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TRANSPORTERS AND ION CHANNELS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of
transporters and ion
channels and to the use of these sequences in the diagnosis, treatment, and
prevention of transport,
neurological, muscle; immunological, and cell proliferative disorders, and in
the assessment of the
effects of exogenous compounds on the expression of nucleic acid and amino
acid sequences of
transporters and ion channels.
BACKGROUND OF THE INVENTION
Eukaryotic cells are surrounded and subdivided into functionally distinct
organelles by
hydrophobic lipid bilayer membranes which are highly impermeable to most polar
molecules. Cells and
organelles require transport proteins to import and export essential nutrients
and metal ions including
K~, NH4~, Pi, SO42-, sugars, and vitamins, as well as various metabolic waste
products. Transport
proteins also play roles in antibiotic resistance, toxin secretion, ion
balance, synaptic neurotransmission,
kidney function, intestinal absorption, tumor growth, and other diverse cell
functions (Griffith, J, and C.
Sansom (1998) The Transporter Facts Book, Academic Press, San Diego CA, pp. 3-
29). Transport
can occur by a passive concentration-dependent mechanism, or can be linked to
an energy source
such as ATP hydrolysis or an ion gradient. Proteins that function in transport
include carrier proteins,
which bind to a specific solute and undergo a conformational change that
translocates the bound solute
across the membrane, and channel proteins, which form hydrophilic pores that
allow specific solutes to
diffuse through the membrane down an electrochemical solute gradient.
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
various thyroid pathologies because it is the molecular basis for radioiodide
thyroid-imaging techniques
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and for specific targeting of radioisotopes to the thyroid gland (Levy, O. et
al. (1997) Proc. Natl.
Acad. Sci. USA 94:5568-5573). SMUT 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; GLUT3 regulates glucose supply to neurons; GLUT4 is
responsible for insulin-
regulated glucose disposal; and GLUTS regulates fructose uptake into skeletal
muscle. Defects in
glucose transporters are involved in a recently identified neurological
syndrome causing infantile
seizures and developmental delay, as well as glycogen storage disease, Fanconi-
Bickel syndrome, and
non-insulin-dependent diabetes mellitus (Mueckler, M. (1994) Eur. J. Biochem.
219:713-725; Longo,
N. and L.J. Elsas (1998) Adv. Pediatr. 45:293-313).
Monocarboxylate anion transporters are proton-coupled symporters with a broad
substrate
specificity that includes L-lactate, pyruvate, and the ketone bodies acetate,
acetoacetate, and
beta-hydroxybutyrate. At least seven isoforms have been identified to date.
The isoforms are
predicted to have twelve transmembrane (TM) helical domains with a large
intracellular loop between
TM6 and TM7, and play a critical role in maintaining intracellular pH by
removing the protons that are
produced stoichiometrically with lactate during glycolysis. The best
characterized
H+-monocarboxylate transporter is that of the erythrocyte membrane, which
transports L-lactate and a
wide range of other aliphatic monocarboxylates. Other cells possess H+-linked
monocarboxylate
transporters with differing substrate and inhibitor selectivities. In
particular, cardiac muscle and tumor
cells have transporters that differ in their KI,1 values for certain
substrates, including stereoselectivity
for L- over D-lactate, and in their sensitivity to inhibitors. There are Na+-
monocarboxylate
cotransporters on the luminal surface of intestinal and kidney epithelia,
which allow the uptake of
lactate, pyruvate, and ketone bodies in these tissues. In addition, there are
specific and selective
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transporters for organic cations and organic anions in organs including the
kidney, intestine and liver.
Organic anion transporters are selective for hydrophobic, charged molecules
with electron-attracting
side groups. Organic cation transporters, such as the ammonium transporter,
mediate the secretion of
a variety of drugs and endogenous metabolites, and contribute to the
maintenance of intercellular pH
(Poole, R.C. and A.P. Halestrap (1993) Am. J. Physiol. 264:C761-C782; Price,
N.T. et al. (1998)
Biochem. J. 329:321-328; and Martinelle, K. and I. Haggstrom (1993) J.
Biotechnol. 30:339-350).
Recently, Yamashita et al. (Yamashita, T. et al. (1997) J. Biol. Chem.
272:10205-10211) have
identified a peptide /histidine transporter (PHT1) in rat, expressed
particularly in brain and retina
tissue. When expressed in Xenopus oocytes, PHTl induces proton-dependent
histidine transport. This
to transport process was inhibited by dipeptides and tripeptides but not free
amino acids such as
glutamate, glycine, leucine, methionine, and aspartate. This transporter is
believed to be a member of
a superfamily of proton-coupled peptide and nitrate transporters.
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 (CF'TR),
or by separate genes.
When encoded by separate genes, each gene product contains a single NBD and
MSD. These "half
molecules" form homo- and heterodimers, such as Tapl and Tap2, the endoplasmic
reticulum-based
major histocompatibility (MHC) peptide transport system. Several genetic
diseases are attributed to
defects in ABC transporters, such as the following diseases and their
corresponding proteins: cystic
fibrosis (CFTR, an ion channel), adrenoleukodystrophy (adrenoleukodystrophy
protein, ALDP),
Zellweger syndrome (peroxisomal membrane protein-70, PMP70), and
hyperinsulinemic hypoglycemia
(sulfonylurea receptor, SUR). Overexpression of the multidrug resistance (MDR)
protein, another
ABC transporter, in human cancer cells makes the cells resistant to a variety
of cytotoxic drugs used
in chemotherapy (Taglicht, D. and S. Michaelis (1998) Meth. Enzymol. 292:130-
162).
A number of metal ions such as iron, zinc, copper, cobalt, manganese,
molybdenum, selenium,
nickel, and chromium are important as cofactors for a number of enzymes. For
example, copper is
involved in hemoglobin synthesis, connective tissue metabolism, and bone
development, by acting as a
cofactor in oxidoreductases such as superoxide dismutase, ferroxidase
(ceruloplasmin), and lysyl
oxidase. Copper and other metal ions must be provided in the diet, and are
absorbed by transporters in
the gastrointestinal tract. Plasma proteins transport the metal ions to the
liver and other target organs,
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where specific transporters move the ions into cells and cellular organelles
as needed. Imbalances in
metal ion metabolism have been associated with a number of disease states
(banks, D.M. (1986) J.
Med. Genet. 23:99-106).
Transport of fatty acids across the plasma membrane can occur by diffusion, a
high capacity,
low affinity process. However, under normal physiological conditions a
significant fraction of fatty
acid transport appears to occur via a high affinity, low capacity protein-
mediated transport process.
Fatty acid transport protein (FATP), an integral membrane protein with four
transmembrane
segments, is expressed in tissues exhibiting high levels of plasma membrane
fatty acid flux, such as
muscle, heart, and adipose. Expression of FATP is upregulated in 3T3-Ll 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).
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 (OMIM)
X275000 Graves Disease).
This class of transporters also includes the mitochondrial uncoupling
proteins, which create
proton leaks across the inner mitochondrial membrane, thus uncoupling
oxidative phosphorylation from
ATP synthesis. The result is energy dissipation in the form of heat.
Mitochondrial uncoupling proteins
have been implicated as modulators of thermoregulation and metabolic rate, and
have been proposed
as potential targets for drugs against metabolic diseases such as obesity
(Ricquier, D. et al. (1999) J.
Int. Med. 245:637-642).
Ion Channels
The electrical potential of a cell is generated and maintained by controlling
the movement of
ions across the plasma membrane. The movement of ions requires ion channels,
which form ion-
selective pores within the membrane. There are two basic types of ion
channels, ion transporters and
gated ion channels. Ion transporters utilize the energy obtained from ATP
hydrolysis to actively
transport an ion against the ion's concentration gradient. Gated ion channels
allow passive flow of an
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ion down the ion's electrochemical gradient under restricted conditions.
Together, these types of ion
channels generate, maintain, and utilize an electrochemical gradient that is
used in 1) electrical impulse
conduction down the axon of a nerve cell, 2) transport of molecules into cells
against concentration
gradients, 3) initiation of muscle contraction, and 4) endocrine cell
secretion.
Ion Transporters
Ion transporters generate and maintain the resting electrical potential of a
cell. Utilizing the
energy derived from ATP hydrolysis, they transport ions against the ion's
concentration gradient.
These transmembrane ATPases are divided into three families. The
phosphorylated (P) class ion
transporters, including Na+-K+ ATPase, 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 Ca2+ are low and
cytosolic concentration of
K+ is high. The vacuolar (V) class of ion transporters includes H+ pumps on
intracellular organelles,
such as lysosomes and Golgi. V-class ion transporters axe responsible for
generating the low pH
within the lumen of these organelles that is required for function. The
coupling factor (F) class
consists of H+ pumps in the mitochondria. F-class ion transporters utilize a
proton gradient to generate
ATP from ADP and inorganic phosphate (P;).
The P-ATPases are hexamers of a 100 kD subunit with ten transmembrane domains
and
several large cytoplasmic regions that may play a role in ion binding
(Scarborough, G.A. (1999) Curr.
Opin. Cell Biol. 11:517-522). The V-ATPases axe composed of two functional
domains: the VI
domain, a peripheral complex responsible for ATP hydrolysis; and the Vo
domain, an integral complex
responsible for proton translocation across the membrane. The F-ATPases are
structurally and
evolutionarily related to the V-ATPases. The F-ATPase Fo domain contains 12
copies of the c
subunit, a highly hydrophobic protein composed of two transmembrane domains
and containing a single
buried carboxyl group in TM2 that is essential for proton transport. The V-
ATPase Vo domain
contains three types of homologous c subunits with four or five transmembrane
domains and the
essential carboxyl group in TM4 or TM3. Both types of complex also contain a
single a subunit that
may be involved in regulating the pH dependence of activity (Forgac, M. (1999)
T. Biol. Chem.
274:12951-12954).
The resting potential of the cell is utilized in many processes involving
carrier proteins and
gated ion chaxmels. 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 Ca2+ out of the cell
with transport of Na+ into the cell (antiport).
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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
channel proteins. The characteristic domain of these channel proteins
comprises six transmembrane
domains (S1-S6), a pore-forming region (P) located between SS and S6, and
intracellular amino and
carboxy termini. In the Na+ and Ca2+ subfamilies, this domain is repeated four
times, while in the K+
channel subfamily, each channel is formed from a tetramer of either identical
or dissimilar subunits.
The P region contains information specifying the ion selectivity for the
channel. In the case of K+
channels, a GYG tripeptide is involved in this selectivity (Ishii, T.M. et al.
(1997) Proc. Natl. Acad.
Sci. USA 94:11651-11656).
Voltage-gated Na+ and K+ channels are necessary for the function of
electrically excitable
cells, such as nerve and muscle cells. Action potentials, which lead to
neurotransmitter release and
muscle contraction, arise from large, transient changes in the permeability of
the membrane to Na+
and K+ ions. Depolarization of the membrane beyond the threshold level opens
voltage-gated Na+
channels. Sodium ions flow into the cell, further depolarizing the membrane
and opening more
voltage-gated Na+ channels, which propagates the depolarization down the
length of the cell.
Depolarization also opens voltage-gated potassium channels. Consequently,
potassium ions flow
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outward, which leads to repolarization of the membrane. Voltage-gated channels
utilize charged
residues in the fourth transmembrane segment (S4) to sense voltage change. The
open state lasts
only about 1 millisecond, at which time the channel spontaneously converts
into an inactive state that
cannot be opened irrespective of the membrane potential. Inactivation is
mediated by the channel's
N-terminus, which acts as a plug that closes the pore. The transition from an
inactive to a closed state
requires a return to resting potential.
Voltage-gated Na+ channels are heterotrimeric complexes composed of a 260 kDa
pore-
forming a subunit that associates with two smaller auxiliary subunits, ail and
X32. The (32 subunit is a
integral membrane glycoprotein that contains an extracellular Ig domain, and
its association with a and
X31 subunits correlates with increased functional expression of the channel, a
change in its gating
properties, as well as an increase in whole cell capacitance due to an
increase in membrane surface
area (Isom, L.L. et al. (1995) Cell 83:433-442).
