc 1440
ctgtggcgct ttgcagaagc cagtggcgtg ggccccgggg acagtccctt ggaggagaac 1500
tccctggcta cagagctgac catgctgtct gcacggagcc cccagccccg gtaccactcc 1560
ccactggggc ttctgcgtac ccgagtcacc tggagaaacg ggcttatctt gggcttcagc 1620
tcgctggttg gtggaggcat cagagctagc ttccgccgca gcctggcacc tcaggtgccg 1680
accttctacc tgccctactt cctggaggcc ggcctggagg cggcagcctt ggtcttcctg 1740
ctcctgacgg cagattgctg tggacgccgc cccgtgctgc tgctgggcac catggtcaca 1800
ggcctggcat ccctgctgct cctcgctggg gcccagtatc tgccaggctg gactgtgctg 1860
ttcctctctg tcctggggct cctggcctcc cgggctgtgt ccgcactcag cagcctcttc 1920
gcggccgagg tcttccccac ggtgatcagg ggggccgggc tgggcctggt gctgggggcc 1980
gggttcctgg gccaggcagc cggccccctg gacaccctgc acggccggca gggcttcttc 2040
ctgcaacaag tcgtcttcgc ctcccttgct gtccttgccc tgctgtgtgt cctgctgctg 2100
cctgagagcc gaagccgggg gctgccccag tcactgcagg acgccgaccg cctgcgccgc 2160
tccccactcc tgcggggccg cccccgccag gaccacctgc ctctgctgcc gccctccaac 2220
tcctactggg ccggccacac ccccgagcag cactagtcct gcctggtggc cctgggagcc 2280
aggatgggac caaagtcaag gcctggggca tggctgagta ccccagacgt ctggtccagg 2340
gcagacacat tcctctcaga agcccgtgtc tcagtgcagg tggagccgtg gggacagcgt 2400
gaaggtgtct ccagccaggc cccaggcact gggaggccct gggtctcccc ccagccacac 2460
ccagtaggtg tggaggataa aggcttctgt ggaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2520
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2580
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa acaaaacaaa caacaaaaac aaaaaacaaa 2640
aaaaacacac aaaaaaaaaa aaagaaaaga agaagatcac agcgacgata taagcgtgaa 2700
tcacagagag agacaggggg acagagcgtg aactgcacac cgcagcgatg gtacgcgcag 2760
ccagctgagc gagggcgacc gtggcgcgca gcccagccga ccagcgcggt acgcgagaga 2820
gagagacgag ctagactgcc accgaaacgc agccaacaac aagagggaac acatagaagt 2880
aggtgataca gcgcacacag acaaggacga agaagaagac agcagggagg tagagcaata 2940
aacaaaaaca taaaagaaac gaaaaaaata acaaaaaaaa aaaaaaacta taaactaata 3000
caatagataa aacacattaa caatctaacc aataaaaaat aaagcgccat aaaacatatt 3060
aagcaatata taagataaaa tcgcacacaa ttaactaatc cctacatact acagaatcca 3.120
acaaacatat aaacaataca tatat 3145
<210> 29
<211> 763
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1700438CB1
<400> 29
ttctcgctct gtcaccaggc tggagtgcag tggcgcgatc tcgactcact gcaagctccg 60
cctcccggat tcacgccatt gtcccgcctc agcctctcga gtagctggga atacaggcgc 120
ccaccaccat gcccggctga tgttttgtat ttttagtaga gacggggttt caccgtgtta 180
gccaggatgg cctcgatctc ctgacctcgt gatccgtctg cctcggcctc ccaaagtgct 240
gggattatag gcatgagcca ctgcgcccgg cctccttttc acctttaaaa catgcaaagg 300
ctgctctgca tgtgccatga gggatgggaa gcctactgca ggcagatggt cttcttagct 360
ggcttgtgct tggtcttcct ctacatgaca gttctggggt ctggcggcat catcactggc 420
tatgcctgta cccagggggt tggagactcc ctgcttagca tcctcacggc cctttcagct 480
ctctctggcc tgatgggcac cgttctcttc acccagctta gggggcacta tggcttggtc 540
acaactggag tcatatctag ccagctccat ttaggctgtc taatgctctg catgttttct 600
43/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
gtcttggctc ctggaaattc ttttgatctg gctgttttct cacttccatt aagtaaaaat 660
ccttcaaact atgagttatt ggtccagtgg atggaagaac agtccagagg aatggcttgg 720
ttcaggtttc tttcaaaggg ataaacatgg gtctggttct ctt 763
<210> 30
<211> 2720
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 535939CB1
<400> 30
tgtgaattat aggcagctgt gatgtcgcgt gcgcgcgttc gttagctcgg tcgttcggtg 60
cgagggaaag atgcggcgaa tcaggaaccc cagagcgcgg tggctagacc gggctccgcc 120
gcctccccca cagccccttt cctaatcgtt cagacggagc ctggtcgact tcgccggaga 180
ctgccagatc tcgttcctct tccctgtgtc atcttcttaa ttataaataa tgggggatga 240
agataaaaga attacatatg aagattcaga accatccaca ggaatgaatt acacgccctc 300
catgcatcaa gaagcacagg aggagacagt tatgaagctc aaaggtatag atgcaaatga 360
accaacagaa ggaagtattc ttttgaaaag cagtgaaaaa aagctacaag aaacaccaac 420
tgaagcaaat cacgtacaaa gactgagaca aatgctggct tgccctccac atggtttact 480
ggacagggtc ataacaaatg ttaccatcat tgttcttctg tgggctgtag tttggtcaat 540
tactggcagt gaatgtcttc ctggaggaaa cctatttgga attataatcc tattctattg 600
tgccatcatt ggtggtaaac ttttggggct tattaagtta cctacattgc ctccactgcc 660
ttctcttctt ggcatgctgc ttgcagggtt tctcatcaga aatatcccag tcatcaacga 720
taatgtgcag atcaagcaca agtggtcttc ctctttgaga agcatagccc tgtctatcat 780
tctggttcgt gctggccttg gtctggattc aaaggccctg aagaagttaa agggcgtttg 840
tgtaagactg tccatgggtc cctgtattgt ggaggcgtgc acatctgctc ttcttgccca 900
ttacctgctg ggtttaccat ggcaatgggg atttatactg ggttttgttt taggtgctgt 960
atctccagct gttgtggtgc cttcaatgct ccttttgcag ggaggaggct atggtgttga 1020
gaagggtgtc ccaaccttgc tcatggcagc tggcagcttc gatgacattc tggccatcac 1080
tggcttcaac acatgcttgg gcatagcctt ttccacaggc tctactgtct ttaatgtcct 1140
cagaggagtt ttggaggtgg taattggtgt ggcaactgga tctgttcttg gatttttcat 1200
tcagtacttt ccaagccgtg accaggacaa acttgtgtgt aagagaacat tccttgtgtt 1260
ggggttgtct gtgctagctg tgttcagcag tgtgcatttt ggtttccctg gatcaggagg 1320
actgtgcacg ttggtcatgg ctttccttgc aggcatggga tggaccagcg aaaaggcaga 1380
ggttgaaaag ataattgcag ttgcctggga catttttcag ccccttcttt ttggactaat 1440
tggagcagag gtatctattg catctctcag accagaaact gtaggccttt gtgttgccac 1500
cgtaggcatt gcagtattga tacgaatttt gactacattt ctgatggtgt gttttgctgg 1560
ttttaactta aaagaaaaga tatttatttc ttttgcatgg cttccaaagg ccacagttca 1620
ggctgcaata ggatctgtgg ctttggacac agcaaggtca catggagaga aacaattaga 1680
agactatgga atggatgtgt tgacagtggc atttttgtcc atcctcatca cagccccaat 1740
tggaagtctg cttattggtt tactgggccc caggcttctg cagaaagttg aacatcaaaa 1800
taaagatgaa gaagttcaag gagagacttc tgtgcaagtt tagaggtgaa aagagagagt 1860
gctgaacata atgtttagaa agctgctact tttttcaaga tgcatattga aatatgtaat 1920
gtttaagctt aaaatgtaat agaaccaaaa gtgtagctgt ttctttaaac agcattttta 1980
gcccttgctc tttccatgtg ggtggtaatg atctatatca ccaaccttaa tctctctgcc 2040
ttttttttca aacacccctt catcatccat cttaatttgc ataaggacat atctacttta 2100
atgtactacc acagtttaca gttaatgtgg gaaagaccag cttcagtatc ctcttcagct 2160
aggattgccc taacttttaa ctttcacagt ttcctgattc atatttgccc aggctctgat 2220
gccttgaatt ggttttggct ctcttttttg gatctgtttt tgttgttaaa catcataatg 2280
cagtctctca ttaattttta ccatcattta ccctgataat ctgcctcttc tccatttctc 2340
cttcccttac tacctttctt tgaattactg taactgattg gtcccaccaa aattttaaag 2400
tacatgaagt atcttcattg gttcatcctc ttgccccctc cagatgtcaa aaaactttat 2460
cctgccccct agctgaccac ccaggttcct ttatttcagt ggcccatgtg agtctacctt 2520
44/65
tctttggcta t
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
cccctaagga gtgccctaat ccagcccttt ttttgtttct tatgacccat atctttaggc 2580
tcttcccatt tctaggtggg agataggtaa gtttcaaatc tatgccagtc ttatgaatat 2640
tacattaggg taatgtgcta taatgaagaa ataaaaaata cagtgcttaa aagaaaataa 2700
aattctattt ctgtctaatg 2720
<210> 31
<211> 4464
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 55118067CB1
<400> 31
ggcagctgag ttgggctgag gtgtccctag ctggctctgc ggctcttccg ggtctgggct 60
cggagattca caggcggccc gcgaggccga gcgagggacg catggccctg aggcggccgc 120
agggcttggc ggggtccgga ggttgacctc gcccccgcag ccggccttcg aggctgcctc 180
ctccaggcag cctctggggc ccgcgcccgc gcctgctcag gctcccgtgt tcaggctgcc 240
catcccctcc ccaccggcgt cccggacgtt gggacctgtg accgtggcct cgggctgggc 300
ttccaaagcc ggccgcagcc cggcgacccc cgaggcctct cgccccgggc ccctagacct 360
ctcactatga ccgcggccgc cgcctccaac tgggggctga tcacgaacat cgtgaacagc 420
atcgtagggg tcagtgtcct caccatgccc ttctgcttca aacagtgcgg catcgtcctg 480
ggggcgctgc tcttggtctt ctgctcatgg atgacgcacc agtcgtgcat gttcttggtg 540
aagtcggcca gcctgagcaa gcggaggacc tacgccggcc tggcattcca cgcctacggg 600
aaggcaggca agatgctggt ggagaccagc atgatcgggc tgatgctggg cacctgcatc 660
gccttctacg tcgtgatcgg cgacttgggg tccaacttct ttgcccggct gttcgggttt 720
caggtgggcg gcaccttccg catgttcctg ctgttcgccg tgtcgctgtg catcgtgctc 780
ccgctcagcc tgcagcggaa catgatggcc tccatccagt ccttcagcgc catggccctc 840
ctcttctaca ccgtgttcat gttcgtgatc gtgctctcct ctctcaagca cggcctcttc 900
agtgggcagt ggctgcggcg ggtcagctac gtccgctggg agggcgtctt ccgctgcatc 960
cccatcttcg gcatgtcctt cgcctgccag tcccaggtgc tgcccaccta cgacagcctg 1020
gatgagccgt cagtgaaaac catgagctcc atatttgctt cctcccttaa tgtggtcacc 1080
accttctacg tcatggtggg gtttttcggc tacgtcagct tcaccgaggc cacggccggc 1140
aacgtgctca tgcactttcc ctccaacctg gtgacggaga tgctccgtgt gggcttcatg 1200
atgtcagtgg ctgtgggctt ccccatgatg atcctgccat gcaggcaggc cctgagcacg 1260
ctgctgtgtg agcagcagca aaaagatggc acctttgcag cagggggcta catgccccct 1320
ctccggttta aagcacttac cctctctgtg gtgtttggaa ccatggttgg tggcatcctt 1380
atccccaacg tggagaccat cctgggcctc acaggagcga ccatgggaag cctcatctgc 1440
ttcatctgcc cggcgctgat ctacaagaaa atccacaaga acgcactttc ctcccaggtg 1500
gtgctgtggg tcggcctggg cgtcctggtg gtgagcactg tcaccacact gtctgtgagc 1560
gaggaggtcc ccgaggactt ggcagaggaa gcccctggcg gccggcttgg agaggccgag 1620
ggtttgatga aggtggaggc agcgcggctc tcagcccagg atccggttgt ggccgtggct 1680
gaggatggcc gggagaagcc gaagctgccg aaggagagag aggagctgga gcaggcccag 1740
atcaaggggc ccgtggatgt gcctggacgg gaagatggca aggaggcacc ggaggaggca 1800
cagctcgatc gccctgggca agggattgct gtgcctgtgg gcgaggccca ccgccacgag 1860
cctcctgttc ctcacgacaa ggtggtggta gatgaaggcc aagaccgaga ggtgccagaa 1920
gagaacaaac ctccatccag acacgcgggc ggaaaggctc caggggtcca gggccagatg 1980
gcgccgcctc tgcccgactc agaaagagag aaacaagagc cggagcaggg agaggttggg 2040
aagaggcctg gacaggccca ggccttggag gaggcgggtg atcttcctga agatccccag 2100
aaagttccag aagcagatgg tcagccagct gtccagcctg caaaggagga cctggggcca 2160
ggagacaggg gcctgcatcc tcggccccag gcagtgctgt ctgagcagca gaacggcctg 2220
gcggtgggtg gaggggaaaa ggccaagggg ggaccgccgc caggcaacgc cgccggggac 2280
acagggcagc ccgcagagga cagcgaccac ggtgggaagc ctcccctccc agcggagaag 2340
ccggctccag ggcctgggct gccgcccgag cctcgcgagc agagggacgt ggagcgagcg 2400
ggtggaaacc aggcggccag ccagctggag gaagctggca gggcggagat gctggaccac 2460
45/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
gccgtcctgc ttcaggtgat caaagaacag caggtgcagc aaaagcgctt gctggaccag 2520
caggagaagc tgctggcggt gatcgaggag cagcacaagg agatccacca gcagaggcag 2580
gaggacgagg aggataaacc caggcaggtg gaggtgcatc aagagcccgg ggcagcggtg 2640
cccagaggcc aggaggcccc tgaaggcaag gccagggaga cggtggagaa tctgcctccc 2700
ctgcctttgg accctgtcct cagagctcct gggggccgcc ctgctccatc ccaggacctt 2760
aaccagcgct ccctggagca ctctgagggg cctgtgggca gagaccctgc tggccctcct 2820
gacggcggcc ctgacacaga gcctcgggca gcccaggcca agctgagaga tggccagaag 2880
gatgccgccc ccagggcagc tggcactgtg aaggagctcc ccaagggccc ggagcaggtg 2940
cccgtgccag accccgccag ggaagccggg ggcccagagg agcgcctcgc agaggaattc 3000
cctgggcaaa gtcaggacgt tactggcggt tcccaagaca ggaaaaaacc tgggaaggag 3060
gtggcagcca ctggcaccag cattctgaag gaagccaact ggctcgtggc agggccagga 3120
gcagagacgg gggaccctcg catgaagccc aagcaagtga gccgagacct gggccttgca 3180
gcggacctgc ccggtggggc ggaaggagca gctgcacagc cccaggctgt gttacgccag 3240
ccggaactgc gggtcatctc tgatggcgag cagggtggac agcagggcca ccggctggac 3300
catggcggtc acctggagat gagaaaggcc cgcggggggg accatgtgcc tgtgtcccac 3360
gagcagccga gaggcgggga ggacgctgct gtccaggagc ccaggcagag gccagagcca 3420
gagctggggc tcaaacgagc tgtcccgggg ggccagaggc cggacaatgc caagcccaac 3480
cgggacctga aactgcaggc tggctccgac ctccggaggc gacggcggga ccttggccct 3540
catgcagagg gtcagctggc cccgagggat ggggtcatca ttggccttaa ccccctgcct 3600
gatgtccagg tgaacgacct ccgtggcgcc ctggatgccc agctccgcca ggctgcgggg 3660
ggagctctgc aggtggtcca cagccggcag cttagacagg cgcctgggcc tccagaggag 3720
tcctagcacc tgctggccat gagggccacg ccagccactg ccctcctcgg ccagcagcag 3780
gtctgtctca gccgcatccc agccaaactc tggaggtcac actcgcctct ccccagggtt 3840
tcatgtctga ggccctcacc aagtgtgagt gacagtataa aagattcact gtggcatcgt 3900
ttccagaatg ttcttgctgt cgttctgttg cagctcttag tctgaggtcc tctgacctct 3960
agactctgag ctcactccag cctgtgagga gaaacggcct ccgctgcgag ctggctggtg 4020
cactcccagg ctcaggctgg ggagctgctg cgtctgtggt caggcctcct gctcctgcca 4080
gggagcacgc gtggtcttcg ggttgagctc ggccgtgcgt ggaggtgcgc atggctgctc 4140
atggtcccaa cacaggctac tgtgagagcc agcatccaac cccacgcttg cagtgactca 4200
gaatgataat tattatgact gtttatcgat gcttcccaca gtgtggtaga aagtcttgaa 4260
taaacacttt tgccttcacc cagcctcggt ggatgctgtt tggtgtccag gaagcacagg 4320
gagcaggggc cagacaagcc gggtgtccag gggcactggc cggtgccgcc ctttgcactc 4380
ctcatggttg gcccagccct ccattgtctg tgtttttcaa aatccaattg tggctttttt 4440
taaaaagtaa aaaacaccac tgtg 4464
<210> 32
<211> 3135
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7502087CB1
<220>
<221> unsure
<222> 2961
<223> a, t, c, g, or other
<400> 32
ggcggagacg ccgggagcca gtggcgcctg tggctccggg caggggccgc ggccgaaaga 60
tgccggtccg caggggccac gtcgctcccc aaaacactta cctggacacc atcatccgca 120
agttcgaggg ccaaagtcgg aagttcctga ttgccaatgc tcagatggag aactgcgcca 180
tcatttactg caacgacggc ttctgcgaac tcttcggcta ctcccgagtg gaggtgatgc 240
agcaaccctg cacctgcgac ttcctcacag gccccaacac accaagcagc gccgtgtccc 300
gcctagcgca ggccctgctg ggggctgagg agtgcaaggt ggacatcctc tactaccgca 360
46/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
aggatgcctc cagcttccgc tgcctggtag atgtggtgcc cgtgaagaac gaggacgggg 420
ctgtcatcat gttcattctc aacttcgagg acctggccca gctcctggcc aagtgcagca 480
gccgcagctt gtcccagcgc ctgttgtccc agagcttcct gggctccgag ggctctcatg 540
gcaggccagg cggaccaggg ccaggcacag gcaggggcaa gtacaggacc atcagccaga 600
tcccacagtt cacgctcaac ttcgtggagt tcaacttgga gaagcaccgc tccagctcca 660
ccacggagat tgagatcatc gcgccccata aggtggtgga gcggacacag aacgtcactg 720
agaaggtcac ccaggtcctg tccctgggcg cggatgtgct gccggagtac aagctgcagg 780
cgccgcgcat ccaccgctgg accatcctgc actacagccc cttcaaggcc gtgtgggact 840
ggctcatcct gctgctggtc atctacacgg ctgtcttcac gccctactca gccgccttcc 900
tgctcagcga ccaggacgaa tcacggcgtg gggcctgcag ctatacctgc agtcccctca 960
ctgtggtgga tctcatcgtg gacatcatgt tcgtcgtgga catcgtcatc aacttccgca 1020
ccacctatgt caacaccaat gatgaggtgg tcagccaccc ccgccgcatc gccgtccact 1080
acttcaaggg ctggttcctc attgacatgg tggccgccat ccctttcgac ctcctgatct 1140
tccgcactgg ctccgatgag accacaaccc tgattgggct attgaagaca gcgcggctgc 1200
tgcggctggt gcgcgtagca cggaagctgg actgctactc tgagtatggg gcggctgtgc 1260
tcttcttgct catgtgcacc ttcgcgctca tagcgcactg gctggcctgc atctggtacg 1320
ccatcggcaa tgtggagcgg ccctacctag aacacaagat cggctggctg gacagcctgg 1380
gtgtgcagct tggcaagcgc tacaacggca gcgacccagc ctcgggcccc tcggtgcagg 1440
acaagtatgt cacagccctc tacttcacct tcagcagcct caccagcgtg ggcttcggca 1500
atgtctcgcc caacaccaac tccgagaagg tcttctccat ctgcgtcatg ctcatcggct 1560
ccctgatgta cgccagcatc ttcgggaacg tgtccgcgat catccagcgc ctgtactcgg 1620
gcaccgcgcg ctaccacacg cagatgctgc gtgtcaagga gttcatccgc ttccaccaga 1680
tccccaaccc actgcgccag cgcctggagg agtatttcca gcacgcctgg tcctacacca 1740
atggcattga catgaacgcg gtgctgaagg gcttccccga gtgcctgcag gctgacatct 1800
gcctgcacct gcaccgcgca ctgctgcagc actgcccagc tttcagcggc gccggcaagg 1860
gctgcctgcg cgcgctagcc gtcaagttca agaccaccca cgcgccgcct ggggacacgc 1920
tggtgcacct cggcgacgtg ctctccaccc tctacttcat ctcccgaggc tccatcgaga 1980
tcctgcgcga cgacgtggtc gtggccatcc taggaaagaa tgacatcttt ggggaacccg 2040
tcagcctcca tgcccagcca ggcaagtcca gtgcagacgt gcgggctctg acctactgcg 2100
acctgcacaa gatccagcgg gcagatctgc tggaggtgct ggacatgtac ccggcctttg 2160
cggagagctt ctggagtaag ctggaggtca ccttcaacct gcgggacgct cctggcagcc 2220
aagaccacca aggtttcttt ctcagtgaca accagtcaga tgcagcccct cccctgagca 2280
tctcagatgc atctggcctc tggcctgagc tactgcagga aatgccccca aggcacagcc 2340
cccaaagccc tcaggaagac ccagattgct ggcctctgaa gctgggctcc aggctagagc 2400
agctccaggc ccagatgaac aggctggagt cccgcgtgtc ctcagacctc agccgcatct 2460
tgcagctcct ccagaagccc atgccccagg gccacgccag ctacattctg gaagcccctg 2520
cctccaatga cctggccttg gttcctatag cctcggagac gacgagtcca gggcccaggc 2580
tgccccaggg ctttctgcct cctgcacaga ccccaagcta tggagacttg gatgactgta 2640
gtccaaagca caggaactcc tcccccagga tgcctcacct ggctgtggca atggacaaaa 2700
ctctggcacc atcctcagaa caggaacagc ctgaggggct ctggccaccc ctagcctcac 2760
ctctacatcc cctggaagta caaggactca tctgtggtcc ctgcttctcc tccctccctg 2820
aacaccttgg ctctgttccc aagcagctgg acttccagag acatggctca gatcctggat 2880
ttgcagggag ttggggccac tgaactccaa gataaagaca ccatgagggg actgaaggtg 2940
ggcaagggga tttcctttag nctgggcatg gtggcgggcg cctgtaatcc cagctactca 3000
ggaggctgaa gcaagagaat cacttgaacc ctaaaggcag aggttgcagt gagccgagat 3060
agtgccactg cactacagcc cgggcgacag agtgagactc catctcaaaa ataaaataca 3120
attaacaaaa aaaga 3135
<210> 33
<211> 843
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7500819CB1
47/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
<400> 33
ctcctcggga ctcggcgggt cctcctggga gtctcggagg ggaccggctg tgcagacgcc 60
atggagttgg tgctggtctt cctctgcagc ctgctggccc ccatggtcct ggccagtgca 120
gctgaaaagg agaaggaaat ggaccctttt cattatgatt accagaccct gaggattggg 180
ggactggtgt tcgctgtggt cctcttctcg gttgggatcc tccttatcct aagtcgcagg 240
tgcaagtgca gcttctactc tgcccctggg gaatgtgtcc cctgcatatc ttctcagcaa 300
taactccatg ggctctggga ccctacccct tccaaccttc cctgcttctg agacttcaat 360
ctacagccca gctcatccag atgcagacta cagtccctgc aattgggtct ctggcaggca 420
atagttgaag gactcctgtt ccgttggggc cagcacaccg ggatggatgg agggagagca 480
gaggcctttg cttctctgcc tacgtcccct tagatgggca gcagaggcaa ctcccgcatc 540
ctttgctctg cctgtcagtg gtcagagcgg tgagcgaggt gggttggaga ctcagcaggc 600
tccgtgcagc ccttgggaac agtgagaggt tgaaggtcat aacgagagtg ggaactcaac 660
ccagatcccg cccctcctgt cctctgtgtt cccgcggaaa ccaaccaaac cgtgcgctgt 720
gacccattgc tgttctctgt atcgtgatct atcctcaaca acaacagaaa aaaggaataa 780
aatatccttt gtttcctaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 840
aaa 843
<210> 34
<211> 3159
<212> DNA
<213> Homo sapiens
<220>
<22~> misc_feature
<223> Incyte ID No: 7503413CB1
<400> 34
gcgcgatggc ctcggcgctg agctatgtct ccaagttcaa gtccttcgtg atcttgttcg 60
tcaccccgct cctgctgctg ccactcgtca ttctgatgcc cgccaagttt gtcaggtgtg 120
cctacgtcat catcctcatg gccatttact ggtgcacaga agtcatccct ctggctgtca 180
cctctctcat gcctgtcttg cttttcccac tcttccagat tctggactcc aggcaggtgt 240
gtgtccagta catgaaggac accaacatgc tgttcctggg cggcctcatc gtggccgtgg 300
ctgtggagcg ctggaacctg cacaagagga tcgccctgcg cacgctcctc tgggtggggg 360
ccaagcctgc acggctgatg ctgggcttca tgggcgtcac agccctcctg tccatgtgga 420
tcagtaacac ggcaaccacg gccatgatgg tgcccatcgt ggaggccata ttgcagcaga 480
tggaagccac aagcgcagcc accgaggccg gcctggagct ggtggacaag ggcaaggcca 540
aggagctgcc agggagtcaa gtgatttttg aaggccccac tctggggcag caggaagacc 600
aagagcggaa gaggttgtgt aaggccatga ccctgtgcat ctgctacgcg gccagcatcg 660
ggggcaccgc caccctgacc gggacgggac ccaacgtggt gctcctgggc cagatgaacg 720
agttgtttcc tgacagcaag gacctcgtga actttgcttc ctggtttgca tttgcctttc 780
ccaacatgct ggtgatgctg ctgttcgcct ggctgtggct ccagtttgtt tacatgagat 840
tcaattttaa aaagtcctgg ggctgcgggc tagagagcaa gaaaaacgag aaggctgccc 900
tcaaggtgct gcaggaggag taccggaagc tggggccctt gtccttcgcg gagatcaacg 960
tgctgatctg cttcttcctg ctggtcatcc tgtggttctc ccgagacccc ggcttcatgc 1020
ccggctggct gactgttgcc tgggtggagg aaaggaaaac tccattttat ccccctcccc 1080
tgctggattg gaaggtaacc caggagaaag tgccctgggg catcgtgctg ctactagggg 1140
gcggatttgc tctggctaaa ggatccgagg cctcggggct gtccgtgtgg atggggaagc 1200
agatggagcc cttgcacgca gtgcccccgg cagccatcac cttgatcttg tccttgctcg 1260