Non voltage-gated Na+ channels include the members of the amiloride-sensitive
Na+
channel/degenerin (NaC/DEG) family. Channel subunits of this family are
thought to consist of two
transmembrane domains flanking a long extracellular loop, with the amino and
carboxyl termini located
within the cell. The NaC/DEG family includes the epithelial Na+ channel (ENaC)
involved in Na+
reabsorption in epithelia including the airway, distal colon, cortical
collecting duct of the kidney, and
exocrine duct glands. Mutations in ENaC result in pseudohypoaldosteronism type
1 and Liddle's
syndrome (pseudohyperaldosteronism). The NaC/DEG family also includes the
recently characterized
H+-gated cation 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
neurodegenera~ion. 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 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
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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 dysrythmia syndrome
(Cmran, M.E. (1998)
Curr. Opin. Biotechnol. 9:565-572; Kaczorowski, G.J. and M.L. Garcia (1999)
C~.uT. Opin. Chem.
Biol. 3:448-458).
A second superfamily of K+ channels is composed of the inward rectifying
channels (Kir).
Kir channels have the property of preferentially conducting K+ currents in the
inward direction. These
proteins consist of a single potassium selective pore domain and two
transmembrane domains, which
correspond to the fifth and sixth transmembrane domains of voltage-gated K+
channels. Kir subunits
also associate as tetramers. The Kir family includes ROMKl, mutations in which
lead to Bartter
syndrome, a renal tubular disorder. Kir channels are also involved in
regulation of cardiac pacemaker
activity, seizures and epilepsy, and insulin regulation (Doupnik, C.A. et al.
(1995) Curr. Opin.
Neurobiol. 5:268-277; Curran, supra).
The recently recognized TWIK K+ channel family includes the mammalian TWIK-l,
TREK-1
and TASK proteins. Members of this family possess an overall structure with
four transmembrane
domains and two P domains. These proteins are probably involved in controlling
the resting potential
in a large set of cell types (Duprat, F. et al. (1997) EMBO J 16:5464-5471).
The voltage-gated Ca2+ channels have been classified into several subtypes
based upon their
electrophysiological and pharmacological characteristics. L-type Ca 2+
channels are predominantly
expressed in heart and skeletal muscle where they play an essential role in
excitation-contraction
coupling. T-type channels are important for cardiac pacemaker activity, while
N-type and P/Q-type
channels are involved in the control of neurotransmitter release in the
central and peripheral nervous
system. The L-type and N-type voltage-gated Ca2+ 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 al, one a28, and four (3 genes. A
fourth subunit, y, has
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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 transient receptor family (Trp) of calcium ion channels are thought to
mediate
capacitative calcium entry (CCE). CCE is the Ca2+ influx into cells to
resupply 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 Ca2+ channels in the S3 through S6 regions. This suggests that Trp
andlor related proteins may
form mammalian CCC entry 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, and
whose expression in melanoma cells is inversely correlated with melanoma
aggressiveness in vivo.
The human cDNA transcript corresponds to a 1533-amino acid protein having
homology to members
of the Trp family. It has been proposed that the combined use of malastatin
mRNA expression status
and tumor thickness might allow for the determination of subgroups of patients
at both low and high
risk fox developing metastatic disease (Duncan, L.M. et al (2001) J. Clin.
Oncol. 19:568-576).
Chloride channels are necessary in endocrine secretion and in regulation of
cytosolic and
organelle pH. In secretory epithelial cells, Cl- enters the cell across a
basolateral membrane through
an Na+, K*lCl- 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
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
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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 (B K) 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 chaxged 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 (Kaczorowslu, supra;
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
canon channels involved in vision. Both systems involve ligand-mediated
activation of a G-protein
coupled receptor which then alters the level of cyclic nucleotide within the
cell. CNG channels also
represent a major pathway for Ca2+ entry into neurons, and play roles in
neuronal development and
plasticity. CNG channels axe 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
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intracellular signalling proteins. Many channels have sites for
phosphorylation by one or more protein
kinases including protein kinase A, protein kinase C, tyrosine kinase, and
casein kinase II, all of which
regulate ion channel activity in cells. I~ir channels are activated by the
binding of the G(3~y 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 MAGUI~s (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 Mockers.
Mutations in muscle sodium and calcium channels cause forms of periodic
paralysis, while mutations in
the sarcoplasmic calcium release channel, T-tubule calcium channel, and muscle
sodium channel
cause malignant hyperthermia. Cardiac arrythmia disorders such as the long QT
syndromes and
idiopathic ventricular fibrillation are caused by mutations in potassium and
sodium channels (Cooper,
E.C. and L.Y. Jan (1998) Proc. Natl. Acad. Sci. USA 96:4759-4766). All four
known human
idiopathic epilepsy genes code for ion channel proteins (Berkovic, S.F. and
LE. Scheffer (1999) Curr.
Opin. Neurology 12:177-182). Other neurological disorders such as ataxias,
hemiplegic migraine and
hereditary deafness can also result from mutations in ion channel genes (Jen,
J. (1999) Curr. Opin.
Neurobiol. 9:274-280; Cooper, supra).
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), congenital hyperbilruginemia (MOAT), Stargart's
disease, which
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causes defective vision in children (RIM/ABCR) and hyperinsulinemic
hypoglycemia (sulfonylurea
receptor, SUR) (Holland, B. and Blight, M.A. (1999) J. Mol. Biol. 293:381-
399). Overexpression of
the multidrug resistance (MDR) protein in human cancer cells makes the cells
resistant to a variety of
cytotoxic drugs used in chemotherapy (Taglight, D. and Michaelis, S. (1998)
Meth. Enzymol. 292:131-
163).
Two monomeric ABC transporters have been identified in the human peroxisome
membrane:
the adrenoleukodystrophy protein (ALDP) and the 70-kDa peroxisomal membrane
protein (PMP70).
Mutations in the adrenoleukodystrophy gene cause X-linked
adrenoleukodystrophy, an inborn error of
peroxisomal (3-oxidation of very long chain fatty acids. Mutations in the
PMP70 genes have been
found in patients with Zellweger syndrome, an inborn error of peroxisome
biogenesis. The
sulfonylurea receptor, an ABC transporter, regulates the function of
pancreatic ATP-sensitive K+
channels, and sulphonylureas are widely used to treat non-insulin dependent
diabetes mellitus
(Demolombe, S. and Escande, D. (1996) Trends Pharmacol. Sci. 17:273-275).
Multidrug-resistance
(MDR) results from overproduction of another member of the ABC transporter
family,
P-glycoprotein. MDR is primarily caused by increased drug extrusion from the
resistant cells by P-
glycoprotein. The P-glycoproteins have 2 homologous halves, each with 6
hydrophobic segments
adjacent to a consensus sequence for nucleotide binding. The hydrophobic
segments may form a
membrane channel, whereas the nucleotide binding site may be involved in
providing energy for drug
transport (Saurin, W. et al. (1994) Mol. Microbiol. 12:993-1004; Shani, N., et
al. (1996) J. Biol. Chem.
271:8725-8730; and Koster, W., and Bohm, B. (1992) Mol. & Gen. Genet. 232:399-
407).
Ion channels have been the target for many drug therapies. Neurotransmitter-
gated channels
have been targeted in therapies for treatment of insomnia, anxiety,
depression, and schizophrenia.
Voltage-gated channels have been targeted in therapies for arrhythmia,
ischemic stroke, head trauma,
and neurodegenerative disease (Taylor, C.P. and L.S. Narasimhan (1997) Adv.
Pharmacol. 39:47-98).
Various classes of ion channels also play an important role in the perception
of pain, and thus are
potential targets for new analgesics. These include the vanilloid-gated ion
channels, which are
activated by the vanilloid capsaicin, as well as by noxious heat. Local
anesthetics such as lidocaine
and mexiletine which blockade voltage-gated Na+ channels have been useful in
the treatment of
neuropathic pain (Eglen, supra).
Ion channels in the immune system have recently been suggested as targets for
immunomodulation. T-cell activation depends upon calcium signaling, and a
diverse set of T-cell
specific ion channels has been characterized that affect this signaling
process. Channel blocking
agents can inhibit secretion of lymphokines, cell proliferation, and killing
of target cells. A peptide
antagonist of the T-cell potassium channel Kv1.3 was found to suppress delayed-
type hypersensitivity
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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).
The discovery of new transporters and ion channels and the polynucleotides
encoding them
satisfies a need in the art by providing new compositions which are useful in
the diagnosis, prevention,
and treatment of transport, neurological, muscle, immunological, and cell
proliferative disorders, and in
the assessment of the effects of exogenous compounds on the expression of
nucleic acid and amino
acid sequences of transporters and ion channels.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, transporters and ion channels,
referred to
collectively as "TRICH" and individually as "TRICH-l," "TRICH-2," "TRICH-3,"
"TRICH-4,"
"TRICH-5," "TRICH-6," "TRICH-7," "TRICH-8," "TRICH-9," "TRICH-10," "TRICH-11,"
"TRICH-12," "TRICH-13," "TRICH-14," "TRICH-15," "TRICH-16," "TRICH-17," "TRICH-
18,"
"TRICH-19," "TRICH-20," "TRICH-21," "TRICH-22," "TRICH-23," "TRICH-24," "TRICH-
25,"
"TRICH-26," and "TRICH-27." In one aspect, the invention provides an isolated
polypeptide selected
from the group consisting of a) a polypeptide comprising an amino acid
sequence selected from the
group consisting of SEQ ID N0:1-27, b) a naturally occurring polypeptide
comprising an amino acid
sequence at least 90% identical to an amino acid sequence selected from the
group consisting of SEQ
ID N0:1-27, c) a biologically active fragment of a polypeptide having an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-27, and d) an immunogenic fragment of
a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
N0:1-27. In one
alternative, the invention provides an isolated polypeptide comprising the
amino acid sequence of SEQ
ID NO:1-27.
The invention further provides an isolated polynucleotide encoding a
polypeptide selected from
the group consisting of a) a polypeptide comprising an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-27, b) a naturally occurnng polypeptide comprising
an amino acid
sequence at least 90% identical to an amino acid sequence selected from the
group consisting of SEQ
ID N0:1-27, c) a biologically active fragment of a polypeptide having an amino
acid sequence selected
from the group consisting of SEQ ID N0:1-27, and d) an immunogenic fragment of
a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
N0:1-27. In one
alternative, the polynucleotide encodes a polypeptide selected from the group
consisting of SEQ ID
NO:l-27. In another alternative, the polynucleotide is selected from the group
consisting of SEQ ID
N0:28-54.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
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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 ID N0:1-27, b) a naturally occurring polypeptide comprising an amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ ID NO:1-27, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID NO:l-27, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ID N0:1-27. In one
alternative, the
invention provides a cell transformed with the recombinant polynucleotide. In
another alternative, the
invention provides a transgenic organism comprising the recombinant
polynucleotide.
The invention also provides a method for producing a polypeptide selected from
the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ID NO:1-27, b) a naturally occurring polypeptide comprising an amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ ID NO:1-27, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group.
consisting of SEQ ID N0:1-27, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ID NO:1-27. The method
comprises a)
culturing a cell under conditions suitable for expression of the polypeptide,
wherein said cell is
transformed with a recombinant polynucleotide comprising a promoter sequence
operably linked to a
polynucleotide encoding the polypeptide, and b) recovering the polypeptide so
expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid sequence .
selected from the group consisting of SEQ ID N0:1-27, b) a naturally occurring
polypeptide
comprising an amino acid sequence at least 90% identical to an amino acid
sequence selected from
the group consisting of SEQ ID N0:1-27, c) a biologically active fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID NO:1-27, and
d) an immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
ID N0:1-27.
The invention further provides an isolated polynucleotide selected from the
group consisting of
a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of SEQ
ID N0:28-54, b) a naturally occurring polynucleotide comprising a
polynucleotide sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ ID N0:28-54,
c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide complementary to
the polynucleotide of b), and e) an RNA equivalent of a)-d). In one
alternative, the polynucleotide
comprises at least 60 contiguous nucleotides.
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Additionally, the invention provides a method for detecting a target
polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide selected from
the group consisting of
a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of SEQ
ID N0:28-54, b) a naturally occurring polynucleotide comprising a
polynucleotide sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ ID N0:28-54,
c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide complementary to
the polynucleotide of b), and e) an RNA equivalent of a)-d). The method
comprises a) hybridizing the
sample with a probe comprising at least 20 contiguous nucleotides comprising a
sequence
complementary to said target polynucleotide in the sample, and which probe
specifically hybridizes to
said target polynucleotide, under conditions whereby a hybridization complex
is formed between said
probe and said target polynucleotide or fragments thereof, and b) detecting
the presence or absence of
said hybridization complex, and optionally, if present, the amount thereof. In
one alternative, the probe
comprises at least 60 contiguous nucleotides.