ttgccgtgtt cactgagtgc acaagcaacg tggccaccac caccttgttc ctgcccatct 1320
ttgcctccat gtctcgctcc atcggcctca atccgctgta catcatgctg ccctgtaccc 1380
tgagtgcctc ctttgccttc atgttgcctg tggccacccc tccaaatgcc atcgtgttca 1440
cctatgggca cctcaaggtt gctgacatgg tgaaaacagg agtcataatg aacataattg 1500
gagtcttctg tgtgtttttg gctgtcaaca cctggggacg ggccatattt gacttggatc 1560
atttccctga ctgggctaat gtgacacata ttgagactta ggaagagcca caagaccaca 1620
cacacagccc ttaccctcct caggactacc gaaccttctg gcacaccttg tacagagttt 1680
tggggttcac accccaaaat gacccaacga tgtccacaca ccaccaaaac ccagccaatg 1740
48/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
ggccacctct tcctccaagc ccagatgcag agatggtcat gggcagctgg agggtaggct 1800
cagaaatgaa gggaacccct cagtgggctg ctggacccat ctttcccaag ccttgccatt 1860
atctctgtga gggaggccag gtagccgagg gatcaggatg caggctgctg tacccgctct 1920
gcctcaagca tcccccacac agggctctgg ttttcactcg cttcgtccta gatagtttaa 1980
atgggaatca gatcccctgg ttgagagcta agacaaccac ctaccagtgc ccatgtccct 2040
tccagctcac cttgagcagc ctcagatcat ctctgtcact ctggaaggga caccccagcc 2100
agggacggaa tgcctggtct tgagcaacct cccactgctg gagtgcgagt gggaatcaga 2160
gcctcctgaa gcctctggga actcctcctg tggccaccac caaaggatga ggaatctgag 2220
ttgccaactt caggacgaca cctggcttgc cacccacagt gcaccacagg ccaacctacg 2280
cccttcatca cttggttctg ttttaatcga ctggccccct gtcccacctc tccagtgagc 2340
ctccttcaac tccttggtcc cctgttgtct gggtcaacat ttgccgagac gccttggctg 2400
gcaccctctg gggtccccct tttctcccag gcaggtcatc ttttctggga gatgcttccc 2460
ctgccatccc caaatagcta ggatcacact ccaagtatgg gcagtgatgg cgctctgggg 2520
gccacagtgg gctatctagg ccctccctca cctgaggccc agagtggaca cagctgttaa 2580
tttccactgg ctatgccact tcagagtctt tcatgccagc gtttgagctc ctctgggtaa 2640
aatcttccct ttgttgactg gccttcacag ccatggctgg tgacaacaga ggatcgttga 2700
gattgagcag cgcttggtga tctctcagca aacaacccct gcccgtgggc caatctactt 2760
gaagttactc ggacaaagac cccaaagtgg ggcaacaact ccagagaggc tgtgggaatc 2820
ttcagaagcc cccctgtaag agacagacat gagagacaag catcttcttt cccccgcaag 2880
tccattttat ttccttcttg tgctgctctg gaagagaggc agtagcaaag agatgagctc 2940
ctggatggca ttttccaggg caggagaaag tatgagagcc tcaggaaacc ccatcaagga 3000
ccgagtatgt gtctggttcc ttgggtggga cgattcctga ccacactgtc cagctcttgc 3060
tctcattaaa tgctctgtct cccgcggaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3120
aaaaaaaaaa aaattctcgg acgcaaggaa ttcagctgg 3159
<210> 35
<211> 1883
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7500007CB1
<400> 35
ccacgcgtcc gtaaggtggg atggatagca gggtctcagg cacaaccagt aatggagaga 60
caaaaccagt gtatccagtc atggaaaaga aggaggaaga tggcaccctg gagcgggggc 120
actggaacaa caagatggag tttgtgctgt cagtggctgg ggagatcatt ggcttaggca 180
acgtctggag gtttccctat ctctgctaca aaaatggggg agaacactgt atggagttcc 240
agaagaccaa cggctccctg aatggtacct ctgagaatgc cacctctcct gtcatcgagt 300
tctgggagcg gcgggtcttg aagatctctg atgggatcca gcacctgggg gccctgcgct 360
gggagctggc tctgtgcctc ctgctggcct gggtcatctg ctacttctgc atctggaagg 420
gggtgaagtc cacaggcaag gtggtgtact tcacggccac atttccttac ctcatgctgg 480
tggtcctgtt aattcgaggg gtgacgttgc ctggggcagc ccaaggaatt cagttttacc 540
tgtacccaaa cctcacgcgt ctgtgggatc cccaggtgtg gatggatgca ggcacccaga 600
tattcttctc cttcgccatc tgtcttgggt gcctgacagc cctgggcagc tacaacaagt 660
accacaacaa ctgctacagg gactgcatcg ccctctgctt cctcaacagc ggcaccagct 720
ttgtggccgg ctttgccatc ttctccatcc tgggcttcat gtctcaggag cagggggtgc 780
ccatttctga ggtggccgag tcaggccctg gcctggcttt catcgcttac ccgcgggctg 840
tggtgatgct gcccttctct cctctctggg cctgctgttt cttcttcatg gtcgttctcc 900
tgggactgga tagccagttt gtgtgtgtag aaagcctggt gacagcgctg gtggacatgt 960
accctcacgt gttccgcaag aagaaccgga gggaagtcct catccttgga gtatctgtcg 1020
tctccttcct tgtggggctg atcatgctca cagagggcgg aatgtacgtg ttccagctct 1080
ttgactacta tgcggccagt ggcatgtgcc tcctgttcgt ggccatcttc gagtccctct 1140
gtgtggcttg ggtttacgga gccaagcgct tctacgacaa catcgaagac atgattgggt 1200
acaggccatg gcctcttatc aaatactgtt ggctcttcct cacaccagct gtgtgcacag 1260
49/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
ccacctttct cttctccctg ataaagtaca ctccgctgac ctacaacaag aagtacacgt 1320
acccgtggtg gggcgatgcc ctgggctggc tcctggctct gtcctccatg gtctgcattc 1380
ctgcctggag cctctacaga ctcggaaccc tcaagggccc cttcagagag agaatccgtc 1440
agctcatgtg cccagccgag gacctgcccc agcggaaccc agcaggaccc tcggctcccg 1500
ccacccccag gacctcactg ctcagactca cagagctaga gtctcactgc tagggggcag 1560
gcccttggat ggtgcctgtg tgcctggcct tggggatggc tgtggaggga acgtggcaga 1620
agcagcccca tgtgcttccc tgcccccgac ctggagtgga taagacaaga ggggtatttt 1680
ggagtccacc tgctgagctg gaggcctccc actgcaactt ttcagctcag gggttgttga 1740
acagatgtga aaggccagtg ccaagagtgt ccctctgaga cccttgggaa gctgggtggg 1800
ggctggtagg tggggcgaga cttgctggct tcgggccctc tcatccttca ttccattaaa 1860
tccacattct tcccgctgaa aaa 1883
<210> 36
<211> 2746
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7500025CB1
<400> 36
cgcctgtggc tccgggcagg ggccgcggcc gaaagatgtc ggtccgcagg ggccacgtcg 60
ctccccaaaa cacttacctg gacaccatca tccgcaagtt cgagggccaa agtcggaagt 120
tcctgattgc caatgctcag atggagaact gcgccatcat ttactgcaac gacggcttct 180
gcgaactctt cggctactcc cgagtggagg tgatgcagca accctgcacc tgcgacttcc 240
tcacaggccc caacacacca agcagcgccg tgtcccgcct agcgcaggcc ctgctggggg 300
ctgaggagtg caaggtggac atcctctact accgcaagga tgcctccagc ttccgctgcc 360
tggtagatgt ggtgcccgtg aagaacgagg acggggctgt catcatgtcc attctcaact 420
tcgaggacct ggcccagctc ctggccaagt gcagcagccg cagcttgtcc cagcgcctgt 480
tgtcccagag cttcctgggc tccgagggct ctcatggcag gccaggcgga ccaggcccag 540
gcacaggcag gggcaagtac aggaccatca gccagatccc acagttcacg ctcaacttcg 600
tggagttcaa cttggagaag caccgctcca gctccaccac ggagattgag atcatcgcgc 660
cccataaggt ggtggagcgg acacagaacg tcactgagaa ggtcacccag gtcctgtccc 720
tgggcgcaga tgtgctgccg gagtacaagc tgcaggcgcc gcgcatccac cgctggacca 780
tcctgcacta cagccccttc aaggccgtgt gggactggct catcccgctg ctggtcatct 840
acacggctgt cttcacgccc tactcagccg ccttcctgct cagcgatcag gacgaatcac 900
ggcgtggggc ctgcagctat acctgcagtc ccctcactgt ggtggatctc atcgtggaca 960
tcatgttcgt cgtggacatc gtcatcaact tccgcaccac ctatgtcaac accaatgatg 1020
aggtggtcag ccacccccgc cgcatcgccg tccactactt caagggctgg ttcctcattg 1080
acatggttJgc cgccatccct ttcgacctcc tgatcttccg cactggctcc gatgagacca 1140
caaccctgat tgggctattg aagacagcgc ggctgctgcg gctggtgcgc gtagcacgga 1200
agctggaccg ctactctgag tatggggcgg ctgtgctctt cttgctcatg tgcaccttcg 1260
cgctcatagc gcactggctg gcctgcatct gcagcctcac cagcgtgggc ttcggcaatg 1320
tctcgcccaa caccaactcc gagaaggtct tctccatctg cgtcatgctc atcggctccc 1380
tgatgtacgc cagcatcttc gggaacgtgt ccgcgatcat ccagcgcctg tactcgggca 1440
ccgcgcgcta ccacacgcag atgctgcgtg tcaaggagtt catccgcttc caccagatcc 1500
ccaacccact gcgccagcgc ctggaggagt atttccagca cgcctggtcc tacaccaatg 1560
gcattgacat gaacgcggtg ctgaagggct tccccgagtg cctgcaggct gacatctgcc 1620
tgcacctgca ccgcgcactg ctgcagcact gcccagcttt cagcggcgcc ggcaagggct 1680
gcctgcgcgc gctagccgtc aagttcaaga ccacccacgc gccgcctggg gacacgctgg 1740
tgcacctcgg cgacgtgctc tccaccctct acttcatctc ccgaggctcc atcgagatcc 1800
tgcgcgacga cgtggtcgtg gccatcctag gaaagaatga catctttggg gaacccgtca 1860
gcctccatgc ccagccaggc aagtccagtg cagacgtgcg ggctctgacc tactgcgacc 1920
tgcacaagat ccagcgggca gatctgctgg aggtgctgga catgtacccg gcctttgcgg 1980
agagcttctg gagtaagctg gaggtcacct tcaacctgcg ggacgctcct ggcagccaag 2040
50/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
accaccaagg tttctttctc agtgacaacc agtcagatgc agcccctccc ctgagcatct 2100
cagatgcatc tggcctctgg cctgagctac tgcaggaaat gcccccaagg cacagccccc 2160
aaagccctca ggaagaccca gattgctggc ctctgaagct gggctccagg ctagagcagc 2220
tccaggccca gatgaacagg ctggagtccc gcgtgtcctc agacctcagc cgcatcttgc 2280
agctcctcca gaagcccatg ccccagggcc acgccagcta cattctggaa gcccctgcct 2340
ccaatgacct ggccttggtt cctatagcct cggagacgac gagtccaggg cccaggctgc 2400
cccagggctt tctgcctcct gcacagaccc caagctatgg agacttggat gactgtagtc 2460
caaagcacag gaactcctcc cccaggatgc ctcacctggc tgtggcaatg gacaaaactc 2520
tggcaccatc ctcagaacag gaacagcctg aggggctctg gccaccccta gcctcacctc 2580
tacatcccct ggaagtacaa ggactcatct gtggtccctg cttctcctcc ctccctgaac 2640
accttggctc tgttcccaag cagctggact tccagagaca tggctcagat cctggatttg 2700
cagggagttg gggccactga actccaagat aaagacacca tgaggg 2746
<210> 37
<211> 2868
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7502736CB1
<400> 37
caaaacactt acctggacac catcatccgc aagttcgagg gccaaagtcg gaagttcctg 60
attgccaatg ctcagatgga gaactgcgcc atcatttact gcaacgacgg cttctgcgaa'120
ctcttcggct actcccgagt ggagcctcca gcttccgctg cctggtagat gtggtgcccg 180
tgaagaacga ggacggggct gtcatcatgt tcattctcaa cttcgaggac ctggcccagc 240
tcctggccaa gtgcagcagc cgcagcttgt cccagcgcct gttgtcccag agcttcctgg 300
gctccgaggg ctctcatggc aggccaggcg gaccagggcc aggcacaggc aggggcaagt 360
acaggaccat cagccagatc ccacagttca cgctcaactt cgtggagttc aacttggaga 420
agcaccgctc cagctccacc acggagattg agatcatcgc gccccataag gtggtggagc 480
ggacacagaa cgtcactgag aaggtcaccc aggtcctgtc cctgggcgcg gatgtgctgc 540
cggagtacaa gctgcaggcg ccgcgcatcc accgctggac catcctgcac tacagccccc 600
ttcaaggccg tgtgggactg gctcatcctg ctgctggtca tctacacggc tgtcttcacg 660
ccctactcag ccgccttcct gctcagcgat caggacgaat cacggcgtgg ggcctgcagc 720
tatacctgca gtcccctcac tgtggtggat ctcatcgtgg acatcatgtt cgtcgtggac 780
atcgtcatca acttccgcac cacctatgtc aacaccaatg atgaggtggt cagccacccc 840
cgccgcatcg ccgtccacta cttcaagggc tggttcctca ttgacatggt ggccgccatc 900
cctttcgacc tcctgatctt ccgcactggc tccgatgaga ccacaaccct gattgggcta 960