The invention further provides a method for detecting a taxget polynucleotide
in a sample, said
target polynucleotide having a sequence of a polynucleotide selected from the
group consisting of a) a
polynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ ID
NO:28-54, b) a naturally occurring polynucleotide comprising a polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:28-54, c) a
polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
complementary to the
polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises
a) amplifying said
target polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b)
detecting the presence or absence of said amplified target polynucleotide or
fragment thereof, and,
optionally, if present, the amount thereof.
The invention further provides a composition comprising an effective amount of
a polypeptide
selected from the group consisting of a) a polypeptide comprising an amino
acid sequence selected
from the group consisting of SEQ ID NO:l-27, b) a naturally occurring
polypeptide comprising an
amino acid sequence at least 90% identical to an amino acid sequence selected
from the group
consisting of SEQ ID N0:1-27, c) a biologically active fragment of a
polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID N0:1-27, and d) an
immunogenic fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID N0:1-27,
and a pharmaceutically acceptable excipient. In one embodiment, the
composition comprises an amino
acid sequence selected from the group consisting of SEQ ID NO:1-27. The
invention additionally
provides a method of treating a disease or condition associated with decreased
expression of
functional TRICH, comprising administering to a patient in need of such
treatment the composition.
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The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:l-27, b) a
naturally occurring
polypeptide comprising an amino acid sequence at least 90% identical to an
amino acid sequence
selected from the group consisting of SEQ ID NO:l-27, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID N0:1-27,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID N0:1-27. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting agonist activity in the sample. In
one alternative, the
invention provides a composition comprising an agonist compound identified by
the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with decreased expression of
functional TRICH, comprising
administering to a patient in need of such treatment the composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID N0:1-27, b) a
naturally occurring
polypeptide comprising an amino acid sequence at least 90% identical to an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-27, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1-27,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID N0:1-27. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting antagonist activity in the sample.
In one alternative, the
invention provides a composition comprising an antagonist compound identified
by the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with overexpression of functional
TRICH, comprising
administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that
specifically binds to
a polypeptide selected from the group consisting of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-27, b) a naturally
occurring polypeptide
comprising an amino acid sequence at least 90% identical to an amino acid
sequence selected from
the group consisting of SEQ ID NO:1-27, c) a biologically active fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID N0:1-27, and
d) an immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
ID N0:1-27. The method comprises a) combining the polypeptide with at least
one test compound
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under suitable conditions, and b) detecting binding of the polypeptide to the
test compound, thereby
identifying a compound that specifically binds to the polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ m N0:1-27, b) a
naturally occurring
polypeptide comprising an amino acid sequence at least 90% identical to an
amino acid sequence
selected from the group consisting of SEQ m NO:1-27, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:l-27,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID N0:1-27. The method comprises a) combining the
polypeptide with at
least one test compound under conditions permissive for the activity of the
polypeptide, b) assessing
the activity of the polypeptide in the presence of the test compound, and c)
comparing the activity of
the polypeptide in the presence of the test compound with the activity of the
polypeptide in the
absence of the test compound, wherein a change in the activity of the
polypeptide in the presence of
the test compound is indicative of a compound that modulates the activity of
the polypeptide.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a sequence
selected from the group consisting of SEQ m N0:28-54, the method comprising a)
exposing a sample
comprising the target polynucleotide to a compound, and b) detecting altered
expression of the target
polynucleotide.
The invention further provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound; b)
hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide selected from the group consisting
of i) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:28-54, ii) a
naturally occurring polynucleotide comprising a polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ m N0:28-54,
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 m N0:28-
54, ii) a naturally
occurnng polynucleotide comprising a polynucleotide sequence at least 90%
identical to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:28-54,
iii) a polynucleotide
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WO 01/92304 PCT/USO1/17065
complementary to the polynucleotide of i), iv) a polynucleotide complementary
to the polynucleotide of
ii), and v) an RNA equivalent of i)-iv). Alternatively, the target
polynucleotide comprises a fragment
of a polynucleotide sequence selected from the group consisting of i)-v)
above; c) quantifying the
amount of hybridization complex; and d) comparing the amount of hybridization
complex in the treated
biological sample with the amount of hybridization complex in an untreated
biological sample, wherein
a difference in the amount of hybridization complex in the treated biological
sample is indicative of
toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the present invention.
Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog for polypeptides of the invention. The probability score for the match
between each
polypeptide and its GenBankhomolog is also shown.
Table 3 shows structural features of polypeptide sequences of the invention,
including
predicted motifs and domains, along with the methods, algorithms, and
searchable databases used for
analysis of the polypeptides.
Table 4 lists the cDNA and/or genomic DNA fragments which were used to
assemble
polynucleotide sequences of the invention, along with selected fragments of
the polynucleotide
sequences.
Table 5 shows the representative cDNA library for polynucleotides of the
invention.
Table 6 provides an appendix which describes the tissues and vectors used for
construction of
the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which will
be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
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reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods axe now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing the
cell lines, protocols, reagents and vectors which are reported in the
publications and which might be
used in connection with the invention. Nothing herein is to be construed as an
admission that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
DEFINITIONS
"TRICH" refers to the amino acid sequences of substantially purified TRICH
obtained from
any species, particularly a mammalian species, including bovine, ovine,
porcine, marine, equine, and
human, and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
TRICH. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of TRICH either by
directly interacting with
TRICH or by acting on components of the biological pathway in which TRICH
participates.
An "allelic variant" is an alternative form of the gene encoding TRICH.
Allelic variants may
result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in
polypeptides whose structure or function may or may not be altered. A gene may
have none, one, or
many allelic variants of its naturally occurring form. Common mutational
changes which give rise to
allelic variants are generally ascribed to natural deletions, additions, or
substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the
others, one or more times
in a given sequence.
"Altered" nucleic acid sequences encoding TRICH include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as TRICH or a
polypeptide with at least one functional characteristic of TRICH. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe of
the polynucleotide encoding TRICH, and improper or unexpected hybridization to
allelic variants, with
a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding TRICH.
The encoded protein may also be "altered," and may contain deletions,
insertions, or substitutions of
amino acid residues which produce a silent change and result in a functionally
equivalent TRICH.
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Deliberate amino acid substitutions may be made on the basis of similarity in
polarity, charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues,
as long as the biological
or immunological activity of TRICH is retained. For example, negatively
charged amino acids may
include aspaxtic acid and glutamic acid, and positively charged amino acids
may include lysine and
arginine. Amino acids with uncharged polar side chains having similar
hydrophilicity values may
include: asparagine and glutamine; and serine and threonine. Amino acids with
uncharged side chains
having similar hydrophilicity values may include: leucine, isoleucine, and
valine; glycine and alanine;
and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or synthetic
molecules. Where "amino acid sequence" is recited to refer to 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 sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well known
in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of TRICH. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of TRICH either by
directly interacting with TRICH or by acting on components of the biological
pathway in which
TRICH participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')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
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
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to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA; RNA;
peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages
such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
designation "positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally 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 sequence" and a "composition
comprising a
given amino acid sequence" refer broadly to any composition containing the
given polynucleotide or
amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding TRICH or fragments
of TRICH may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCI), detergents
(e.g., sodium dodecyl sulfate;
SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm
DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to
repeated DNA sequence analysis to resolve uncalled bases, extended using the
XI,-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
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and assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of the
protein is conserved and not significantly changed by such substitutions. The
table below shows amino
acids which may be substituted for an original amino acid in a protein and
which are regaxded as
conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
l0 Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val 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, andlor (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide can include, for example,
replacement of hydrogen by an
alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a
polypeptide which retains
at least one biological or immunological function of the natural molecule. A
derivative polypeptide is
one modified by glycosylation, pegylation, or any similar process that retains
at least one biological or
immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
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"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.
A "fragment" is a unique portion of TRICH or the polynucleotide encoding TRICH
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example, a
fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10, 15,
16, 20, 25, 30, 40, 50, 60, 75> 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 25 % or 50%) of a polypeptide
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ ID N0:28-54 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:28-54, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:28-54 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish SEQ
ID N0:28-54 from related polynucleotide sequences. The precise length of a
fragment of SEQ ID
N0:28-54 and the region of SEQ ~ N0:28-54 to which the fragment corresponds
are routinely
determinable by one of ordinary skill in the art based on the intended purpose
for the fragment.
A fragment of SEQ ID N0:1-27 is encoded by a fragment of SEQ 1D N0:28-54. A
fragment of SEQ ID N0:1-27 comprises a region of unique amino acid sequence
that specifically
identifies SEQ ID NO:1-27. For example, a fragment of SEQ ID N0:1-27 is useful
as an
immunogenic peptide for the development of antibodies that specifically
recognize SEQ ID NO:1-27.
The precise length of a fragment of SEQ D7 N0:1-27 and the region of SEQ ID
N0:1-27 to which
the fragment corresponds are routinely determinable by one of ordinary skill
in the art based on the
intended purpose for the fragment.
A "full length" polynucleotide sequence is one containing at least a
translation initiation codon
(e.g., methionine) followed by an open reading frame and a translation
termination codon. A "full
length" polynucleotide sequence encodes a "full length" polypeptide sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between two
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or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide
sequences, refer to
the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps in
the sequences being compared in order to optimize alignment between two
sequences, and therefore
achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison Wl). 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
is provided by the National Center for Biotechnology Information (NCB17 Basic
Local Alignment
Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410),
which is available from
several sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence analysis
programs including "blastn," that is used to align a known polynucleotide
sequence with other
polynucleotide sequences from a variety of databases. Also available is a tool
called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http:l/www.ncbi.nlm.nih.gov/gorflbl2.html. The
"BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLO,SUM62
Reward for match: 1
Penalty for mismatch: -2
Opefi Gap: 5 and Extefasion Gap: 2 penalties
Gap x drop-off SO
Expect: 10
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Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a parfiicular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken fxom a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length supported
by the sequences shown herein, in the tables, figures, or Sequence Listing,
may be used to describe a
length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some alignment.
methods take into account conservative amino acid substitutions. Such
conservative substitutions,
explained in more detail above, generally preserve the charge and
hydrophobicity at the site of
substitution, thus preserving the structure (and therefore function) of the
polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version
2Ø12 (April-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Open Gap: l l and Extension Gap: I penalties
Gap x drop-off 50
Expect: 10
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Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ m number, or may be measured over
a shorter length,
for example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Speciftc hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly importantin
determining the
stringency of the hybridization process, with more stringent conditions
allowing less non-specific
binding, i.e., binding between pairs of nucleic acid strands that are not
perfectly matched. Permissive
conditions for annealing of nucleic acid sequences are routinely determinable
by one of ordinary skill in
the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
1 % (w/v) SDS, and about 100 ~ g/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (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 penectly 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 Laborator'r Manual, 2nd ed., vol. 1-3, Cold Spring Harbor
Press, Plainview NY;
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specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present invention
include wash conditions of 68°C in the presence of about 0.2 x SSC and
about 0.1% SDS, for 1 hour.
Alternatively, temperatures of about 65°C, 60°C, 55°C, or
42°C may be used. SSC concentration may
be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1 %.
Typically, blocking
reagents are used to block non-specific hybridization. Such blocking reagents
include, for instance,
sheared and denatured salmon sperm DNA at about 100-200 ~ g/ml. Organic
solvent, such as
formamide at a concentration of about 35-60% vlv, may also be used under
particular circumstances,
such as for RNA:DNA hybridizations. Useful variations on these wash conditions
will be readily
apparent to those of ordinary skill in the art. Hybridization, particularly
under high stringency
conditions, may be suggestive of evolutionary similarity between the
nucleotides. Such similarity is
strongly indicative of a similar role for the nucleotides and their encoded
polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate .
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"hnmune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
An "imtnunogenic fragment" is a polypeptide or oligopeptide fragment of TRICH
which is
capable of eliciting an immune response when introduced into a living
organism, for example, a
mammal. The term "immunogenic fragment" also includes any polypeptide or
oligopeptide fragment of
TRICH which is useful in any of the antibody production methods disclosed
herein or known in the art.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides,
polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of TRICH. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other biological,
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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 finked 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
by cell type depending on the enzymatic milieu of TRICH.
"Probe" refers to nucleic acid sequences encoding TRICH, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents,
and enzymes. "Primers"
are short nucleic acids, usually DNA oligonucleotides, which may be annealed
to a target
polynucleotide by complementary base-pairing. The primer may then be extended
along the target
DNA strand by a DNA polymerase enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence, e.g., by the polymerase chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
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example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences; New York NY; Innis, M. et
al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to 5,000
nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection
programs have incorporated additional features for expanded capabilities. For
example, the PrimOU
primer selection program (available to the public from the Genome Center at
University of Texas
South West Medical Center, Dallas TX) is capable of choosing specific primers
from megabase
sequences and is thus useful for designing primers on a genome-wide scope. The
Primer3 primer
selection program (available to the public from the Whitehead Institute/MIT
Center for Genome
Research, Cambridge MA) allows the user to input a "mispriming library," in
which sequences to
avoid as primer binding sites are user-specified. Primer3 is useful, in
particular, for the selection of
oligonucleotides for microarrays. (The source code for the latter two primer
selection programs may
also be obtained from their respective sources and modified to meet the user's
specific needs.) The
PrimeGen program (available to the public from the UK Human Genome Mapping
Project Resource
Centre, Cambridge UK) designs primers based on multiple sequence alignments,
thereby allowing
selection of primers that hybridize to either the most conserved or least
conserved regions of aligned
nucleic acid sequences. Hence, this program is useful for identification of
both unique and conserved
oligonucleotides and polynucleotide fragments. The oligonucleotides and
polynucleotide fragments
identified by any of the above selection methods are useful in hybridization
technologies, for example,
as PCR or sequencing primers, microarray elements, or specific probes to
identify fully or partially
complementary polynucleotides in a sample of nucleic acids. Methods of
oligonucleotide selection are
not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, s-upra. The term recombinant includes
nucleic acids that have
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been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter sequence.
Such a recombinant nucleic acid may be part of a vector that is used, for
example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions
(ITTRs). 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.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of
the nitrogenous base thymine are replaced with uracil, and the sugar backbone
is composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing TRICH,
nucleic acids encoding TRICH, or fragments thereof may comprise a bodily
fluid; an extract from a
cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic
DNA, RNA, or cDNA,
in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular structure
of the protein, e.g., the antigenic determinant or epitope, recognized by the
binding molecule. For
example, if an antibody is specific for epitope "A," the presence of a
polypeptide comprising the
epitope A, or the presence of free unlabeled A, in a reaction containing free
labeled A and the
antibody will reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 75% free, and most preferably at least 90% free from other
components with
which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides
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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" 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 the type of host cell being transformed and may include, but is not limited
to, bacteriophage or viral
infection, electroporation, heat shock, lipofection, and particle bombardment.
The term "transformed
cells" includes stably transformed cells in which the inserted DNA is capable
of replication either as
an autonomously replicating plasmid or as part of the host chromosome, as well
as transiently
transformed cells which express the inserted DNA or RNA for limited periods of
time.
A "transgenic organism," as used herein, is any organism, including but not
limited to animals
and plants, in which one or more of the cells of the organism contains
heterologous nucleic acid
introduced by way of human intervention, such as by transgenic techniques well
known in the art. The
nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor of the cell,
by way of deliberate genetic manipulation, such as by microinjection or by
infection with a
recombinant virus. The term genetic manipulation does not include classical
cross-breeding, or in vitro
fertilization, but rather is directed to the introduction of a recombinant DNA
molecule. The transgenic
organisms contemplated in accordance with the present invention include
bacteria, cyanobacteria,
fungi, plants and animals. The isolated DNA of the present invention can be
introduced into the host
by methods known in the art, for example infection, transfection,
transformation or transconjugation.
Techniques for transferring the DNA of the present invention into such
organisms are widely known
and provided in references such as Sambrook et al. (1989), supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91
%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99% or greater
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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 alternative splicing of exons during mRNA processing.
The corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotide sequences that vary
from one species to
another. The resulting polypeptides will generally have significant amino acid
identity relative to each
other. A polymorphic variant is a variation in the polynucleotide sequence of
a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07- .
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at.
least 60.%, at least 70%, at least 80%, at least 90%, at least 91 %, at least
92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human transporters and ion
channels
(TRICH), the polynucleotides encoding TRICH, and the use of these compositions
for the diagnosis,
treatment, or prevention of transport, neurological, muscle, immunological,
and cell proliferative
disorders.
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to a
single Incyte project identification number (Incyte Project ID). Each
polypeptide sequence is denoted
by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:)
and an Incyte
polypeptide sequence number (Incyte Polypeptide ID) as shown. Each
polynucleotide sequence is
denoted by both a polynucleotide sequence identification number
(Polynucleotide SEQ ID NO:) and an
Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as
shown.
Table 2 shows sequences with homology to the polypeptides of the invention as
identified by
BLAST analysis against the GenBankprotein (genpept) database. Columns l and 2
show the
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polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ff~) for polypeptides of the
invention. Column 3
shows the GenBank identification number (Genbank ID NO:) of the nearest
GenBank homolog.
Column 4 shows the probability score for the match between each polypeptide
and its GenBank
homolog. Column 5 shows the annotation of the GenB ank homolog along with
relevant citations
where applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the
invention. Columns 1 and
2 show the polypeptide sequence identification number (SEQ ID 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 xesidues 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 structurelfunction 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 )D NO:1 is 88% identical to rat ABC transporter (GenBank ID 82982567) as
determined by the
Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST
probability score is 0.0
(scores are rounded down to zero if they are extremely small, e.8, less than
10'3°°), which indicates
the probability of obtaining the observed polypeptide sequence alignment by
chance. SEQ ID N0:1
also contains an ABC transporter active site domain and transmembrane domain
as determined by
searching for statistically significant matches in the hidden Markov model
(HMM)-based PFAM
database of conserved protein family domains. (See Table 3.) Results from
BLIMPS, MOTIFS, and
PROFILESCAN analyses provide further corroborative evidence that SEQ )D NO:1
is an ABC
transporter. In an alternative example, SEQ >D N0:4 is 87% identical to human
mitochondria)
ornithine transporter (GenBank ID 85565862) as determined by the Basic Local
Alignment Search
Tool (BLAST). (See Table 2.) The BLAST probability scoxe is 8.1 e-141, which
indicates the
probability of obtaining the observed polypeptide sequence alignment by
chance. SEQ ID N0:4 also
contains a mitochondria) carrier proteins domain as determined by searching
for statistically significant
matches in the hidden Markov model (HMM)-based PFAM database of conserved
protein family
domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses
provide
further corroborative evidence that SEQ ID N0:4 is a mitochondria) carrier
protein. In an alternative
example, SEQ ID N0:8 is 88% identical to rat peptide/histidine transporter
(GenBank ID 82208839)
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as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.)
The BLAST
probability score is 1.8e-262, which indicates the probability of obtaining
the observed polypeptide
sequence alignment by chance. SEQ ID N0:8 also contains a PTR2 proton-
dependent oligopeptide
transport (POT) family peptide transporter signature as determined by
searching for statistically
significant matches in the hidden Markov model (HMM)-based PFAM database of
conserved protein
family domains. (See Table 3.) Data from BLAST-DOMO, BLAST-PRODOM, BLIMPS, and
MOTIFS analyses provide further corroborative evidence that SEQ ID N0:8 is a
transmembrane
PTR2 POT family transporter. In an alternative example, SEQ ID N0:15 is 51 %
identical from
amino acid residues 117 to 742 to rat sodiumlglucose cotransporter (GenBank ID
8286259) as
determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.)
The BLAST
probability score is 8.9e-174, which indicates the probability of obtaining
the observed polypeptide
sequence alignment by chance. SEQ ID N0:15 also contains a sodiumaolute
symporter family
domain as determined by searching for statistically significant matches in the
hidden Markov model
(HMM)-based PFAM database of conserved protein family domains. (See Table 3.)
Data from
BLIMPS, MOTIFS, and PROFILESCAN analyses provide further corroborative
evidence that SEQ
ID N0:15 is a sodium/glucose cotransporter. In an alternative example, SEQ ID
N0:18 is 94%
identical from amino acids 300 to 1771 to mouse ATP-binding cassette 2
transporter (GenBank ID
8495259) as determined by the Basic Local Alignment Search Tool (BLAST). (See
Table 2.) The
BLAST probability score is 0.0, which indicates the probability of obtaining
the observed polypeptide
sequence alignment by chance. SEQ ID N0:18 also contains an ABC transporter
domain as
determined by searching for statistically significant matches in the hidden
Markov model (HMM)-
based PFAM database of conserved protein family domains. (See Table 3.) Data
from MOTIFS, and
PROFILESCAN analyses provide further corroborative evidence that SEQ ID N0:18
is an ABC
transporter. SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7,
SEQ ID
N0:9, SEQ ID NO:10, SEQ >D NO:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14,
SEQ ID
N0:16, SEQ ID N0:17, SEQ ID N0:19, SEQ ID N0:20, SEQ ID N0:21, SEQ ID N0:22,
SEQ ID
N0:23, SEQ ID NO:24, SEQ ID N0:25, SEQ ID NO:26, and SEQ ID N0:27 were
analyzed and
annotated in a similar manner. The algorithms and parameters for the analysis
of SEQ ID N0:1-27
are described in Table 7.
As shown in Table 4, the full length polynucleotide sequences of the present
invention were
assembled using cDNA sequences or coding (exon) sequences derived from genomic
DNA, or any
combination of these two types of sequences. Columns 1 and 2 list the
polynucleotide sequence
identification number (Polynucleotide SEQ ID NO:) and the corresponding Incyte
polynucleotide
consensus sequence number (Incyte Polynucleotide ID) for each polynucleotide
of the invention.
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Column 3 shows the length of each polynucleotide sequence in basepairs. Column
4 lists fragments of
the polynucleotide sequences which are useful, for example, in hybridization
or amplification
technologies that identify SEQ ID N0:28-54 or that distinguish between SEQ ID
N0:28-54 and
related polynucleotide sequences. Column 5 shows identification numbers
corresponding to cDNA
sequences, coding sequences (exons) predicted from 8enomic DNA, and/or
sequence assemblages
comprised of both cDNA and 8enomic DNA. These sequences were used to assemble
the full length
polynucleotide sequences of the invention. Columns 6 and 7 of Table 4 show the
nucleotide start (5')
and stop (3') positions of the cDNA and/or genomic sequences in column 5
relative to their respective
full length sequences.
The identification numbers in Column 5 of Table 4 may refer specifically, for
example, to
Incyte cDNAs along with their corresponding cDNA libraries. For example,
7249756H2 is the
identification number of an Incyte cDNA sequence, and PROSTMY01 is the cDNA
library from
which it is derived. Incyte cDNAs for which cDNA libraries are not indicated
were derived from
pooled cDNA libraries (e.8., 71753989V1). Alternatively, the identification
numbers in column 5 may
refer to GenBank cDNAs or ESTs (e.8., 87457275) which contributed to the
assembly of the full
length polynucleotide sequences. Alternatively, the identification numbers in
column 5 may refer to
coding regions predicted by Genscan analysis of 8enomic DNA. For example,
GNN.g7160536_000034 002 is the identification number of a Genscan-predicted
coding sequence,
with 87160536 being the GenBank identification number of the sequence to which
Genscan was
applied. The Genscan-predicted coding sequences may have been edited prior to
assembly. (See
Example IV.) Alternatively, the identification numbers in column 5 may refer
to assemblages of both
cDNA and Genscan-predicted exons brought together by an "exon stitching"
algorithm. For example,
FL180719_00001 represents a "stitched" sequence in which 180719 is the
identification number of the
cluster of sequences to which the algorithm was applied, and 00001 is the
number of the prediction
generated by the algorithm. (See Example V.) Alternatively, the identification
numbers in column 5
may refer to assemblages of both cDNA and Genscan-predicted exons brought
together by an "exon-
stretching" algorithm. For example, FL7472537_g5815493_g7406950 is the
identification number of a
"stretched" sequence, with 7472537 being the Incyte project identification
number, 85815493 being the
GenBank identification number of the human 8enomic sequence to which the "exon-
stretching"
algorithm was applied, and 87406950 being the GenBank identification number of
the nearest
GenBank protein homolo8. (See Example V.) In some cases, Incyte cDNA coverage
redundant with
the sequence covera8e shown in column 5 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 len8th
polynucleotide
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sequences which were assembled using Incyte cDNA sequences. The representative
cDNA library
is the Incyte cDNA library which is most frequently represented by the Incyte
cDNA sequences
which were used to assemble and confirm the above polynucleotide sequences.