ttgaagacag cgcggctgct gcggctggtg cgcgtagcac ggaagctgga ccgctactct 1020
gagtatgggg cggctgtgct cttcttgctc atgtgcacct tcgcgctcat agcgcactgg 1080
ctggcctgca tctggtacgc catcggcaat gtggagcggc cctacctaga acacaagatc 1140
ggctggctgg acagcctggg tgtgcagctt ggcaagcgct acaacggcag cgacccagcc 1200
tcgggcccct cggtgcagga caagtatgtc acagccctct acttcacctt cagcagcctc 1260
accagcgtgg gcttcggcaa tgtctcgccc aacaccaact ccgagaaggt cttctccatc 1320
tgcgtcatgc tcatcggctc cctgatgtac gccagcatct tcgggaacgt gtccgcgatc 1380
atccagcgcc tgtactcggg caccgcgcgc taccacacgc agatgctgcg tgtcaaggag 1440
ttcatccgct tccaccagat ecccaaccca ctgcgccagc gcctggagga gtatttccag 1500
cacgcctggt cctacaccaa tggcattgac atgaacgcgg tgctgaaggg cttccccgag 1560
tgcctgcagg ctgacatctg cctgcacctg caccgcgcac tgctgcagca ctgcccagct 1620
ttcagcggcg ccggcaaggg ctgcctgcgc gcgctagccg tcaagttcaa gaccacccac 1680
gcgccgcctg gggacacgct ggtgcacctc ggcgacgtgc tctccaccct ctacttcatc 1740
tcccgaggct ccatcgagat cctgcgcgac gacgtggtcg tggccatcct aggaaagaat 1800
gacatctttg gggaacccgt cagcctccat gcccagccag gcaagtccag tgcagacgtg 1860
cgggctctga cctactgcga cctgcacaag atccagcggg cagatctgct ggaggtgctg 1920
gacatgtacc cggcctttgc ggagagcttc tggagtaagc tggaggtcac cttcaacctg 1980
51/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
cgggacgctc ctggcagcca agaccaccaa ggtttctttc tcagtgacaa ccagtcagat 2040
gcagcccctc ccctgagcat ctcagatgca tctggcctct ggcctgagct actgcaggaa 2100
atgcccccaa ggcacagccc ccaaagccct caggaagacc cagattgctg gcctctgaag 2160
ctgggctcca ggctagagca gctccaggcc cagatgaaca ggctggagtc ccgcgtgtcc 2220
tcagacctca gccgcatctt gcagctcctc cagaagccca tgccccaggg ccacgccagc 2280
tacattctgg aagcccctgc ctccaatgac ctggccttgg ttcctatagc ctcggagacg 2340
acgagtccag ggcccaggct gccccagggc tttctgcctc ctgcacagac cccaagctat 2400
ggagacttgg atgactgtag tccaaagcac aggaactcct cccccaggat gcctcacctg 2460
gctgtggcaa tggacaaaac tctggcacca tcctcagaac aggaacagcc tgaggggctc 2520
tggccacccc tagcctcacc tctacatccc ctggaagtac aaggactcat ctgtggtccc 2580
tgcttctcct ccctccctga acaccttggc tctgttccca agcagctgga cttccagaga 2640
catggctcag atcctggatt tgcagggagt tggggccact gaactccaag ataaagacac 2700
catgagggga ctgaaggtgg gcaaggggat ttcctttagc tgggcatggt ggcgggcgcc 2760
tgtaatccca gctactcagg aggctgaagc aagagaatca cttgaaccct aaaggcagag 2820
gttgcagtga gccgagatag tgccactgca ctacagcccg ggcgacag 2868
<210> 38
<211> 1906
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503570CB1
<400> 38
ccttaagggg cgggccgggg cggggctccg ctgccccttc ccagaggccg cgcctgctgc 60
tgagcagatg cagtagccga aactgcgcgg aggcacagag gccggggaga gcgttctggg 120
tccgagggtc caggtagggg ttgagccacc atctgaccgc aagctgcgtc gtgtcgccgg 180
ttctgcaggc accatgagcc aggacaccga ggtggatatg aaggaggtgg agctgaatga 240
gttagagccc gagaagcagc cgatgaacgc ggcgtctggg gcggccatgt ccctggcggg 300
agccgagaag aatggtctgg tgaagatcaa ggtggcggaa gacgaggcgg aggcggcagc 360
cgcggctaag ttcacgggcc tgtccaagga ggagctgctg aaggtggcag gcagccccgg 420
ctgggtacgc acccgctggg cactgctgct gctcttctgg ctcggctggc tcggcatgct 480
tgctggtgcc gtggtcataa tcgtgcgagc gccgcgttgt cgcgagctac cggcgcagaa 540
gtggtggcac acgggcgccc tctaccgcat cggcgacctt caggccttcc agggccacgg 600
cgcgggcaac ctggcgggtc tgaaggggcg tctcgattac ctgagctctc tgaaggtgaa 660
gggccttgtg ctgggtccaa ttcacaagaa ccagaaggat gatgtcgctc agactgactt 720
gctgcagatc gaccccaatt ttggctccaa ggaagatttt gacagtctct tgcaatcggc 780
taaaaaaaag agcatccgtg tcattctgga ccttactccc aactaccggg gtgagaactc 840
gtggttctcc actcaggttg acactgtggc caccaaggtg aaggatgctc tggagttttg 900
gctgcaagct ggcgtggatg ggttccaggt tcgggacata gagaatctga aggatgcatc 960
ctcattcttg gctgagtggc aaaatatcac caagggcttc agtgaagaca ggctcttgat 1020
tgcggggact aactcctccg accttcagca gatcctgagc ctactcgaat ccaacaaaga 1080
cttgctgttg actagctcat acctgtctga ttctggttct actggggagc atacaaaatc 1140
cctagtcaca cagtatttga atgccactgg caatcgctgg tgcagctgga gtttgtctca 1200
ggcaaggctc ctgacttcct tcttgccggc tcaacttctc cgactctacc agctgatgct 1260
cttcaccctg ccagggaccc ctgttttcag ctacggggat gagattggcc tggatgcagc 1320
tgcccttcct ggacagggcc agagtgaaga ccctggctcc ctcctttcct tgttccggcg 1380
gctgagtgac cagcggagta aggagcgctc cctactgcat ggggacttcc acgcgttctc 1440
cgctgggcct ggactcttct cctatatccg ccactgggac cagaatgagc gttttctggt 1500
agtgcttaac tttggggatg tgggcctctc ggctggactg caggcctccg acctgcctgc 1560
cagcgccagc ctgccagcca aggctgacct cctgctcagc acccagccag gccgtgagga 1620
gggctcccct cttgagctgg aacgcctgaa actggagcct cacgaagggc tgctgctccg 1680
cttcccctac gcggcctgac ttcagcctga catggaccca ctacccttct cctttccttc 1740
ccaggccctt tggcttctga tttttctctt ttttaaaaac aaacaaacaa actgttgcag 1800
52/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
attatgagtg aacccccaaa tagggtgttt tctgccttca aataaaagtc acccctgcat 1860
ggtgaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa attcgg 1906
<210> 39
<211> 2506
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7504008CB1
<400> 39
cggctctagc tcgagcagca ggagcagccc gcaccggaca acttgcgagc catggggctg 60
gcggatgcgt cgggaccgag ggacacacag gcactgctgt ctgcaacaca agcaatggac 120
ctgcggaggc gagactacca catggaacgg ccgctgctga accaggagca tttggaggag 180
ctggggcgct ggggctcagc acctaggacc caccagtggc ggacctggtt gcagtgctcc 240
cgtgctcggg cctatgccct tctgctccaa cacctcccgg ttttggtctg gttaccccgg 300
tatcctgtgc gtgactggct cctgggtgac ctgttatccg gcctgagtgt ggccatcatg 360
cagcttccgc agggcttggc ctacgccctc ctggctggat tgccccccgt gtttggcctc 420
tatagctcct tctaccctgt cttcatctac ttcctgtttg gcacttcccg gcacatctcc 480
gtggggacct ttgctgtcat gtctgtgatg gtgggcagtg tgacagaatc cctggccccg 540
caggccttga acgactccat gatcaatgag acagccagag atgctgcccg ggtacaggtg 600
gcctccacac tcagtgtcct ggttggcctc ttccaggtgg ggctgggcct gatccacttc 660
ggcttcgtgg tcacctacct gtcagaacct cttgtccgag gctataccac agctgcagct 720
gtgcaggtct tcgtctcaca gctcaagtat gtgtttggcc tccatctgag cagccactct 780
gggccactgt ccctcatcta tacagtgctg gaggtctgct ggaagctgcc ccagagcaag 840
ctcatcgggg ccacaggcat ctcctatggc atgggtctaa agcacagatt tgaggtagat 900
gtcgtgggca acatccctgc agggctggtg cccccagtgg cccccaacac ccagctgttc 960
tcaaagctcg tgggcagcgc cttcaccatc gctgtggttg ggtttgccat tgccatctca 1020
ctggggaaga tcttcgccct gaggcacggc taccgggtgg acagcaacca ggagctggtg 1080
gccctgggcc tcagtaacct tatcggaggc atcttccagt gcttccccgt gagttgctct 1140
atgtctcgga gcctggtaca ggagagcacc gggggcaact cgcaggttgc tggagccatc 1200
tcttcccttt tcatcctcct catcattgtc aaacttgggg aactcttcca tgacctgccc 1260
aaggcggtcc tggcagccat catcattgtg aacctgaagg gcatgctgag gcagctcagc 1320
gacatgcgct ccctctggaa ggccaatcgg gcggatctgc ttatctggct ggtgaccttc 1380
acggccacca tcttgctgaa cctggacctt ggcttggtgg ttgcggtcat cttctccctg 1440
ctgctcgtgg tggtccggac acagatgccc cactactctg tcctggggca ggtgccagac 1500
acggatattt acagagatgt ggcagagtac tcagaggcca aggaagtccg gggggtgaag 1560
gtcttccgct cctcggccac cgtgtacttt gccaatgctg agttctacag tgatgcgctg 1620
aagcagaggt gtggtgtgga tgtcgacttc ctcatctccc agaagaagaa actgctcaag 1680
aagcaggagc agctgaagct gaagcaactg cagaaagagg agaagcttcg gaaacaggct 1740
gcctccccca agggcgcctc agtttccatt aatgtcaaca ccagccttga agacatgagg 1800
agcaacaacg ttgaggactg caagatgatg caggtgagct caggagataa gatggaagat 1860
gcaacagcca atggtcaaga agactccaag gccccagatg ggtccacact gaaggccctg 1920
ggcctgcctc agccagactt ccacagcctc atcctggacc tgggtgccct ctcctttgtg 1980
gacactgtgt gcctcaagag cctgaagaat attttccatg acttccggga gattgaggtg 2040
gaggtgtaca tggcggcctg ccacagccct gtggtcagcc agcttgaggc tgggcacttc 2100
ttcgatgcat ccatcaccaa gaagcatctc tttgcctctg tccatgatgc tgtcaccttt 2160
gccctccaac acccgaggcc tgtccccgac agccctgttt cggtcaccag actctgaaca 2220
tgctacatcc tgcccaagac tgcacctctg gaggtgcagg gcacccttga gaagcccctc 2280
acccctaggc cgcctccagg tgctacccag gagtcccctc catgtacaca cacacaactc 2340
agggaaggag gtcctgggac tccaagttca gcgctccagg tctgggacag ggcctgcatg 2400
cagtcaggct ggcagtggcg cggtacaggg agggaactgg tgcatatttt agcctcagga 2460
ataaagattt gtctgctcaa aaaaaaaaaa aaaaaaaacc gcggtc 2506
53/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
<210> 40
<211> 3836
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503559CB1
<400> 40
ctggaccttt aatccactgt aggtatggac agggaagaaa ggaagaccat caatcagggt 60
caagaagatg aaatggagat ttatggttac aatttgagtc gctggaagct tgccatagtt 120
tctttaggag tgatttgctc tggtgggttt ctcctcctcc tcctctattg gatgcctgag 180
tggcgggtga aagcgacctg tgtcagagct gcaattaaag actgtgaagt agtgctgctg 240
aggactactg atgaattcaa aatgtggttt tgtgcaaaaa ttcgcgttct ttctttggaa 300
acttacccag tttcaagtcc aaaatctatg tctaataagc tttcaaatgg ccatgcagtt 360
tgtttaattg agaatcccac tgaagaaaat aggcacagga tcagtaaata ttcacagact 420
gaatcacaac agattcgtta tttcacccac catagtgtaa aatatttctg gaatgatacc 480
attcacaatt ttgatttctt aaagggactg gatgaaggtg tttcttgtac gtcaatttat 540
gaaaagcata gtgcaggact gacaaagggg atgcatgcct acagaaaact gctttatgga 600
gtaaatgaaa ttgctgtaaa agtgccttct gtttttaagc ttctaattaa agaggttctc 660
aacccatttt acattttcca gctgttcagt gttatactgt ggagcactga tgaatactat 720
tactatgctc tagctattgt ggttatgtcc atagtatcaa tcgtaagctc actatattcc 780
attagaaagc aatatgttat gttgcatgac atggtggcaa ctcatagtac cgtaagagtt 840
tcagtttgta gagtaaatga agaaatagaa gaaatctttt ctaccgacct tgtgccagga 900
gatgtcatgg tcattccatt aaatgggaca ataatgcctt gtgatgctgt gcttattaat 960
ggtacctgca ttgtaaacga aagcatgtta acaggagaaa gtgttccagt gacaaagact 1020
aatttgccaa atccttcagt ggatgtgaaa ggaataggag atgaattata taatccagaa 1080
acacataaac gacatacttt gttttgtggg acaactgtta ttcagactcg tttctacact 1140
ggagaactcg tcaaagccat agttgttaga acaggattta gtacttccaa aggacagctt 1200