The tissues and
vectors which were used to construct the cDNA libraries shown in Table 5 are
described in Table 6.
The invention also encompasses TRICH variants. A preferred TRICH variant is
one which
has at least about 80%, or alternatively at least about 90%, or even at least
about 95% amino acid
sequence identity to the TRICH amino acid sequence, and which contains at
least one functional or
structural characteristic of TRICH.
The invention also encompasses polynucleotides which encode TRICH. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:28-54, which encodes TRICH. The
polynucleotide
sequences of SEQ ID N0:28-54, as presented in the Sequence Listing, embxace
the equivalent RNA
sequences, wherein occurrences of the nitrogenous base thymine are replaced
with uracil, and the
sugar backbone is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding
TRICH. In
particular, such a variant polynucleotide sequence will have at least about
70%, or alternatively at least
about 85%, or even at least about 95% polynucleotide sequence identity to the
polynucleotide
sequence encoding TRICH. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID N0:28-
54 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
1D N0:28-54. Any one of the polynucleotide variants described above can encode
an amino acid
sequence which contains at least one functional or structural characteristic
of TRICH.
It will be appreciated by those skilled in the art that as a result of the
degeneracy of the
genetic code, a multitude of polynucleotide sequences encoding TRICH, some
bearing minimal
similarity to the polynucleotide sequences of any known and naturally
occurring gene, may be
produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
sequence that could be made by selecting combinations based on possible codon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring TRICH, and all such variations
are to be considered as
being specifically disclosed.
Although nucleotide sequences which encode TRICH and its variants are
generally capable of
hybridizing to the nucleotide sequence of the naturally occurring TRICH under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding TRICH or
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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 DNA sequences which encode TRICH
and
TRICH derivatives, or fragments thereof, entirely by synthetic chemistry.
After production, the
synthetic sequence may be inserted into any of the many available expression
vectors and cell systems
using reagents well known in the art. Moreover, synthetic chemistry may be
used to introduce
mutations into a sequence encoding TRICH or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ 1D
N0:28-54 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods
Enzymol. 152:507-
511.) Hybridization conditions, including annealing and wash conditions, are
described in "Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(Applied
Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerases and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
(Applied Biosystems). Sequencing is then carried out using either the ABI 373
or 377 DNA
sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing
system
(Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The
resulting sequences
are analyzed using a variety of algorithms which are well known in the art.
(See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY,
unit 7.7; Meyers,
R.A. (1995) Molecular Biology d Biotechnoloev, Wiley VCH, New York NY, pp. 856-
853.)
The nucleic acid sequences encoding TRICH may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
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restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unknown
sequence from a circularized template. The template is derived from
restriction fragments comprising
a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et
al. (1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments adjacent
to known sequences in human and yeast artificial chromosome DNA. (See, e.g.,
Lagerstrom, M. et
al: (1991) PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme digestions and
ligations may be used to insert an engineered double-stranded sequence into a
region. of unknown
sequence before performing PCR. Other methods which may be used to retrieve
unknown sequences
are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids
Res. 19:3055-3060).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries
(Clontech, Palo
Alto CA) to walk genomic DNA. This procedure avoids the need to screen
libraries and is useful in
finding intronlexon junctions. For all PCR-based methods, primers may be
designed using
commercially available software, such as OLIGO 4.06 primer analysis software
(National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30
nucleotides in length;
to have a GC content of about 50% or more, and to anneal to the template at
temperatures of about
68°C to 72°C.
When screening for full length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence
into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof which
encode TRICH may be cloned in recombinant DNA molecules that direct expression
of TRICH, or
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fragments or functional equivalents thereof, in appropriate host cells. Due to
the inherent degeneracy
of the genetic code, other DNA sequences which encode substantially the same
or a functionally
equivalent amino acid sequence may be produced and used to express TRICH.
The nucleotide sequences of the present invention can be engineered using
methods generally
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
l0 sites, alter glycosylation patterns, change codon preference, produce
splice variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
Number
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or improve
the biological properties of TRICH, such as its biological or enzymatic
activity or its ability to bind to
other molecules or compounds. DNA shuffling is a process by which a library of
gene variants is
produced using PCR-mediated recombination of gene fragments. The library is
then subjected to
selection or screening procedures that identify those gene variants with the
desired properties. These
preferred variants may then be pooled and further subjected to recursive
rounds of DNA shuffling and
selection/screening. Thus, genetic diversity is created through "artificial"
breeding and rapid molecular
evolution. For example, fragments of a single gene containing random point
mutations may be
recombined, screened, and then reshuffled until the desired properties are
optimized. Alternatively,
fragments of a given gene may be recombined with fragments of homologous genes
in the same gene
family, either from the same or different species, thereby maximizing the
genetic diversity of multiple
naturally occurring genes in a directed and controllable manner.
In another embodiment, sequences encoding TRICH may be synthesized, in whole
or in part,
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
al. (1980) Nucleic Acids
Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.) Alternatively,
TRICH itself or a fragment thereof may be synthesized using chemical methods.
For example,
peptide synthesis can be performed using various solution-phase or solid-phase
techniques. (See, e.g.,
Creighton, T. (1984) Proteins, Structures and Molecular Pr~erties, WH Freeman,
New York NY, pp.
55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated
synthesis may be achieved
using the ABI 431A peptide synthesizer (Applied Biosystems). Additionally, the
amino acid sequence
of TRICH, or any part thereof, may be altered during direct synthesis and/or
combined with
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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. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by sequencing.
(See, e.g., Creighton, supra, pp. 28-53.)
In order to express a biologically active TRICH, the nucleotide sequences
encoding TRICH or
derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which contains
the necessary elements for transcriptional and translational control of the
inserted coding sequence in
a suitable host. These elements include regulatory sequences, such as
enhancers, constitutive and
inducible promoters, and 5' and 3' untranslated regions in the vector and in
polynucleotide sequences
encoding TRICH. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
TRICH. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding TRICH and its initiation codon and upstream regulatory
sequences are inserted
into the appropriate expression vector, no additional transcriptional or
translational control signals may
be needed. However, in cases where only coding sequence, or a fragment
thereof, is inserted,
exogenous translational control signals including an in-frame ATG initiation
codon should be provided
by the vector. Exogenous translational elements and initiation codons may be
of various origins, both
natural and synthetic. The efficiency of expression may be enhanced by the
inclusion of enhancers
appropriate for the particular host cell system used. (See, e.g., Scharf, D.
et al. (1994) Results Probl.
Cell Differ. 20:125-162.)
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding TRICH and appropriate transcriptional
and translational control
elements. These methods include in vitro recombinant DNA techniques, synthetic
techniques, and in
vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
Cloning, A Laboratory
Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel,
F.M. et al. (1995)
Current Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY, ch. 9,
13, and 16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding TRICH. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV, or
tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or
CA 02410084 2002-11-20
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animal cell systems. (See, e.g., Sambrook, supra; Ausubel, su ra; Van Heeke,
G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.I~. 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. 6:307-31 l; 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; and
Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors
derived from retroviruses,
adenoviruses, or herpes or vaccinia viruses, or from various bacterial
plasmids, may be used for
delivery of nucleotide sequences to the targeted organ, tissue, or cell
population. (See, e.g., Di Nicola,
M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc.
Natl. Acad. Sci. USA
l0 90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al. (1994)
Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature 389:239-
242.) The
invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding TRICH. For
example, routine cloning,
subcloning, and propagation of polynucleotide sequences encoding TRICH can be
achieved using a
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORT1
plasmid (Life Technologies). Ligation of sequences encoding TRICH into the
vector's multiple
cloning site disrupts the lacZ gene, allowing a colorimetric screening
procedure for identification of
transformed bacteria containing recombinant molecules. In addition, these
vectors may be useful for
in vitro transcription, dideoxy sequencing, single strand rescue with helper
phage, and creation of
nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M.
Schuster (1989) J. Biol.
Chem. 264:5503-5509.) When large quantities of TRICH are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of TRICH may be used.
Fox example, vectors
containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of TRICH. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomvces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable integration
of foreign sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, 1995, supra;
Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et
al. (1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of TRICH. Transcription of
sequences
encoding TRICH may be driven by viral promoters, e.g., the 35S and 19S
promoters of CaMV used
alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
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6:307-311). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-
1680; Brogue, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105.) These
constructs can be introduced into plant cells by direct DNA transformation or
pathogen-mediated
transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technolo~y
(1992) McGraw Hill,
New York NY, pp. 191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding TRICH
may be ligated into
an adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential El or E3 region of the viral genome may
be used to obtain
infective virus which expresses TRICH in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalian host
cells. SV40 or EBV-
based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino polymers;
or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J. et al.
(1997) Nat. Genet. 15:345-
355.)
For long term production of recombinant proteins in mammalian systems, stable
expression of
TRICH in cell lines is preferred. For example, sequences encoding TRICH can be
transformed into
cell lines using expression vectors which may contain viral origins of
replication andlor endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media before
being switched to selective media. The purpose of the selectable marker is to
confer resistance to a
selective agent, and its presence allows growth and recovery of cells which
successfully express the
introduced sequences. Resistant clones of stably transformed cells may be
propagated using tissue
culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
'include, but are not limited to, the herpes simplex virus thymidine kinase
and adenine
phosphoribosyltransferase genes, for use in tk and Apr. cells, respectively.
(See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic, or
herbicide resistance can be used as the basis for selection. For example, dhfr
confers resistance to
methotrexate; rzeo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
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confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. (1981)
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and laiSD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech),13 glucuronidase and its substrate 13-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presencelabsence of marker gene expression suggests that the gene
of interest
is also present, the presence and expression of the gene may need to be
confirmed. For example, if
the sequence encoding TRICH is inserted within a marker gene sequence,
transformed cells
containing sequences encoding TRICH can be identified by the absence of marker
gene function.
Alternatively, a marker gene can be placed in tandem with a sequence encoding
TRICH under the
control of a single promoter. Expression of the marker gene in response to
induction or selection
usually indicates expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding TRICH
and that express
TRICH may be identified by a variety of procedures known to those of skill in
the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations,
PCR
amplification, and protein bioassay or immunoassay techniques which include
membrane, solution, or
chip based technologies for the detection and/or quantification of nucleic
acid or protein sequences.
Immunological methods for detecting and measuring the expression of TRICH
using either
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques
include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),
and
fluorescence activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on TRICH is
preferred, but a
competitive binding assay may be employed. These and other assays are well
known in the art. (See,
e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN,
Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Immunolo~y, Greene
Pub. Associates and
Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical
Protocols, Humana
Press, Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled hybridization
or PCR probes for detecting sequences related to polynucleotides encoding
TRICH include
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oligolabeling, nick translation, end-labeling, or PCR amplification using a
labeled nucleotide.
Alternatively, the sequences encoding TRTCH, or any fragments thereof, may be
cloned into a vector
for the production of an mRNA probe. Such vectors are known in the art, are
commercially available,
and may be used to synthesize RNA probes in vitro by addition of an
appropriate RNA polymerase
such as T7, T3, or SP6 and labeled nucleotides. These procedures may be
conducted using a variety
of commercially available kits, such as those provided by Amersham Pharmacia
Biotech, Promega
(Madison WI), and US Biochemical. Suitable reporter molecules or labels which
may be used for
ease of detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic
agents, as well as substrates, cofactors, inhibitors, magnetic particles, and
the like.