gttcgttcca tattgtatcc caaaccaact gattttaaac tctacagaga tgcctacttg 1260
tttctactat gtcttgtggc agttgctggc attgggttta tctacactat tattaatagc 1320
attttaaatg aggtacaagt tggggtcata attatcgagt ctcttgatat tatcacaatt 1380
actgtgcccc ctgcacttcc tgctgcaatg actgctggta ttgtgtatgc tcagagaaga 1440
ctgaaaaaaa tcggtatttt ctgtatcagt cctcaaagaa taaatatttg tggacagctc 1500
aatcttgttt gctttgacaa gactggaact ctaactgaag atggtttaga tctttggggg 1560
attcaacgag tggaaaatgc acgatttctt tcaccagaag aaaatgtgtg caatgagatg 1620
ttggtaaaat cccagtttgt tgcttgtatg gctacttgtc attcacttac aaaaattgaa 1680
ggagtgctct ctggtgatcc acttgatctg aaaatgtttg aggctattgg atggattctg 1740
gaagaagcaa ctgaagaaga aacagcactt cataatcgaa ttatgcccac agtggttcgt 1800
cctcccaaac aactgcttcc tgaatctacc cctgcaggaa accaagaaat ggagctgttt 1860
gaacttccag ctacttatga gataggaatt gttcgccagt tcccattttc ttctgctttg 1920
caacgtatga gtgtggttgc cagggtgctg ggggatagga aaatggacgc ctacatgaaa 1980
ggagcgcccg aggccattgc cggtctctgt aaacctgaaa cagttcctgt cgattttcaa 2040
aacgttttgg aagacttcac taaacagggc ttccgtgtga ttgctcttgc acacagaaaa 2100
ttggagtcaa aactgacatg gcataaagta cagaatatta gcagagatgc aattgagaac 2160
aacatggatt ttatgggatt aattataatg cagaacaaat taaagcaaga aacccctgca 2220
gtacttgaag atttgcataa agccaacatt cgcaccgtca tggtcacagg tgacagtatg 2280
ttgactgctg tctctgtggc cagagattgt ggaatgattc tacctcagga taaagtgatt 2340
attgctgaag cattacctcc aaaggatggg aaagttgcca aaataaattg gcattatgca 2400
gactccctca cgcagtgcag tcatccatca gcaattgacc cagaggctat tccggttaaa 2460
ttggtccatg atagcttaga ggatcttcaa atgactcgtt atcattttgc aatgaatgga 2520
aaatcattct cagtgatact ggagcatttt caagaccttg ttcctaagtt gatgttgcat 2580
ggcaccgtgt ttgcccgtat ggcacctgat cagaagacac agttgataga agcattgcaa 2640
aatgttgatt attttgttgg gatgtgtggt gatggcgcaa atgattgtgg tgctttgaag 2700
agggcacacg gaggcatttc cttatcggag ctcgaagctt cagtggcatc tccctttacc 2760
54/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
tctaagactc ctagtatttc ctgtgtgcca aaccttatca gggaaggccg tgctgcttta 2820
ataacttcct tctgtgtgtt taaattcatg gcattgtaca gcattatcca gtacttcagt 2880
gttactctgc tgtattctat cttaagtaac ctaggagact tccagtttct cttcattgat 2940
ctggcaatca ttttggtagt ggtatttaca atgagtttaa atcctgcctg gaaagaactt 3000
gtggcacaaa gaccaccttc gggtcttata tctggggccc ttctcttctc cgttttgtct 3060
cagattatca tctgcattgg atttcaatct ttgggttttt tttgggtcaa acagcaacct 3120
tggtatgaag tgtggcatcc aaaatcagat gcttgtaata caacaggaag cgggttttgg 3180
aattcttcac acgtagacaa tgaaaccgaa cttgatgaac ataatataca aaattatgaa 3240
aataccacag tgttttttat ttccagtttt cagtacctca tagtggcaat tgccttttca 3300
aaaggaaaac ccttcaggca accttgctac aaaaattatt tttttgtttt ttctgtgatt 3360
tttttatata tttttatatt attcatcatg ttgtatccag ttgcctctgt tgaccaggtt 3420
cttcagatag tgtgtgtacc atatcagtgg cgtgtaacta tgctcatcat tgttcttgtc 3480
aatgcctttg tgtctatcac agtggaggag tcagtggatc ggtggggaaa atgctgctta 3540
ccctgggccc tgggctgtag aaagaagaca ccaaaggcaa agtacatgta tctggcgcag 3600
gagctcttgg ttgatccaga atggccacca aaacctcaga caaccacaga agctaaagct 3660
ttagttaagg agaatggatc atgtcaaatc atcaccataa catagcagtg aatcagtctc 3720
agtggtattg ctgatagcag tattcaggaa tatgtgattt taggagtttc tgatcctgtg 3780
tgtcagaatg gcactagttc agtttatgtc ccttctgata tagtagctta tttgac 3836
<210> 41
<211> 2240
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6243872CB1
<400> 41
agcgggtttt gcctcctgcc ctaggagaaa cgttcgctct ggaggcttgg gcggcaagag 60
ccccttgtgg ccaccgagtc ctccgacgcc ctcggccagg ctggcctttg ggttggccca 120
ggcaggacgg gcagccgaga gcactcgggc cgcgtcgcca ggagccgccc agggtgagcc 180
atgttcgtag gcgtcgcccg gcactctggg agccaggatg aagtctcaag gggagtagag 240
ccgctggagg ccgcgcgggc ccagcctgct aaggacagga gggccaaggg aaccccgaag 300
tcctcgaagc ccgggaaaaa acaccggtat ctgagactac ttccagaggc cttgataagg 360
ttcggcggtt tccgaaaaag gaaaaaagcc aagtcctcag tttccaagaa gccgggagaa 420
gtggatgaca gtttggagca gccctgtggt ttgggctgct tagtcagcac ctgctgtgag 480
tgttgcaata acattcgctg cttcatgatt ttctactgca tcctgctcat atgtcaaggt 540.
gtggtgtttg gtcttataga tgtcagcatt ggtgattttc agaaggaata tcaactgaaa 600
accattgaga agttggcatt ggaaaagagt tacgatattt catctggcct gactgtgcag 660
ggaatagcag gaatgcctct ttatatcctt ggaataacct ttattgatga gaatgttgct 720
acacactcag ctggtatcta tttaggtatt gcagaatgta catcaatgat tggatatgct 780
ctgggttatg tgctaggagc accactagtt aaagtccctg agaatactac ttctgcaaca 840
aacactacag tcaataatgg tagtccagaa tggctatgga cttggtggat taattttctt 900
tttgccgctg tcgttgcatg gtgtacatta ataccattgt catgctttcc aaacaatatg 960
ccaggttcaa cacggataaa agctaggaaa cgtaaacagc ttcatttttt tgacagcaga 1020
cttaaagatc tgaaacttgg aattaatatc aaggatttat gtgctgctct ttggattctg 1080
atgaagaatc cagtgctcat atgcctagct ctgtcaaaag ctacagaata tttagttatt 1140
attggagctt ctgaattttt gcctatatat ttagaaaatc agtttatatt aacacccact 1200
gtggcaacta cacttgcagg acttgtttta attccaggag gtgcacttgg ccagcttctg 1260
ggaggtgtca ttgtttccac attagaaatg tcttgtaaag cccttatgag atttataatg 1320
gttacatctg tgatatcact tatactgctt gtgtttatta tttttgtacg ctgtaatcca 1380
gtgcaatttg ctgggatcaa tgaagattat gatggaacag ggaagttggg aaacctcacg 1440
gctccttgca atgaaaaatg tagatgctca tcttcaattt attcttctat atgtggaaga 1500
gatgatattg aatatttttc tccctgcttt gcaggtattg tttcctgtct tcaatactca 1560
cagatgtact acaattgttc ttgcattaaa gaaggattaa taactgcaga tgcagaaggt 1620
55/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
gattttattg atgccagacc cgggaaatgt gatgcaaagt gctataagtt acctttgttc 1680
attgctttta tcttttctac acttatattt tctggttttt ctggtgtacc aatcgtcttg 1740
gccatgacgc gggttgtacc tgacaaactg cgttctctgg ccttgggtgt aagctatgtg 1800
attttgagaa tatttgggac tattcctgga ccatcaatct ttaaaatgtc aggagaaact 1860
tcttgtattt tacgggatgt taataaatgt ggacacacag gacgttgttg gatatataac 1920
aagacaaaaa tggctttctt attggtagga atatgttttc tttgcaaact atgcactatc 1980
atcttcacta ctattgcatt tttcatatac aaacgtcgtc taaatgagaa cactgacttc 2040
ccagatgtaa ctgtgaagaa tccaaaagtt aagaaaaaag aagaaactga cttgtaactg 2100
gatcatcatt gtgattgcag atcatttgag gatcagagtg tgaaaaggag tttctctttt 2160
acagattctc caagatttgt ttctgtgccc aactttcaga agaggaaaat cacacattat 2220
gtttacataa gtagcaaaaa 2240
<210> 42
<211> 2807
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 90011608CB1
<400> 42
gggagtcaga agtcaagtga actcagcccg cctctgtgta ctttgcactt ttccatttcc 60
cttggtacca ggcactttca tacttaatcc atagtggagc tgtcacagtg agcaactctg 120
acaatgacag cttctacccc agaggcgacc ccaaacatgg agctaaaggc tccagctgca 180
ggaggtctta atgctggccc tgtcccccca gctgccttgt ccacgcagag acttcggaat 240
gaagactacc acgactacag ctccacggac gtgagccctg aggagagccc gtcggaaggc 300
ctcaacaacc tctcctcccc gggctcctac cagcgctttg gtcaaagcaa tagcacaaca 360
tggttccaga ccttgatcca cctgttaaaa ggcaacattg gcacaggact cctgggactc 420
cctctggcgg tgaaaaatgc aggcatcgtg atgggtccca tcagcctgct gatcataggc 480
atcgtggccg tgcactgcat gggtatcctg gtgaaatgtg ctcaccactt ctgccgcagg 540
ctgaataaat cctttgtgga ttatggtgat actgtgatgt atggactaga atccagcccc 600
tgctcctggc tccggaacca cgcacactgg ggaagacgtg ttgtggactt cttcctgatt 660
gtcacccagc tgggattctg ctgtgtctat tttgtgtttc tggctgacaa ctttaaacag 720
gtgatagaag cggccaatgg gaccaccaat aactgccaca acaatgagac ggtgattctg 780
acgcctacca tggactcgcg actctacatg ctctccttcc tgcccttcct ggtgctgctg 840
gttttcatca ggaacctccg agccctgtcc atcttctccc tgttggccaa catcaccatg 900
ctggtcagct tggtcatgat ctaccagttc attgttcaga ggatcccaga ccccagccac 960
ctccccttgg tggccccttg gaagacctac cctctcttct ttggcacagc gattttttca 1020
tttgaaggca ttggaatggt tctgcccctg gaaaacaaaa tgaaggatcc tcggaagttc 1080
ccactcatcc tgtacctggg catggtcatc gtcaccatcc tctacatcag cctggggtgt 1140
ctggggtacc tgcaatttgg agctaatatc caaggcagca taaccctcaa cctgcccaac 1200
tgctggttgt accagtcagt taagctgctg tactccatcg ggatcttttt cacctacgca 1260
ctccagttct acgtcccggc tgagatcatc atccccttct ttgtgtcccg agcgcccgag 1320
cactgtgagt tagtggtgga cctgtttgtg cgcacagtgc tggtctgcct gacatgcatc 1380
ttggccatcc tcatcccccg cctggacctg gtcatctccc tggtgggctc cgtgagcagc 1440
agcgccctgg ccctcatcat cccaccgctc ctggaggtca ccaccttcta ctcagagggc 1500
atgagccccc tcaccatctt taaggacgcc ctgatcagca tcctgggctt cgtgggcttt 1560
gtggtgggga cctatgaggc tctctatgag ctgatccagc caagcaatgc tcccatcttc 1620
atcaattcca cctgtgcctt catataggga tctgggttcg tctctgcagc tgcctacccc 1680
tgccccatgt gtcccccgtt acctgtcctc agagcctcag gtatggtcca ggctctgagg 1740
aaagtcaggg ttgctgtgtg ggaacccctc tgcctggcac ctggataccc tgggccaggt 1800
aacctgaggg caggggagag gtggggtggc agacacgcag aagtgctact agtgacaggg 1860
ctgccatcgc tcacctgtac ctatttacac ccagaacttt ccagctcccc ctcatcatgc 1920
ctcctccttc ctacctgcct cccctctgct ggtgcacctc gcccaactca ttcttactgc 1980
acagttcact ttatttaaca attttcatgt cccccacctc atgttttcac cttttctggg 2040
56/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
ccaggcatag attaagtaac tgggaacgcc ccctctttat aaagctgggc ttctttctca 2100
tctctctccc aaatgttgta tctcagtatt cttcctattc gagtctccag ggggtggctg 2160
gacctacctg gtcatttgaa acaggccccc aagctggagt tttttaatct ggactctctg 2220
gcttgctgtg acccctaagg caatgcttct cttccctgga ttccttagtg tgggtcacag 2280
tactgtgttc ttagttgctt tagctcttaa aacatacgaa gtgttgccta aactgaaaat 2340
atttatcttt tatttaaaat cagatttttg tttttagact gtcttagatc tggggctatt 2400
acgaatcact tcttcttcag taaactttga ctcaacttct cctgctgaaa agaagctcgc 2460
tccagatgtc tgcatgggtc ctcggcactc ttggctgagg actcaaaggt tttaatcagg 2520
atcgtctaaa aatgtacctc ggtgaggagg cacagatttt gcctcctgtt gaccagcctg 2580
gtttcatacc gaaaagacat tgaaggactg cagaaatgta tgggtgcacc gggccgaggg 2640