Host cells transformed with nucleotide sequences encoding TRICH may be
cultured under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the sequence
and/or the vector used. As will be understood by those of skill in the art,
expression vectors containing
polynucleotides which encode TRTCH may be designed to contain signal sequences
which direct
secretion of TRICH through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of
the polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which cleaves a
"prepro" or "pro" form of the
protein may also be used to specify protein targeting, folding, and/or
activity. Different host cells
which have specific cellular machinery and characteristic mechanisms for post-
translational activities
(e.g., CHO, HeLa, MDCK, HEK293, and WI3S) are available from the American Type
Culture
Collection (ATCC, Manassas VA) and may be chosen to ensure the correct
modification and
processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding TRICH may be ligated to a heterologous sequence resulting
in translation of a
fusion protein in any of the aforementioned host systems. For example, a
chimeric TRICH protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of TRICH
activity. Heterologous protein and
peptide moieties may also facilitate purification of fusion proteins using
commercially available affinity
matrices. Such moieties include, but are not limited to, glutathione S-
transferase (GST), maltose
binding protein (MBP), thioredoxin (Trx), calinodulin 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,
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respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity
purification of fusion
proteins using commercially available monoclonal and polyclonal antibodies
that specifically recognize
these epitope tags. A fusion protein may also be engineered to contain a
proteolytic cleavage site
located between the TRICH encoding sequence and the heterologous protein
sequence, so that
TRICH may be cleaved away from the heterologous moiety following purification.
Methods for
fusion protein expression and purification are discussed in Ausubel (1995,
supra, ch. 10). A variety of
commercially available kits may also be used to facilitate expression and
purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled TRICH may
be achieved in
vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These
systems couple transcription and translation of protein-coding sequences
operably associated with the
T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
precursor, for example, 35S-methionine.
TRICH of the present invention or fragments thereof may be used to screen for
compounds
that specifically bind to TRICH. At least one and up to a plurality of test
compounds may be screened
for specific binding to TRICH. Examples of test compounds include antibodies,
oligonucleotides,
proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the
natural ligand of
TRICH, e.g., a ligand or fragment thereof, a natural substrate, a structural
or functional mimetic, or a
natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current
Protocols in Immunolo~y 1(2,):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which TRICH
binds, or to at least a fragment of the receptor, e.g., the ligand binding
site. In either case, the
compound can be rationally designed using known techniques. In one embodiment,
screening for
these compounds involves producing appropriate cells which express TRICH,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosonhila, 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.
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TRICH of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of TRICH. Such compounds may include agonists,
antagonists, or partial or
inverse agonists. In one embodiment, an assay is performed under conditions
permissive for TRICH
activity, wherein TRICH is combined with at least one test compound, and the
activity of TRICH in
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 Number 5,175,383 and U.S. Patent Number
5,767,337.) For .
example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from
the early mouse
embryo and grown in culture. The ES cells are transformed with a vector
containing the gene of
interest disrupted by a marker gene, e.g., the neomycin phosphotransferase
gene (neo; Capecchi,
M.R. (1989) Science 244:1288-1292). The vector integrates into the
corresponding region of the host
genome by homologous recombination. Alternatively, homologous recombination
takes place using the
Cre-loxP system to knockout a gene of interest in a tissue- or developmental
stage-specific manner
(Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U.~et al. (1997)
Nucleic Acids Res.
25:4323-4330). Transformed ES cells are identified and microinjected into
mouse cell blastocysts such
as those from the C57BL/6 mouse strain. The blastocysts are surgically
transferred to
pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred
to produce
heterozygous or homozygous strains. Transgenic animals thus generated may be
tested with potential
therapeutic or toxic agents.
Polynucleotides encoding TRICH may also be manipulated in vitro in ES cells
derived from
human blastocysts. Human ES cells have the potential to differentiate into at
least eight separate cell
lineages including endoderm, mesoderm, and ectodermal cell types. These cell
lineages differentiate
into, for example, neural cells, hematopoietic lineages, and cardiomyocytes
(Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding TRICH can also be used to create "knockin" humanized
animals
(pigs) or transgenic animals (mice or rats) to model human disease. With
knockin technology, a region
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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. (I998) Biotechnol. Annu.
Rev. 4:55-74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists between
regions of TRICH and transporters and ion channels. In addition, the
expression of~TRICH is closely
associated with normal tissues such as liver, ileum, skin, brain, dorsal root
ganglion, breast, kidney,
lung, pancreas, small intestine, seminal vesicle and placental tissues; normal
cells such as
promonocytes and bone maxrow cells; and tumor tissues such as prostate,
frontal lobe, pancreatic,
ilea), colon and spleen tumor tissues. Therefore, TRICH appears to play a xole
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 ox
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,
myotonic dystrophy, catatonia, tardive dyskinesia, dystonias, peripheral
neuropathy, cerebral
neoplasms, prostate cancer, cardiac disorders associated with transport, e.g.,
angina, bradyarrythmia,
tachyarrythmia, hypertension, Long QT syndrome, myocarditis, cardiomyopathy,
nemaline myopathy,
centronuclear myopathy, lipid myopathy, mitochondria) myopathy, thyrotoxic
myopathy, ethanol
myopathy, dermatomyositis, inclusion body myositis, infectious myositis,
polymyositis, neurological
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
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disease, Addison's disease, glucose-galactose malabsorption syndrome,
hypercholesterolemia,
adrenoleukodystrophy, Zellweger syndrome, Menkes disease, occipital horn
syndrome, von Gierke
disease, cystinuria, iminoglycinuria, Hariup disease, and Fanconi disease; a
neurological disorder such
as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms,
Alzheimer's disease, Pick's
disease, Huntington's disease, dementia, Parkinson's disease and other
extrapyramidal disorders,
amyotrophic lateral sclerosis and other motor neuron disorders, progressive
neural muscular atrophy,
retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other
demyelinating diseases, bacterial
and viral meningitis, brain abscess, subdural empyema, epidural abscess,
suppurative intracranial
thrombophlebitis, myelitis and radiculitis, viral central nervous system
disease, prion diseases including
kuru, Creutzfeldt-Jakob disease, and 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,
dermatomyositas 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, 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, nemafine 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, and acid
maltase deficiency (AMD,
also known as Pompe's disease); 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
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thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis,
myasthenia gravis,
myocardial or pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis, polymyositis, psoriasis,
Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome,
systemic anaphylaxis,
systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura,
ulcerative colitis, uveitis,
Werner syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral,
bacterial, fungal, parasitic, protozoal, and helminthic infections, and
trauma; and a cell proliferative
disorder such as actinic keratosis, arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis, mixed
connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal
hemoglobinuria, polycythemia
vera, psoriasis, primary thrombocythemia, and cancers including
adenocarcinoma, leukemia,
l0 lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular,
cancers of the adrenal
gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,
ganglia, gastrointestinal tract,
heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,
prostate, salivary glands, skin,
spleen, testis, thymus, thyroid, and uterus.
In another embodiment, a vector capable of expressing TRICH or a fragment or
derivative .
thereof may be administered to a subject to treat or prevent a disorder
associated with decreased
expression or activity of TRICH including, but not limited to, those described
above.
In a further embodiment, a composition comprising a substantially purified
TRICH in
conjunction with a suitable pharmaceutical carrier may be administered to a
subject to treat or prevent
a disorder associated with decreased expression or activity of TRICH
including, but not limited to,
those provided above.
In still another embodiment, an agonist which modulates the activity of TRICH
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of TRICH including, but not limited to, those listed above.
In a further embodiment, an antagonist of TRICH may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of TRICH.
Examples of such
disorders include, but are not limited to, those transport, neurological,
muscle, immunological, and cell
proliferative disorders described above. In one aspect, an antibody which
specifically binds TRICH
may be used directly as an antagonist or indirectly as a targeting or delivery
mechanism for bringing a
pharmaceutical agent to cells or tissues which express TRICH.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding TRICH may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of TRICH including, but not limited to, those
described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be administered in combination with
other appropriate
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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
R
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.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, 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 paivum are especially
preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
TRICH have an amino acid sequence consisting of at least about 5 amino acids,
and generally will
consist of at least about 10 amino acids. It is also preferable that these
oligopeptides, peptides, or
fragments are identical to a portion of the amino acid sequence of the natural
protein. Short stretches _
of TRICH amino acids may be fused with those of another protein, such as KLH,
and antibodies to
the chimeric molecule may be produced.
Monoclonal antibodies to TRICH may be prepared using any technique which
provides for the
production of antibody molecules by continuous cell lines in culture. These
include, but are not limited
to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-
hybridoma
technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; and
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
CA 02410084 2002-11-20
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Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and Takeda,
S. et al. ( 1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the art, to produce
TRICH-specific single
chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be
generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g., Burton,
D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte population
or by screening immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in
the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.
USA 86:3833-3837; Winter,
l0 G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for TRICH may also be
generated.
For example, such fragments include, but are not limited to, F(ab~2 fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab~2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
TRICH and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive
to two non-interfering TRICH epitopes is generally used, but a competitive
binding assay may also be
employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay techniques
may be used to assess the affinity of antibodies for TRICH. Affinity is
expressed as an association
constant, I~, 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 I~
determined for a preparation of polyclonal antibodies, which are heterogeneous
in their affinities for
multiple TRICH epitopes, represents the average affinity, or avidity, of the
antibodies for TRICH.
The I~ determined for a preparation of monoclonal antibodies, which are
monospecific for a particular
TRICH epitope, represents a true measure of affinity. High-affinity antibody
preparations with I~
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 I~
ranging from about 106 to 10' L/mole are preferred for use in
immunopurification and similar
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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/ml, preferably 5-10 mg
specific antibody/ml, is generally employed in procedures requiring
precipitation of TRICH-antibody
complexes. Procedures for evaluating antibody specificity, titer, and avidity,
and guidelines for
antibody quality and usage in various applications, are generally available.
(See, e.g., Catty, supra, and
Coligan et al. supra.)
In another embodiment of the invention, the polynucleotides encoding TRICH, or
any
fragment or complement thereof, may be used for therapeutic purposes. In one
aspect, modifications
of gene expression can be achieved by designing complementary sequences or
antisense molecules
(DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory
regions of the gene
encoding TRICH. Such technology is well known in the art, and antisense
oligonucleotides or larger
fragments can be designed from various locations along the coding or control
regions of sequences
encoding TRICH. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics,
Humana Press Inc.,
Totawa NJ.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. (1998) J. Allergy Cli. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. (1990) Blood
76:271; Ausubel, suQra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull. 51
(1):217-225; Boado, R.J. et
3o al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al. (1997)
Nucleic Acids Res.
25(I4):2730-2736.)
In another embodiment of the invention, polynucleotides encoding TRICH may be
used for
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCID)-Xl disease
characterized by X-
52
CA 02410084 2002-11-20
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linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672),
severe combined
iminunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-
242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites (e.g.,
against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore, D. (1988)
Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA.
93:11395-11399),
hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans
and Paracoccidioides
brasiliensis; and protozoan parasites such as Plasmodium falcipanun 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 vectors (Invitrogen,
Carlsbad CA),
PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF,
PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). TRICH may be
expressed
using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV),
Rous sarcoma virus
(RSV), SV40 virus, thymidine kinase (TK), or (3-actin genes), (ii) an
inducible promoter (e.g., the
tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl.
Acad. Sci. USA
89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V.
and H.M. Blau (1998)
Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen)); the
ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the
53
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible
promoter (Rossi, F.M.V.
and Blau, H.M. 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 LIPS
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of these
l0 standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to TRICH expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding TRICH under the control of an independent promoter or
the retrovirus long
terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and
(iii) a Rev-responsive
element (RRE) along with additional retrovirus cis-acting RNA sequences and
coding sequences
required for efficient vector propagation. Retrovirus vectors (e.g., PFB and
PFBNEO) are
commercially available (Stratagene) and are based on published data (Riviere,
I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The
vector is propagated in
an appropriate vector producing cell line (VPCL) that expresses an envelope
gene with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R. et
al. (1998) J. Virol. 72:9873-9880). U.S. Patent Number 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant") discloses
a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by reference.
Propagation of retrovirus vectors, transduction of a population of cells
(e.g., CD4+ T-cells), and the
return of transduced cells to a patient are procedures well known to persons
skilled in the art of gene
therapy and have been well documented (Ranga, U. et al. (1997) J. Virol.
71:7020-7029; Bauer, G. et
al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-4716;
Ranga, U. et al. (1998)
Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding TRICH to cells which have one or more genetic
abnormalities with respect
to the expression of TRICH. The construction and packaging of adenovirus-based
vectors are well
known to those with ordinary skill in the art. Replication defective
adenovirus vectors have proven to
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CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
be versatile for importing genes encoding immunoregulatory proteins into
intact islets in the pancreas
(Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful
adenoviral vectors are
described in U.S. Patent Number 5,707,618 to Armentano ("Adenovirus vectors
for gene therapy"),
hereby incorporated by reference. For adenoviral vectors, see also Antinozzi,
P.A. et al. (1999)
Annu. Rev. Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature
18:389:239-242, both
incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding TRICH to target cells which have one or more genetic
abnormalities with
respect to the expression of TRICH. The use of herpes simplex virus (HSV)-
based vectors may be
especially valuable for introducing TRICH to cells of the central nervous
system, for which HSV has
a tropism. The construction and packaging of herpes-based vectors are well
known to those with
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
type 1-based vector has
been used to deliver a reporter gene to the eyes of primates (Liu, X. et al.