aagggtggct gagtgagagg catataaaat ggggctgtgt gcatgcaggc ccatgtttca 2700
gcctcagccc acgccaggtg aaaggatcag caatgctctg tcgccatcgt gctgggacga 2760
caccagctct attgccaccg atgagtagct gaggtcagtg tgcacag 2807
<210> 43
<211> 3201
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 90024583CB1
<400> 43
atggcctcgg cgctgagcta tgtctccaag ttcaagtcct tcgtgatctt gttcgtcacc 60
ccgctcctgc tgctgccact cgtcattctg atgcccgcca agtttgtcag gtgtgcctac 120
gtcatcatcc tcatggccat ttactggtgc acagaagtca tccctctggc tgtcacctct 180
ctcatgcctg tcttgctttt cccactcttc cagattctgg actccaggca ggtatgtgtc 240
cagtacatga aggacaccaa catgctgttc ctgggcggcc tcatcgtggc cgtggctgtg 300
gagcgctgga acctgcacaa gaggatcgcc ctgcgcacgc tcctctgggt gggggccaag 360
cctgcacggc tgatgctggg cttcatgggc gtcacagccc ccctgtccat gtggatcagt 420
aacacggcaa ccacggccat gatggtgccc atcgtggagg ccatattgca gcagatggaa 480
gccacaagcg cagccaccga ggccggcctg gagctggtgg acaagggcaa ggccaaggag 540
ctgccaggga gtcaagtgat ttttgaaggc cccactctgg ggcagcagga agaccaagag 600
cggaagaggt tgtgtaaggc catgaccctg tgcatctgct acgcggccag catcgggggc 660
accgccaccc tgaccgggac gggacccaac gtggtgctcc tgggccagat gaacgagttg 720
tttcctgaca gcaaggacct cgtgaacttt gcttcctggt ttgcatttgc ctttcccaac 780
atgctggtga tgctgctgtt cgcctggctg tggctccagt ttgtttacat gagattcaat 840
tttaaaaagt cctggggctg cgggctagag agcaagaaaa acgagaaggc tgccctcaag 900
gtgctgcagg aggagtaccg gaagctgggg cccttgtcct tcgcggagat caacgtgctg 960
atctgcttct tcctgctggt catcctgtgg~ttctcccgag accccggctt catgcccggc 1020
tggctgactg ttgcctgggt ggagggtgag acaaagtatg tctccgatgc cactgtggcc 1080
atctttgtgg ccaccctgct attcattgtg ccttcacaga agcccaagtt taacttccgc 1140
agccagactg aggaagaaag gaaaactcca ttttatcecc ctcccctgct ggattggaag 1200
gtaacccagg agaaagtgcc ctggggcatc gtgctgctac tagggggcgg atttgctctg 1260
gctaaaggat ccgaggcctc ggggctgtcc gtgtggatgg ggaagcagat ggagcccttg 1320
cacgcagtgc ccccggcagc catcaccttg atcttgtcct tgctcgttgc cgtgttcact 1380
gagtgcacaa gcaacgtggc caccaccacc ttgttcctgc ccatctttgc ctccatgtct 1440
cgctccaacg gcctcaatcc gctgtacatc atgctgccct gtaccctgag tgcctccttt 1500
gccttcatgt tgcctgtggc cacccctcca aatgccatcg tgttcaccta tgggcacctc 1560
aaggttgctg acatggtgaa aacaggagtc ataatgaaca taattggagt cttctgtgtg 1620
tttttggctg tcaacacctg gggacgggcc atatttgact tggatcattt ccctgactgg 1680
gctaatgtga cacatattga gacttaggaa gagccacaag accacacaca cagcccttac 1740
cctcctcagg actaccgaac cttctggcac accttgtaca gagttttggg gttcacaccc 1800
caaaatgacc caacgatgtc cacacaccac caaaacccag ccaatgggcc acctcttcct 1860
ccaagcccag atgcagagat ggtcatgggc agctggaggg taggctcaga aatgaaggga 1920
57/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
acccctcagt gggctgctgg acccatcttt cccaagcctt gccattatct ctgtgaggga 1980
ggccaggtag ccgagggatc aggatgcagg ctgctgtacc cgctctgcct caagcatccc 2040
ccacacaggg ctctggtttt cactcgcttc gtcctagata gtttaaatgg gaatcggatc 2100
ccctggttga gagctaagac aaccacctac cagtgcccat gtcccttcca gctcaccttg 2160
agcagcctca gatcatctct gccactctgg aagggacacc ccagccaggg acggaatgcc 2220
tggtcttgag caacctccca ctgctggagt gcgagtggga atcagagcct cctgaagcct 2280
ctgggaactc ctcctgtggc caccaccaaa ggatgaggaa tctgagttgc caacttcagg 2340
acgacacctg gcttgccacc cacagtgcac cacaggccaa cctacgccct tcatcacttg 2400
gttctgtttt aatcgactgg ccccctgtcc~cacctctcca gtgagcctcc ttcaactcct 2460
tggtcccctg ttgtctgggt caacatttgc cgagacgcct tggctggcac cctctggggt 2520
cccccttttc tcccaggcag gtcatctttt ctgggagatg cttcccctgc catccccaaa 2580
tagctaggat cacactccaa gtatgggcag tgatggcgct ctgggggcca cagtgggcta 2640
tctaggccct ccctcacctg aggcccagag tggacacagc tgttaatttc cactggctat 2700
gccacttcag agtctttcat gccagcgttt gagctcctct gggtaaaatc ttccctttgt 2760
tgactggcct tcacagccat ggctggtgac aacagaggat cgttgagatt gagcagcgct 2820
tggtgatctc tcagcaaaca acccctgccc gtgggccaat ctacttgaag ttactcggac 2880
aaagacccca aagtggggca acaactccag agaggctgtg ggaatcttca gaacccccct 2940
gtaagagaca gacatgagag acaagcatct tctttccccc gcaagtccat tttatttcct 3000
tcttgtgctg ctctggaaga gaggcagtag caaagagatg agctcctgga tggcattttc 3060
cagggcagga gaaagtatga gagcctcagg aaaccccatc aaggaccgag tatgtgtctg 3120
gttccttggg tgggacgatt cctgaccaca ctgtccagct cttgctctca ttaaatgctc 3180
tgtctcccgc ggaaaaaaaa a 3201
<210> 44
<211> 3688
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 90113658CB1
<400> 44
gctgtgtcca agaaaggagt cagcaaaggg tgca~tgggatt atcattagtt cttataggtt 60
tgggataggc ggtggagtta ggagcaattt tttataggca gggggtggat ctcacaaagt 120
acattctcaa gggcggggag aatattacaa agtaccttct taagggcggg ggagtatgac 180
aaagtatatt attgcaaggg cggggagggg gtattgtcat aaggtcaatt gatcagttag 240
ggtgggtagg aacagatcac aatggtggaa tgttattttt tgtggttctt catttgcttc 300
aggtcatctg gatgtatatg tgcaggtcac aggagatgtg atggcttagc ttgggctcag 360
aggcctgaca gtttgtatcc tcgtctccgt aatggtgcaa tgatccttcc ttcgcgtagg 420
gtaccatgtg tctcagggct ggaggaccac ctggccttgc tggcctctta caccagagcc 480
tgggccatag ctcataattt tgctgatatt ggcatctttt aacctcagaa agtatactct 540
gggtctaata atctgcctcc ttcaggaata ttcattcctc ttgttgcccg ctagcttcca 600
gtcataaaaa aaaatccgtt taataacttt agatttggtt acatccatct gtgtgttttt 660
cttcacctcc atttagaatt agtaggaaaa gcattagaac aaaagggttg tttaaagaac 720
attagtttat ctcatttgaa aagtgaaact tctttcatgt aagctctggt tacttcattg 780
ttctttccac aagcccttct aacaatgatt atctgcagtt tgcccaccca ctgtcaagag 840
ctcagcatcc atacttgaaa ctcaagcttc atatgccaac tttgggatga ttctcatcaa 900
acagaagagg ctgtttccat gctggatccc ggcgctgttc atcggcttca gccagttctc 960
ggactcgttc ctcctggacc agcccaactt ctggtgccgc ggggccggca aaggcaccga 1020
gctggcaggg gtcaccacca caggccgggg cggggacatg ggcaactgga ccagcctccc 1080
caccaccccc ttcgccactg ccccctggga ggctgcgggc aaccggagca acagcagcgg 1140
cgcggacgga ggcgacacac cacccctgcc atcccctccg gacaaggggg acaacgcctc 1200
caactgtgac tgccgcgcat gggactacgg catccgcgcc ggcctcgtcc agaacgtggt 1260
cagcaagtgg gatcttgtgt gtgataatgc ctggaaggtc catatcgcta agttctcctt 1320
actggttgga ttaatctttg gctacctaat aactggatgc attgctgact gggtcggccg 1380
58/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
gcggcctgtg ctgctgtttt ccatcatctt cattctgatc tttggactga ctgtggcact 1440
gtcagtgaat gtgacaatgt tcagcacact caggttcttt gaaggatttt gcctggctgg 1500
aatcattctc accttgtatg ctttacgaat agagctgtgc ccccctggaa aacggttcat 1560
gattacgatg gtggcgagct tcgtggccat ggcgggccag ttcctcatgc ctgggctagc 1620
cgccctgtgc cgggattggc aggtgctgca ggccctcatc atctgcccct tcctgctcat 1680
gctgctctac tggtcgatat tccccgagtc cctccggtgg ctaatggcca cccagcagtt 1740
tgagtctgca aagaggctga tcctccactt cacacagaag aatcgcatga accctgaggg 1800
cgacatcaag ggtgtgatac cagagctgga gaaagagctt tcccggaggc ccaagaaggt 1860
ctgcatcgtg aaggtggtgg ggacacggaa cctgtggaag aacattgtgg tcctgtgtgt 1920
gaactcgctg acggggtacg ggatccacca ctgctttgcc aggagcatga tgggccacga 1980
ggtgaaggtg ccgctcctgg agaacttcta tgctgactac tataccacgg ccagcatcgc 2040
gctggtgtcc tgcctggcca tgtgcgtggt ggtccgattc ctcgggcgca ggggagggct 2100
gctgctcttc atgatcctca ccgccctggc ctcgctcctg cagctcggcc tcctcaacct 2160
gattggaaag tacagccagc acccagactc agggatgagt gacagcgtca aggacaaatt 2220
ttccatcgcg ttttccatcg tgggcatgtt tgcctcccat gcggtgggga gcctcagcgt 2280
gttcttctgt gcggagatca ccccgacggt gataaggtgt ggcgggctgg ggctggtgct 2340
ggccagcgcg ggcttcggca tgctgacggc acccatcatc gagctgcaca accagaaagg 2400
ctacttcctg caccacatca tctttgcctg ctgcacgctc atctgcatca tctgcatcct 2460
cctgctgccc gagagcaggg accagaacct gcctgagaac atttctaacg gggagcacta 2520
cacgcgccag ccgctgctgc cgcacaagaa gggggagcag ccactgctgc tcaccaacgc 2580
cgagctcaag gactactcgg gcctccacga tgccgcagcc gcgggtgaca cactgcccga 2640
ggg.tgccacg gccaacggca tgaaggccat gtagcccggc ctgcggaacc cggggctcca 2700
gggtctgggg cagcttgggc acaggtttac agaccaggga ccgaacacgc agccaggggt 2760
gggaaagctg cctcagccaa gctgagcctc tcaactggtg tggggaaatc ctgtctttcc 2820
aaaagtccaa ggagcgcggg tcggaggaga caaactcttt ggaaataacc ctttcaagac 2880
tttcttttct gccgttaaat gtgtgtattt attttggtca tttttacgag aagcacttta 2940
ttccctctcc ctctcactga tcacaaatgg aatcacctcc ctgggcagcg agacgcagtt 3000
gctctgggaa gatgccacag tgaccagggc catggccggt ccctctgggg agatgggaca 3060
cggctcctgg cagcacccag gagcccccca ggctgcctgc ctgcgtgcag aggcaggaga 3120
ggaccgtgga gcgtttccgg gactgcattt tagacggagt gaaatgtaca tgaatttggc 3180
ttttgctaga gtctgtgtat ggttttttaa ggtccttttt ccctttctgt ttgtaaggta 3240
agagcttctg ttcgtgtgca gggaaagcag ctcacagacg ccgttaaaac cagcttcatc 3300
tttccttcag gatcatcctt ttgtacttga tgctggaagc tcttggagaa aaagcttaaa 3360
catttcacca gaaatcttaa ttgagcagca gtcatatcgc cacagctttg tgagtacaca 3420
gctaacagat gcgtcgagac ctgagatgtg ctcgttttta ttcctcctct cccagattgg 3480
ctccagcaga aaagtccctc gtgcacaggc agctcttgtg ccggcactac ttgaaaacat 3540
ggctactttc taagctacaa accattagaa aataactcaa aaagatggca gaggcaaatc 3600
cacagaaggg gggctgccct ccacacacac acgcccgtca cccacacctt gagcacacac 3660
catgaagatc acctactgag gaggatcc 3688
<210> 45
<211> 2402
<212> DNA
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 3942766CB1
<400> 45
gcccgtaccg ccaggcgatc gcgctgatgg cggcgctggc agcagcggcc aagaaggtgt 60
ggagcgcgcg gcggctgctg gtgctgctgt tcacgccgct cgcgctgctg ccggtggtct 120
tcgccctccc gcccaaggaa ggccgctgct tgtttgtcat cctgctcatg gcggtgtact 180
ggtgcacgga ggccctgccg ctctcagtga cggcgctgct gcccatcgtc ctcttcccct 240
tcatgggcat cttgccctcc aacaaggtct gcccccagta cttcctcgac accaacttcc 300
tcttcctcag tgggctgatc atggccagcg ccattgagga gtggaacctg caccggcgaa 360
59/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
tcgccctcaa gatcctgatg cttgttggag tccagccggc caggctcatc ctggggatga 420
tggtgaccac ctcgttcttg tccatgtggc tgagcaacac cgcctccact gccatgatgc 480
ttcccattgc caatgccatc ctgaaaagtc tctttggcca gaaggaggtt cgaaaggacc 540
ccagccagga gagtgaagag aacacagctg ctgtgcggag aaacggccta cacactgtgc 600
ccacggagat gcagtttctc gccagcacag aagccaaaga ccaccctggg gagacagagg 660
ttccactgga tctgccggct gactccagga aggaggatga atatcgtcgg aacatctgga 720
agggcttcct catctccatc ccctactcag ccagtattgg gggcacagcc acactcacgg 780
gcacagcccc taacctcatc ctgcttggcc agctcaagag tttctttccg cagtgtgacg 840
tggtgaattt cggctcctgg ttcattttcg ccttccctct tatgctgttg ttcctgttgg 900
caggctggct ctggatctcc ttcctgtacg ggggactgag cttcaggggc tggaggaaga 960
ataaatctga gataagaacc aatgcagaag atagggctcg agctgtaatt cgggaagaat 1020
accagaacct ggggcccatc aagtttgccg aacaggctgt tttcatcctt ttctgcatgt 1080
ttgccatcct cctcttcacc cgggacccga agttcatccc tggctgggcc agcctcttca 1140
atcctgggtt tctttctgat gctgtcaccg gcgtggctat tgtcaccatc ttgttcttct 1200
tcccgtccca aaggccctct ctcaagtggt ggtttgactt caaagctccc aacacagaga 1260
cagagccctt gctgacctgg aagaaggccc aggagacagt gccctggaac atcatccttc 1320
tcctgggagg gggcttcgcc atggccaaag gctgtgagga atcggggctg tctgtatgga 1380
ttggtgggca gctgcacccc ctggagaatg tgccccccgc cctggctgtg ctgctcatca 1440
ctgtggtcat cgccttcttc actgagtttg ccagcaacac ggcgaccatc atcatcttcc 1500
tgccggtcct ggcagagctg gccatccgcc tgagagtgca ccccctgtat ctgatgattc 1560
cgggcacagt cggctgctcc tttgccttca tgctcccggt ctcaacgccc cccaactcca 1620
tcgccttcgc ctctggacac ttgctggtca aagacatggt gcggacaggc ctcctgatga 1680
acctgatggg tgtcctgctg ctcagtttgg ctatgaatac ctgggcacag accatcttcc 1740
agctgggcac cttcccggac tgggctgata tgtactcggt caatgtcaca gcattgccac 1800
ccaccttggc caatgacaca tttcggaccc tctgagtccc cctggaggac tcccttgagg 1860
ctgaaccctc tcaaagggct gtcaccatca cctgctgcta ggacactaca aaacaatcaa 1920
ataattcttt tctgtaatcc aatatgcagc aagcaagggt gacctccagt ggcccactca 1980
agtccatgag ccattatcta ggatactttc tctctctttc atgcagttca aagcccaggt 2040
atctctcaga tctgctgcct gagaaataag ctcctttatc agttagctgt tttatcatta 2100
ggatacaaga cagcccagtg tcatcaacag tgagcaaatc tggcatggtg ttgtctcgta 2160
cagtgggata ggaggccatt cattcccatg ggcacagcta acattatccc ccagtgatta 2220
ctttcgatta cactgaagag acacctgtct taaagatcac tttcctgggc tggcatgtcc 2280
aaccagttgt tccagcctgt tcacaccagg gggggattgc ttagttggtg cccatctgcc 2340
cagtgtttac cctgcgatac caggagcaga gatcctttaa tcctgggacc ggaaggcagt 2400
ac 2402
<210> 46
<211> 2410
<212> DNA
<213> Homo Sapiens
<220>
<221> misc-feature
<223> Incyte ID No: 7501987CB1
<400> 46
catccgctca caatgccaca tcaatgatac gagcacgtag cctcactgct tgcacagtgc 60
atggcagagt cggctgcgag caggcgaggt ggcctgaggg aggtcactag gctggctgag 120
ggctttttgc tgtggttctg agccggcctg cttccaggca ccgtgtccat gcgggtaagc 180
ggtctccctg ggtgcccact cttgcgcccg gagatcctga gtttggtcct gtctggccat 240
gaagctcagc ctgctgggag gccacaggga gatgcaggct gggcggcggg tggatggttc 300
cagccggttg ggtccggggc ctggagctca gcctgtgggg tggggaccca gtggtgccct 360
ggagctgccg cttctgctct cagcaggatg atgggcagga cagggagagg ctgacctact 420
tccagaacct gcctgagtct ctgacttccc tcctggtgct gctgaccacg gccaacaacc 480
ccgatgtgat gattcctgcg tattccaaga accgggccta tgccatcttc ttcatagtct 540
tcactgtgat aggaagcctg tttctgatga acctgctgac agccatcatc tacagtcagt 600
60/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
tccggggcta cctgatgaaa tctctccaga cctcgctgtt tcggaggcgg ctgggaaccc 660
gggctgcctt tgaagtccta tcctccatgg tgggggaggg aggagccttc cctcaggcag 720
ttggggtgaa gccccagaac ttgctgcagg tgcttcagaa ggtccagctg gacagctccc 780
acaaacaggc catgatggag aaggtgcgtt cctatggcag tgttctgctc tcagctgagg 840
agtttcagaa gctcttcaac gagcttgaca gaagtgtggt taaagagcac ccgccgaggc 900
ccgagtacca gtctccgttt ctgcagagcg cccagttcct cttcggccac tactactttg 960
actacctggg gaacctcatc gccctggcaa acctggtgtc catttgcgtg ttcctggtgc 1020
tggatgcaga tgtgctgcct gctgagcgtg atgacttcat cctggggatt ctcaactgcg 1080
tcttcattgt gtactacctg ttggagttgc tgctcaaggt ctttgccctg ggcctgcgag 1140
ggtacctgtc ctaccccagc aacgtgtttg acgggctcct caccgttgtc ctgctggagg 1200
ccggagatgg tgggcctgct gtcgctgtgg gacatgaccc gcatgctgaa catgctcatc 1260
gtgttccgct tcctgcgtat catccccagc atgaagccga tggccgtggt ggccagtacc 1320
gtcctgggcc tggtgcagaa catgcgtgcg tttggcggga tcctggtggt ggtctactac 1380
gtatttgcca tcattgggat caacttgttt agaggcgtca ttgtggctct tcctggaaac 1440
agcagcctgg cccctgccaa tggctcggcg ccctgtggga gcttcgagca gctggagtac 1500
tgggccaaca acttcgatga ctttgcggct gccctggtca ctctgtggaa cttgatggtg 1560
gtgaacaact ggcaggtgtt tctggatgca tatcggcgct actcaggccc gtggtccaag 1620
atctattttg tgttgtggtg gctggtgtcg tctgtcatct gggtcaacct gtttctggcc 1680
ctgattctgg agaacttcct tcacaagtgg gacccccgca gccacctgca gccccttgct 1740
gggaccccag aggccaccta ccagatgact gtggagctcc tgttcaggga tattctggag 1800
gagcccgggg aggatgagct cacagagagg ctgagccagc acccgcacct gtggctgtgc 1860
aggtgacgtc cgggctgccg tcccagcagg ggcggcagga gagagaggct ggcctacaca 1920
ggtgcccatc atggaagagg cggccatgct gtggccagcc aggcaggaag agacctttcc 1980
tctgacggac cactaagctg gggacaggaa ccaagtcctt tgcgtgtggc ccaacaacca 2040
tctacagaac agctgctggt gcttcaggga ggcgccgtgc cctccgcttt cttttatagc 2100
tgcttcagtg agaattccct cgtcgactcc acagggacct ttcagacaaa aatgcaagaa 2160
gcagcggcct cccctgtccc ctgcagcttc cgtggtgcct ttgctgccgg cagcccttgg 2220
ggaccacagg cctgaccagg gcctgcacag gttaaccgtc agacttccgg ggcattcagg 2280
tggggatgct ggtggtttga catggagaga accttgactg tgttttatta tttcatggct 2340
tgtatgagtg tgactgggtg tgtttcttta gggttctgat tgccagttat tttcatcaat 2400
aagtcttgca 2410
<210> 47
<211> 968
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 750.3223CB1
<400> 47
gaggagatgg gttccaattt tagggagcag gctgcctaat gaaggagcca ggcttgcaca 60
cagacaattc tagaactggt ggcccgagag ggatgtgaag gcccaaaatg accctcttac 120
cgggagacaa ttctgactac gactacagcg cgctgagctg cacctcggac gcctccttcc 180
acccggcctt cctcccgcag cgccaggcca tcaagggcgc gttctaccgc cgggcgcagc 240
ggctgcggcc gcaggatgag ccccgccagg gctgtcagcc cgaggaccgc cgccgtcgga 300
tcatcatcaa cgtaggcggc atcaagtact cgctgccctg gaccacgctg gacgagttcc 360
cgctgacgcg cctgggccag ctcaaggcct gcaccaactt cgacgacatc ctcaacgtgt 420
gcgatgacta cgacgtcacc tgcaacgagt tcttcttcga ccgcaacccg ggggccttcg 480
gcactatcct gaccttcctg cgcgcgggca agctgcggct gctgcgcgag atgtgcgcgc 540
tgtccttcca ggacagtgac atcttgttcg gaagtgcctc ctcggacacc agagacaata 600
actgagcgcg gaggacacgc ctgccctgcc tgccatctgt ggcccgaagc cattgccatc 660
cactgcagac gcctggagag ggacaggccg cttccgagtg cagtcctggc gcagcaccga 720
ctcccacgca cccggggaag gacaccctca ctcccacacc ccgggaagaa cactagaaca 780
tcagcagagg ggccctgccc ctccgcctgc agccgtgaaa ggaagctggg tcatcagccc 840
61/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
agccccgccc accccagccc ctatgtgtgt ttccctcaat aaggagatgc cttgttcttt 900
tcaccatgca aataacatgc ccagcaaaaa cttgctttat gggtctgcct ggagaaaaaa 960
aaaaaaaa 968
<210> 48
<211> 2267
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503566CB1
<400> 48
gtcttgctaa cagctgccaa tacctcactg agtgcctcac accaacatgg gctccaagtg 60
agtttccttc gtctgggcag actccctccc ctcttccata aaggctgcag gagacctgta 120
gctgtcacag gaccttccct aagagcccgc aggggaagac tgccccagtc cggccatcac 180
catgctccgg accattctgg atgctcccca gcggttgctg aaggagggga gagcgtcccg 240
gcagctggtg ctggtggtgg tattcgtcgc tttgctcctg gacaacatgc tgtttactgt 300
ggtggtgcca attgtgccca ccttcctata tgacatggag ttcaaagaag tcaactcttc 360
tctgcacctc ggccatgccg gaagttcccc acatgccctc gcctctcctg ccttttccac 420
catcttctcc ttcttcaaca acaacaccgt ggctgttgaa gaaagcgtac ctagtggaat 480
agcatggatg aatgacactg ccagcaccat cccacctcca gccactgaag ccatctcagc 540
tcataaaaac aactgcttgc aaggcacagg tttcttggag gaagagacta cccgggtcgg 600
ggttctgttt gcttcaaagg ctgtgatgca acttctggtc aacccattcg tgggccctct 660
caccaacagg attggatatc atatccccat gtttgctggc tttgttatca tgtttctctc 720
cacagttagt cttggaatgc tggccagtgt ctacactgat gaccatgaga gaggacgagc 780
catgggaact gctctggggg gcctggcctt ggggttgctg gtgggagctc cctttggaag 840
tgtaatgtac gagtttgttg ggaagtctgc acccttcctc atcctggcct tcctggcact 900
actggatgga gcactccagc tttgcatcct acagccttcc aaagtctctc ctgagagtgc 960
caaggggact cccctcttta tgcttctcaa agacccttac atcctggtgg ctgcagggtc 1020
catctgcttt gccaacatgg gggtggccat cctggagccc acactgccca tctggatgat 1080
gcagaccatg tgctccccca agtggcagct gggtctagct ttcttgcctg ccagtgtgtc 1140
ctacctcatt ggcaccaacc tctttggtgt gttggccaac aagatgggtc ggtggctgtg 1200
ttccctaatc gggatgctgg tagtaggtac cagcttgctc tgtgttcctc tggctcacaa 1260
tatttttggt ctcattggcc ccaatgcagg gcttggcctt gccataggca tggtggattc 1320
ttctatgatg cccatcatgg ggcacctggt ggatctacgc cacacctcgg tgtatgggag 1380
tgtctacgcc atcgctgatg tggctttttg catgggcttt gctataggtc catccaccgg 1440
tggtgccatt gtaaaggcca tcggttttcc ctggctcatg gtcatcactg gggtcatcaa 1500
catcgtctat gctccactct gctactacct gcggagcccc ccggcaaagg aagagaagct 1560
tgctattctg agtcaggact gccccatgga gacccggatg tatgcaaccc agaagcccac 1620
gaaggaattt cctctggggg aggacagtga tgaggagcct gaccatgagg agtagcagca 1680
gaaggtgctc cttgaattca tgatgcctca gtgaccacct ctttccctgg gaccagatca 1740
ccatggctga gcccacggct cagtgggctt cacatacctc tgcctgggaa tcttctttcc 1800
tcccctccca tggacactgt ccctgatact cttctcacct gtgtaacttg tagctcttcc 1860
tctatgcctt ggtgccgcag tggcccatct tttatgggaa gacagagtga tgcaccttcc 1920
cgctgctgtg aggttgatta aacttgagct gtgacgggtt ctgcaagggg tgactcattg 1980
catagaggtg gtagtgagta atgtgcccct gaaaccagtg gggtgactga caagcctctt 2040
taatctgttg cctgattttc tctggcatag tcccaacaga tcggaagagt gttaccctct 2100
tttcctcaac gtgttctttc ccgggttttc ccagccgagt tgagaaaatg ttctcagcat 2160
tgtcttgctg ccaaatgcca gcttgaagag ttttgttttg ttttttttcc atttattttt 2220