(1999) Exp. Eye Res.
169:385-395). The construction of a HSV-1 virus vector has also been disclosed
in detail in U.S.
Patent Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is
hereby incorporated by reference. U.S. Patent Number 5,804,413 teaches the use
of recombinant
HSV d92 which consists of a genome containing at least one exogenous gene to
be transferred to a
cell under the control of the appropriate promoter for purposes including
human gene therapy. Also
taught by this patent are the construction and use of recombinant HSV strains
deleted for ICP4,
ICP27 and ICP22. For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol.
73:519-532 and Xu,
H. et al. (1994) Dev. Biol. 163:152-161, hereby incorporated by reference. The
manipulation of
cloned herpesvirus sequences, the generation of recombinant virus following
the transfection of
multiple plasmids containing different segments of the large herpesvirus
genomes, the growth and
propagation of herpesvirus, and the infection of cells with herpesvirus are
techniques well known to
those of ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding TRICH to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based
on the SFV genome (Garoff, H. and I~.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During
alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full length
genomic RNA,
resulting in the overproduction of capsid proteins relative to the viral
proteins with enzymatic activity
(e.g., protease and polymerase). Similarly, inserting the coding sequence for
TRICH into the
alphavirus genome in place of the capsid-coding region results in the
production of a large number of
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
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
application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host
range of alphaviruses will
allow the introduction of TRICH into a variety of cell types. The specific
transduction of a subset of
cells in a population may require the sorting of cells prior to transduction.
The methods of
manipulating infectious cDNA clones of alphaviruses, performing alphavirus
cDNA and RNA
transfections, and performing alphavirus infections, are well known to those
with ordinary skill in the
art.
Oligonucleotides derived from the transcription initiation site, e.g., between
about positions -10
and +10 from the start site, may also be employed to inhibit gene expression.
Similarly, inhibition can
be achieved using triple helix base-pairing methodology. Triple helix pairing
is useful because it causes
inhibition of the ability of the double helix to open sufficiently for the
binding of polymerases,
transcription factors, or regulatory molecules. Recent therapeutic advances
using triplex DNA have
been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in
Huber, B.E. and B.I. Carr,
Molecular and Immunolo~ic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-
177.) A
complementary sequence or antisense molecule may also be designed to block
translation of mRNA
by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding TRICH.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
~ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared
by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis.
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CA 02410084 2002-11-20
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Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA
sequences encoding TRICH. Such DNA sequences may be incorporated into a wide
variety of
vectors with suitable RNA polymerase promoters such as T7 or SP6.
Alternatively, these cDNA
constructs that synthesize complementary RNA, constitutively or inducibly, can
be introduced into cell
lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3' ends
of the molecule, or the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages
within the backbone of the molecule. This concept is inherent in the
production of PNAs and can be
extended in all of these molecules by the inclusion of nontraditional bases
such as inosine, queosine,
and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified
forms of adenine, cytidine,
guanine, thymine, and uridine which are not as easily recognized by endogenous
endonucleases.
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
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
57
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
by any method commonly known in the art. Typically, the expression of a
specific nucleotide is
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. Commun.
268:8-13). A particular embodiment of the present invention involves screening
a combinatorial library
of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide
nucleic acids, and modified
oligonucleotides) for antisense activity against a specific polynucleotide
sequence (Bruice, T.W. et al.
(1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No.
6,022,691).
Many methods for introducing vectors into cells or tissues are available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
Biotechno1.15:462-466.)
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,
sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
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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-
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
l0 of an effective dose is well within the capability of those skilled in the
art.
Specialized forms of compositions rnay be prepared for direct intracellular
delivery of
macromolecules comprising TRICH or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of the
macromolecule. Alternatively, TRICH or a fragment thereof may be joined to a
short cationic N-
terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated
have been found to
transduce into the cells of all tissues, including the brain, in a mouse model
system (Schwarze, S.R. et
al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as nuce,
rats, rabbits, dogs, monkeys,
or pigs. An animal model may also be used to determine the appropriate
concentration range and
route of administration. Such information can then be used to determine useful
doses and routes for
administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example
TRICH or fragments thereof, antibodies of TRICH, and agonists, antagonists or
inhibitors of TRICH,
which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity
may be determined
by standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDSO (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDso/EDso ratio. Compositions
which exhibit large
therapeutic indices are preferred. The data obtained from cell culture assays
and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDso
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
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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 same manner as described above for
therapeutics. Diagnostic
assays for TRICH include methods which utilize the antibody and a label to
detect TRICH in human
body fluids or in extracts of cells or tissues. The antibodies may be used
with or without modification,
and may be labeled by covalent or non-covalent attachment of a reporter
molecule. A wide variety of
reporter molecules, several of which are described above, are known in the art
and may be used.
A variety of protocols for measuring TRICH, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
TRICH expression. Normal
or standard values for TRICH expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibodies to TRICH under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of TRICH
expressed in
subject, control, and disease samples from biopsied tissues are compared with
the standard values.
Deviation between standard and subject values establishes the parameters for
diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding TRICH may
be used for
diagnostic purposes. The polynucleotides which may be used include
oligonucleotide sequences,
complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used
to detect
and quantify gene expression in biopsied tissues in which expression of TRICH
may be correlated
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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
polynucleotide
sequences, including genomic sequences, encoding TRICH or closely related
molecules may be used
to identify nucleic acid sequences which encode TRICH. The specificity of the
probe, whether it is
made from a highly specific region, e.g., the 5' regulatory region, or from a
less specific region, e.g., a
conserved motif, and the stringency of the hybridization or amplification will
determine whether the
probe identifies only naturally occurring sequences encoding TRICH, allelic
variants, or.related
sequences.
l0 Probes may also be used for the detection of related sequences, and may
have at least 50%o
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 ID
N0:28-54 or from
genomic sequences including promoters, enhancers, and introns of the TRICH
gene.
Means for producing specific hybridization probes for DNAs encoding TRICH
include the
cloning of polynucleotide sequences encoding TRICH or TRICH derivatives into
vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and may
be used to synthesize RNA probes in vitro by means of the addition of the
appropxiate 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 355,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding TRICH may be used for the diagnosis of
disorders
associated with expression of TRICH. Examples of such disorders include, but
are not limited to, a
transport disorder such as akinesia, amyotrophic lateral sclerosis, ataxia
telangiectasia, cystic fibrosis,
Becker's muscular dystrophy, Bell's palsy, Charcot-Marie Tooth disease,
diabetes mellitus, diabetes
insipidus, diabetic neuropathy, Duchenne muscular dystrophy, hyperkalemic
periodic paralysis,
normokalemic periodic paralysis, Parkinson's disease, malignant hyperthermia,
multidrug resistance,
myasthenia gravis, myotonic dystrophy, catatonia, tardive dyskinesia,
dystonias, peripheral neuropathy,
cerebral neoplasms, prostate cancer, cardiac disorders associated with
transport, e.g., angina,
bradyarrythmia, tachyarrythmia, hypertension, Long QT syndrome, myocarditis,
cardiomyopathy,
nemaline myopathy, centronuclear myopathy, lipid myopathy, mitochondrial
myopathy, thyrotoxic
myopathy, ethanol myopathy, dermatomyositis, inclusion body myositis,
infectious myositis,
polymyositis, neurological disorders associated with transport, e.g.,
Alzheimer's disease, amnesia,
bipolar disorder, dementia, depression, epilepsy, Tourette's disorder,
paranoid psychoses, and
schizophrenia, and other disorders associated with transport, e.g.,
neurofibromatosis, postherpetic
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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,
hypercholesterolemia, adrenoleukodystrophy, Zellweger syndrome, Menkes
disease, occipital horn
syndrome, von Gierke disease, cystinuria, iminoglycinuria, Hartup disease, and
Fanconi disease; a
neurological disorder such as epilepsy, ischemic cerebrovascular disease,
stroke, cerebral neoplasms,
Alzheimer's disease, Pick's disease, Huntington's disease, dementia,
Parkinson's disease and other
extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron
disorders, progressive
neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple
sclerosis and other
demyelinating diseases, bacterial and viral meningitis, brain abscess,
subdural empyema, epidural
abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis,
viral central nervous system
disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and
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, 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 myopathy, lipid myopathy, mitochondria) myopathy,
infectious myositis,
polymyositis, dermatomyositis, inclusion body myositis, thyrotoxic myopathy,
ethanol myopathy, angina,
anaphylactic shock, arrhythmias, asthma, cardiovascular shock, C~shing's
syndrome, hypertension,
hypoglycemia, myocardial infarction, migraine, pheochromocytoma, and
myopathies including
encephalopathy, epilepsy, Kearns-Sayre syndrome, lactic acidosis, myoclonic
disorder,
ophthalmoplegia, and acid maltase deficiency (AMD, also known as Pompe's
disease); 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
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. dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema, episodic
lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum,
atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis,
hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's
syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial,
fungal, parasitic, protozoal, and helminthic infections, and trauma; and a
cell proliferative disorder such
as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis,
hepatitis, mixed connective
tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria,
polycythemia vera,
psoriasis, primary thrombocythemia, and cancers including adenocarcinoma,
leukemia, lymphoma,
melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of
the adrenal gland,
bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract, heart,
kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate,
salivary glands, skin, spleen,
testis, thymus, thyroid, and uterus. The polynucleotide sequences encoding
TRICH may be used in
Southern or northern analysis, dot blot, or other membrane-based technologies;
in PCR technologies; in
dipstick, pin, and multiformat ELISA-like assays; and in microarrays utilizing
fluids or tissues from
patients to detect altered TRICH expression. Such qualitative or quantitative
methods are well known
in the art.
In a particular aspect, the nucleotide sequences encoding TRICH may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding TRICH may be labeled by standard methods and added to a
fluid or tissue sample
from a patient under conditions suitable for the formation of hybridization
complexes. After a suitable
incubation period, the sample is washed and the signal is quantified and
compared with a standard
value. If the amount of signal in the patient sample is significantly altered
in comparison to a control
sample then the presence of altered levels of nucleotide sequences encoding
TRICH in the sample
indicates the presence of the associated disorder. Such assays may also be
used to evaluate the
efficacy of a particular therapeutic treatment regimen in animal studies, in
clinical trials, or to monitor
the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with
expression of
TRICH, a normal or standard profile for expression is established. This may be
accomplished by
combining body fluids or cell extracts taken from normal subjects, either
animal or human, with a
sequence, or a fragment thereof, encoding TRICH, under conditions suitable for
hybridization or
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amplification. Standard hybridization may be quantified by comparing the
values obtained from normal
subjects with values from an experiment in which a known amount of a
substantially purified
polynucleotide is used. Standard values obtained in this manner may be
compared with values
obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard
values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
l0 days to months.
With respect to cancer, the pxesence 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 filrther
progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding TRICH .
may involve the use of PCR. These oligomers may be chemically synthesized,
generated
enzymatically, or produced in vitro. Oligomers will preferably contain a
fragment of a polynucleotide
encoding TRICH, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
TRICH, and will be employed under optimized conditions for identification of a
specific gene or
condition. Oligomers may also be employed under less stringent conditions for
detection or
quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding TRICH may be used to detect single nucleotide polymoiphisms (SNPs).
SNPs are
substitutions, insertions and deletions that are a frequent cause of inherited
or acquired genetic disease
in humans. Methods of SNP detection include, but are not limited to, single-
stranded conformation
polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP,
oligonucleotide primers
derived from the polynucleotide sequences encoding TRICH are used to amplify
DNA using the
polymerase chain reaction (PCR). The DNA may be derived, for example, from
diseased or normal
tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause
differences in the
secondary and tertiary structures of PCR products in single-stranded form, and
these differences are
detectable using gel electrophoresis in non-denaturing gels. In fSCCP, the
oligonucleotide primers are
fluorescently labeled, which allows detection of the amplimers in high-
throughput equipment such as
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DNA sequencing machines. Additionally, sequence database analysis methods,
termed in silico SNP
(isSNP), are capable of identifying polymorphisms by comparing the sequence of
individual
overlapping DNA fragments which assemble into a common consensus sequence.