ttttttaata aagtgagtga tttttctgtg gctaaaaaaa aaaaaaa 2267
<210> 49
<211> 319
<212> DNA
62/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7505122CB1
<400> 49
cggctcgagc tcgaccgaat cggctcgagc ggctcgagtg ctcagcctgg tgaaccacac 60
aggccagcgc tctgacatgc agaaggtgac cctgggcctg cttgtgttcc tggcaggctt 120
tcctgtcctg gacgccaatg acctagaaga taaaaacagt cctttctact atgactggca 180
cagcctccag gttggcgggc tcatctgcgc tggggttctg tgcatggcag ggcctcatct 240
cacctctcgc aagagggtct ctttgttcaa ttttttttaa tctaaaatga ttgtgcctct 300
gcccaaaaaa aaaaaaaaa 319
<210> 50
<211> 2510
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7511620CB1
<400> 50
ggagcagccc gcaccggaca acttgcgagc catggggctg gcggatgcgt cgggaccgag 60
ggacacacag gcactgctgt ctgcaacaca agcaatggac ctgcggaggc gagactacca 120
catggaacgg ccgctgctga accaggagca tttggaggag ctggggcgct ggggctcagc 180
acctaggacc caccagtggc ggacctggtt gcagtgctcc cgtgctcggg cctatgccct 240
tctgctccaa cacctcccgg ttttggtctg gttaccccgg tatcctgtgc gtgactggct 300
cctgggtgac ctgttatccg gcctgagtgt ggccatcatg cagcttccgc agggcttggc 360
ctacgccctc ctggctggat tgccccccgt gtttggcctc tatagctcct tctaccctgt 420
cttcatctac ttcctgtttg gcacttcccg gcacatctcc gtgggccttg aacgactcca 480
tgatcaatga gacagccaga gatgctgccc gggtacaggt ggcctccaca ctcagtgtcc 540
tggttggcct cttccaggtg gggctgggcc tgatccactt cggcttcgtg gtcacctacc 600
tgtcagaacc tcttgtccga ggctatacca cagctgcagc tgtgcaggtc ttcgtctcac 660
agctcaagta tgtgtttggc ctccatctga gcagccactc tgggccactg tccctcatct 720
atacagtgct ggaggtctgc tggaagctgc cccagagcaa ggttggcacc gtggtcactg 780
cagctgtggc tggggtggtg ctcgtggtgg tgaagctgtt gaatgacaag ctgcagcagc 840
agctgcccat gccgataccc ggggagctgc tcacgctcat cggggccaca ggcatctcct 900
atggcatggg tctaaagcac agatttgagg tagatgtcgt gggcaacatc cctgcagggc 960
tggtgccccc agtggccccc aacacccagc tgttctcaaa gctcgtgggc agcgccttca 1020
ccatcgctgt ggttgggttt gccattgcca tctcactggg gaagatcttc gccctgaggc 1080
acggctaccg ggtggacagc aaccaggagc tggtggccct gggcctcagt aaccttatcg 1140
gaggcatctt ccagtgcttc cccgtgagtt gctctatgtc tcggagcctg gtacaggaga 1200
gcaccggggg caactcgcag gttgctggag ccatctcttc ccttttcatc ctcctcatca 1260
ttgtcaaact tggggaactc ttccatgacc tgcccaaggc ggtcctggca gccatcatca 1320
ttgtgaacct gaagggcatg ctgaggcagc tcagcgacat gcgctccctc tggaaggcca 1380
atcgggcgga tctgcttatc tggctggtga ccttcacggc caccatcttg ctgaacctgg 1440
accttggctt ggtggttgcg gtcatcttct ccctgctgct cgtggtggtc cggacacaga 1500
tgccccacta ctctgtcctg gggcaggtgc cagacacgga tatttacaga gatgtggcag 1560
agtactcaga ggccaaggaa gtccgggggg tgaaggtctt ccgctcctcg gccaccgtgt 1620
actttgccaa tgctgagttc tacagtgatg cgctgaagca gaggtgtggt gtggatgtcg 1680
acttcctcat ctcccagaag aagaaactgc tcaagaagca ggagcagctg aagctgaagc 1740
aactgcagaa agaggagaag cttcggaaac aggctgcctc ccccaagggc gcctcagttt 1800
ccattaatgt caacaccagc cttgaagaca tgaggagcaa caacgttgag gactgcaaga 1860
tgatggtgag ctcaggagat aagatggaag atgcaacagc caatggtcaa gaagactcca 1920
63/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
aggccccaga tgggtccaca ctgaaggccc tgggcctgcc tcagccagac ttccacagcc 1980
tcatcctgga cctgggtgcc ctctcctttg tggacactgt gtgcctcaag agcctgaaga 2040
atattttcca tgacttccgg gagattgagg tggaggtgta catggcggcc tgccacagcc 2100
ctgtggtcag ccagcttgag gctgggcact tcttcgatgc atccatcacc aagaagcatc 2160
tctttgcctc tgtccatgat gctgtcacct ttgccctcca acacccgagg cctgtccccg 2220
acagccctgt ttcggtcacc agactctgaa catgctacat cctgcccaag actgcacctc 2280
tggaggtgca gggcaccctt gagaagcccc tcacccctag gccgcctcca ggtgctaccc 2340
aggagtcccc tccatgtaca cacacacaac tcagggaagg aggtcctggg actccaagtt 2400
cagcgctcca ggtctgggac agggcctgca tgcagtcagg ctggcagtgg cgcggtacag 2460
ggagggaact ggtgcatatt ttagcctcag gaataaagat ttgtctgctc 2510
<210> 51
<211> 2241
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7506995CB1
<400> 51
catccgctca caatgccaca tcaatgatac gagcacgtag cctcactgct tgcacagtgc 60
atggcagagt cggctgcgag caggcgaggt ggcctgaggg aggtcactag gctggctgag 120
ggctttttgc tgtggttctg agccggcctg cttccaggca ccgtgtccat gcgggtgagc 180
ggtctccctg ggtgcccact cttgcgcccg gagatcctga gtttggtcct gtctggccat 240
gaagctcagc ctgctgggag gccacaggga gatgcaggct gggcggcggg tggatggttc 300
cagccggttg ggtccggggc ctggagctca gcctgtgggg tggggaccca gtggtgccct 360
ggagctgccg cttctgctct cagcaggatg atgggcagga cagggagagg ctgacctact 420
tccagaacct gcctgagtct ctgacttccc tcctggtgct gctgaccacg gccaacaacc 480
ccgatgtgat gattcctgcg tattccaaga accgggccta tgccatcttc ttcatagtct 540
tcactgtgat aggaagcctg tttctgatga acctgctgac agccatcatc tacagtcagt 600
tccggggcta cctgatgaaa tctctccaga cctcgctgtt tcggaggcgg ctgggaaccc 660
gggctgcctt tgaagtccta tcctccatgg tgggggaggg aggagccttc cctcaggcca 720
cccgccgagg cccgagtacc agtctccgtt tctgcagagc gcccagttcc tcttcggcca 780
ctactacttt gactacctgg ggaacctcat cgccctggca aacctggtgt ccatttgcgt 840
gttcctggtg ctggatgcag atgtgctgcc tgctgagcgt gatgacttca tcctggggat 900
tctcaactgc gtcttcattg tgtactacct gttggagatg ctgctcaagg tctttgccct 960
gggcctgcga gggtacctgt cctaccccag caacgtgttt gacgggctcc tcaccgttgt 1020
cctgctggag gccggagatg gtgggcctgc tgtcgctgtg ggacatgacc cgcatgctga 1080
acatgctcat cgtgttccgc ttcctgcgta tcatccccag catgaagccg,atggccgtgg 1140
tggccagtac cgtcctgggc ctggtgcaga acatgcgtgc ttttggcggg atcctggtgg 1200
tggtctacta cgtatttgcc atcattggga tcaacttgtt tagaggcgtc attgtggctc 1260
ttcctggaaa cagcagcctg gcccctgcca atggctcggc gccctgtggg agcttcgagc 1320
agctggagta ctgggccaac aacttcgatg actttgcggc tgccctggtc actctgtgga 1380
acttgatggt ggtgaacaac tggcaggtgt ttctggatgc atatcggcgc tactcaggcc 1440
cgtggtccaa gatctatttt gtgttgtggt ggctggtgtc gtctgtcatc tgggtcaacc 1500
tgtttctggc cctgattctg gagaacttcc ttcacaagtg ggacccccgc agccacctgc 1560
agccccttgc tgggacccca gaggccacct accagatgac tgtggagctc ctgttcaggg 1620
atattctgga ggagcccggg gaggatgagc tcacagagag gctgagccag cacccgcacc 1680
tgtggctgtg caggtgacgt ccgggctgcc gtcccagcag gggcggcagg agagagaggc 1740
tggcctacac aggtgcccat catggaagag gcggccatgc tgtggccagc caggcaggaa 1800
gagacctttc ctctgacgga ccactaagct ggggacagga accaagtcct ttgcgtgtgg 1860
cccaacaacc atctacagaa cagctgctgg tgcttcaggg aggcgccgtg ccctccgctt 1920
tcttttatag ctgcttcagt gagaattccc tcgtcgactc cacagggacc tttcagacaa 1980
aaatgcaaga agcagcggcc tcccctgtcc cctgcagctt ccgtggtgcc tttgctgccg 2040
gcagcccttg gggaccacag gcctgaccag ggcctgcaca ggttaaccgt cagacttccg 2100
64/65
CA 02458625 2004-02-16
WO 03/016493 PCT/US02/26323
gggcattcag gtggggatgc tggtggtttg acatggagag aaccttgact gtgttttatt 2160
atttcatggc ttgtatgagt gtgactgggt gtgtttcttt agggttctga ttgccagtta 2220
ttttcatcaa taagtcttgc a 2241
<210> 52
<211> 2312
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7506996CB1
<400> 52
catccgctca caatgccaca tcaatgatac gagcacgtag cctcactgct tgcacagtgc 60
atggcagagt cggctgcaga gcaggcgagg tggcctgagg gaggtcacta ggctggctga 120
gggctttttg ctgtggttca tgagccggcc tgcttccagg caccgtgtcc atgcgggtga 180
gcggtctccc tgggtgccca ctcttgcgcc cggagatcct gagtttggtc ctgtctggcc 240
atgaagctca gcctgctggg aggccacagg gagatgcagg ctgggcggcg ggtggatggt 300
tccagccggt tgggtccggg gcctggagct cagcctgtgg ggtggggacc cagtggtgcc 360
ctggagctgc cgcttctgct ctcagcagga tgatgggcag gacagggaga ggctgaccta 420
cttccagaac ctgcctgagt ctctgacttc cctcctggtg ctgctgacca cggccaacaa 480
ccccgatgtg atgattcctg cgtattccaa gaaccgggcc tatgccatct tcttcatagt 540
cttcactgtg ataggaagcc tgtttctgat gaacctgctg acagccatca tctacagtca 600
gttccggggc tacctgatga aatctctcca gacctcgctg tttcggaggc ggctgggaac 660
ccgggctgcc tttgaagtcc tatcctccat ggtgggggag ggaggagcct tccctcaggc 720
agttggggtg aagccccaga acttgctgca ggtgcttcag aaggtccagc tggacagctc 780
ccacaaacag gccatgatgg agaaggtgcg ttcctatggc agtgttctgc tctcagctga 840
ggagtttcag aagctcttca acgagcttga cagaagtgtg gttaaagagc acccgccgag 900
gcccgagtac cagtctccgt ttctgcagag cgcccagttc ctcttcggcc actactactt 960
tgactacctg gggaacctca tcgccctggc aaacctggtg tccatttgcg tgttcctggt 1020
gctggatgca gatgtgctgc ctgctgagcg tgatgacttc atcctgggga ttctcaactg 1080
cgtcttcatt gtgtactacc tgttggagtt gctgctcaag gtctttgccc tgggcctgcg 1140
agggtacctg tcctacccca gcaacgtgtt tgacgggctc ctcaccgttg tcctgctgcc 1200
gatggccgtg gtggccagta ccgtcctggg cctggtgcag aacatgcgtg cttttggcgg 1260
gatcctggtg gtggtctact acgtatttgc catcattggg atcaacttgt ttagaggcgt 1320
cattgtggct cttcctggaa acagcagcct ggcccctgcc aatggctcgg cgccctgtgg 1380
gagcttcgag cagctggagt actgggccaa caacttcgat gactttgcgg ctgccctggt 1440
cactctgtgg aacttgatgg tggtgaacaa ctggcaggtg tttctggatg catatcggcg 1500
ctactcaggc ccgtggtcca agatctattt tgtgttgtgg tggctggtgt cgtctgtcat 1560
ctgggtcaac ctgtttctgg ccctgattct ggagaacttc cttcacaagt gggacccccg 1620
cagccacctg cagccccttg ctgggacccc agaggccacc taccagatga ctgtggagct 1680
cctgttcagg gatattctgg aggagcccgg ggaggatgag ctcacagaga ggctgagcca 1740
gcacccgcac ctgtggctgt gcaggtgacg tccgggctgc cgtcccagca ggggcggcag 1800
gagagagagg ctggcctaca caggtgccca tcatggaaga ggcggccatg ctgtggccag 1860
ccaggcagga agagaccttt cctctgacgg accactaagc tggggacagg aaccaagtcc 1920
tttgcgtgtg gcccaacaac catctacaga acagctgctg gtgcttcagg gaggcgccgt 1980
gccctccgct ttcttttata gctgcttcag tgagaattcc ctcgtcgact ccacagggac 2040
ctttcagaca aaaatgcaag aagcagcggc ctcccctgtc ccctgcagct tccgtggtgc 2100
ctttgctgcc ggcagccctt ggggaccaca ggcctgacca gggcctgcac aggttaaccg 2160
tcagacttcc ggggcattca ggtggggatg ctggtggttt gacatggaga gaaccttgac 2220
tgtgttttat tatttcatgg cttgtatgag tgtgactggg tgtgtttctt tagggttctg 2280
attgccagtt attttcatca ataagtcttg ca 2312
65/65