These computer-
based methods filter out sequence variations due to laboratory preparation of
DNA and sequencing
errors using statistical models and automated analyses of DNA sequence
chromatograms. In the
alternative, SNPs may be detected and characterized by mass spectrometry
using, for example, the
high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
Methods which may also be used to quantify the expression of TRICH include
radiolabeling or
biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives rapid
quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described below. The microarray may also be
used to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, to monitor
progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used
to develop a pharmacogenomic profile of a patient in order to select the most
appropriate and effective
treatment regimen for that patient. For example, therapeutic agents which are
highly effective and
display the fewest side effects may be selected for a patient based on his/her
pharmacogenomic
profile.
In another embodiment, TRICH, fragments of TRICH, or antibodies specific for
TRICH may
be used as elements on a microarray. The microarray may be used to monitor or
measure protein-
protein interactions, drug-target interactions, and gene expression profiles,
as described above.
A particular embodiment relates to the use of the polynucleotides of the
present invention to
generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern of
gene expression by a particular tissue or cell type. Global gene expression
patterns are analyzed by
quantifying the number of expressed genes and their relative abundance under
given conditions and at
a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent Number
CA 02410084 2002-11-20
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5,840,484, expressly incorporated by reference herein.) Thus a transcript
image may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcxipt images may be generated using transcripts isolated from tissues,
cell lines, biopsies,
or other biological samples. The transcript image may thus reflect gene
expression in vivo, as in the
case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the
present invention
may also be used in conjunction with in vitro model systems and preclinical
evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and toxicity
(Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L.
Anderson (2000)
Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein).
If a test compound has a
signature similar to that of a compound with known toxicity, it is likely to
share those toxic properties.
These fingerprints or signatures are most useful and refined when they contain
expression information
from a large number of genes and gene families. Ideally, a genome-wide
measurement of expression
provides the highest quality signature. Even genes whose expression is not
altered by any tested
compounds are important as well, as the levels of expression of these genes
are used to normalize the
rest of the expression data. The normalization procedure is useful for
comparison of expression data
after treatment with different compounds. While the assignment of gene
function to elements of a
toxicant signature aids in interpretation of toxicity mechanisms, knowledge of
gene function is not
necessary for the statistical matching of signatures which leads to prediction
of toxicity. (See, for
example, Press Release 00-02 from the National Institute of Environmental
Health Sciences, released
February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm.)
Therefore, it is
important and desirable in toxicological screening using toxicant signatures
to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological sample
containing nucleic acids with the test compound. Nucleic acids that are
expressed in the treated
biological sample are hybridized with one or more probes specific to the
polynucleotides of the present
invention, so that transcript levels corresponding to the polynucleotides of
the present invention may be ,
quantified. The transcript levels in the treated biological sample are
compared with levels in an
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untreated biological sample. Differences in the transcript levels between the
two samples are
indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a proteome
can be subjected individually to further analysis. Proteome expression
patterns, or profiles, are
analyzed by quantifying the number of expressed proteins and their relative
abundance under given
conditions and at a given time. A profile of a cell's proteome may thus be
generated by separating
and analyzing the polypeptides of a particular tissue or cell type. In one
embodiment, the separation is
achieved using two-dimensional gel electrophoresis, in which proteins from a
sample are separated by
isoelectric focusing in the first dimension, and then according to molecular
weight by sodium dodecyl
sulfate slab gel electrophoresis in the second dimension (Steiner and
Anderson, supra). The proteins
are visualized in the gel as discrete and uniquely positioned spots, typically
by staining the gel with an
agent such as Coomassie Blue or silver or fluorescent stains. The optical
density of each protein spot
is generally proportional to the level of the protein in the sample. The
optical densities of equivalently
positioned protein spots from different samples, for example, from biological
samples either treated or
untreated with a test compound or therapeutic agent, are compared to identify
any changes in protein
spot density related to the treatment. The proteins in the spots are partially
sequenced using, for
example, standard methods employing chemical or enzymatic cleavage followed by
mass
spectrometry. The identity of the protein in a spot may be determined by
comparing its partial
sequence, preferably of at least 5 contiguous amino acid residues, to the
polypeptide sequences of the
present invention. In some cases, further sequence data may be obtained for
definitive protein
identification.
A proteomic profile may also be generated using antibodies specific for TRICH
to quantify
the levels of TRICH expression. In one embodiment, the antibodies are used as
elements on a
microarray, and protein expression levels are quantified by exposing the
microarray to the sample and
detecting the levels of protein bound to each array element (Lueking, A. et
al. (1999) Anal. Biochem.
270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection
may be performed by
a variety of methods known in the art, for example, by reacting the proteins
in the sample with a thiol-
or amino-reactive fluorescent compound and detecting the amount of
fluorescence bound at each
array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
correlation between transcript and protein abundances for some proteins in
some tissues (Anderson,
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N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures may be
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to rapid
degradation of mRNA, so proteomic profiling may be more reliable and
informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
each protein is compared to the amount of the corresponding protein in an
untreated biological sample.
A difference in the amount of protein between the two samples is indicative of
a toxic response to the
test compound in the treated sample. Individual proteins are identified by
sequencing the amino acid
residues of the individual proteins and comparing these partial sequences to
the polypeptides of the
present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are incubated
with antibodies specific to the polypeptides of the present invention. The
amount of protein recognized
by the antibodies is quantified. The amount of protein in the treated
biological sample is compared
with the amount in an untreated biological sample. A difference in the amount
of protein between the
two samples is indicative of a toxic response to the test compound in the
treated sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad.
Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application
W095/251116; Shalom D. et
al. (1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA
94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.)
Various types of
microarrays axe well known and thoroughly described in DNA Microarravs: A
Practical Auproach,
M. Schena, ed. (1999) Oxford University Press, London, hereby expressly
incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding TRICH
may be
used to generate hybridization probes useful in mapping the naturally
occurring genomic sequence.
Either coding or noncoding sequences may be used, and in some instances,
noncoding sequences may
be preferable over coding sequences. For example, conservation of a coding
sequence among
members of a mufti-gene family may potentially cause undesired cross
hybridization during
chromosomal mapping. The sequences may be mapped to a particular chromosome,
to a specific
region of a chromosome, or to artificial chromosome constructions, e.g., human
artificial chromosomes
(HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes
(BACs), bacterial P1
constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. (1997) Nat.
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Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop
genetic linkage maps, for example, which correlate the inheritance of a
disease state with the
inheritance of a particular chromosome region or restriction fragment length
polymorphism (RFLP).
(See, for example, Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci.
USA 83:7353-7357.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic
map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-
968.) Examples of genetic
map data can be found in various scientific journals or at the Online
Mendelian Inheritance in Man
(OMIM) World Wide Web site. Correlation between the location of the gene
encoding TRICH on a
l0 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 11 q22-23, any
sequences mapping to that area may represent associated or regulatory genes
for further investigation.
(See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide
sequence of the 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 TR1CH and the agent being tested may be measured.
Another technique for drng screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. (1984) PCT
application W084103564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with TRICH,
or fragments thereof,
and washed. Bound TRICH is then detected by methods well known in the art.
Purified TRICH can
also be coated directly onto plates for use in the aforementioned drug
screening techniques.
Alternatively, non-neutralizing antibodies can be used to capture the peptide
and immobilize it on a
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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 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,
including U.S. Ser. No. 60!208,424, U.S. Ser. No. 60/209,001, U.S. Ser. No.
60/210,588, U.S. Ser.
No. 60/212,335, U.S. Ser. No. 60/213,747, and U.S. Ser. No. 60/215,391, 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) and shown in Table 4, column 5. Some
tissues were
homogenized and lysed in guanidinium isothiocyanate, while others were
homogenized and lysed in
phenol or in a suitable mixture of denaturants, such as TRIZOL (Life
Technologies), a monophasic
solution of phenol and guanidine isothiocyanate. The resulting lysates were
centrifuged over CsCI
cushions or extracted with chloroform. RNA was precipitated from the lysates
with either isopropanol
or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A)+ RNA was
isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX
latex particles
(QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
Alternatively,
RNA was isolated directly from tissue lysates using other RNA isolation kits,
e.g., the
POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
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libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the
UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life
Technologies), using
the recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra,
units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CI~B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
l0 PBLUESCRIPT plasmid (Stratagene), PSPORTl plasmid (Life Technologies),
PCDNA2.1 plasmid
(Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), or pINCY (Incyte
Genomics, Palo Alto
CA), or derivatives thereof. Recombinant plasmids were transformed into
competent E. coli cells
including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DHSa, DH10B, or
ElectroMAX
DH10B from Life Technologies.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo
excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega); an
AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid,
QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
R.E.A.L. PREP
96 plasmid purification kit from QIAGEN. Following precipitation, plasmids
were resuspended in 0.1
ml of distilled water and stored, with or without lyophilization, at
4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically using
PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence
scanner
(Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
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
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using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as
the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied
Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carned out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics); the
ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction
with standard ABI
protocols and base calling software; or other sequence analysis systems known
in the art. Reading
frames within the cDNA sequences were identified using standard methods
(reviewed in Ausubel,
1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension
using the
techniques disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by
removing
vector, linker, and poly(A) sequences and by masking ambiguous bases, using
algorithms and
programs based on BLAST, dynamic programming, and dinucleotide nearest
neighbor analysis. The
Incyte cDNA sequences or translations thereof were then queried against a
selection of public
databases such as the GenBankprimate, rodent, mammalian, vertebrate, and
eukaryote databases, and
BLOCKS, PRINTS, DOMO, PRODOM, and hidden Markov model (HMM)-based protein
family
databases such as PFAM. (HMM is a probabilistic approach which analyzes
consensus primary
structures of gene families. See, for example, Eddy, S.R. (1996) C~.ur. Opin.
Struct. Biol. 6:361-365.)
The queries were performed using programs based on BLAST, FASTA, BLIMPS, and
HMMER.
The Incyte cDNA sequences were assembled to produce full length polynucleotide
sequences.
Alternatively, GenBankcDNAs, GenBankESTs, stitched sequences, stretched
sequences, or
Genscan-predicted coding sequences (see Examples IV and V) were used to extend
Incyte cDNA
assemblages to full length. Assembly was performed using programs based on
Phred, Phrap, and
Consed, and cDNA assemblages were screened for open reading frames using
programs based on
GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were
translated to derive
the corresponding full length polypeptide sequences. Alternatively, a
polypeptide of the invention may
begin at any of the methionine residues of the full length translated
polypeptide. Full length polypeptide
sequences were subsequently analyzed by querying against databases such as the
GenBank protein
databases (genpept), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and
hidden
Markov model (HMM)-based protein family databases such as PFAM. Full length
polynucleotide
sequences are also analyzed using MACDNASIS PRO software (Hitachi Software
Engineering,
South San Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and
polypeptide
sequence alignments are generated using default parameters specified by the
CLIJSTAL algorithm as
incorporated into the MEGALIGN multisequence alignment program (DNASTAR),
which also
calculates the percent identity between aligned sequences.
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Table 7 summarizes the tools, programs, and algorithms used for the analysis
and assembly of
Incyte cDNA and full length sequences and provides applicable descriptions,
references, and threshold
parameters. The first column of Table 7 shows the tools, programs, and
algorithms used, the second
column provides brief descriptions thereof, the third column presents
appropriate references, all of
which are incorporated by reference herein in their entirety, and the fourth
column presents, where
applicable, the scores, probability values, and other parameters used to
evaluate the strength of a
match between two sequences (the higher the score or the lower the probability
value, the greater the
identity between two sequences).
The programs described above for the assembly and analysis of full length
polynucleotide and
polypeptide sequences were also used to identify polynucleotide sequence
fragments from SEQ ID
N0:28-54. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
amplification technologies are described in Table 4, column 4.
IV. Identification and Editing of Coding Sequences from Genomic DNA
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 (See Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-
94, and Burge, C. and
S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354). The program
concatenates predicted exons to
form an assembled cDNA sequence extending from a methionine to a stop codon.
The output of
Genscan is a FASTA database of polynucleotide and polypeptide sequences. The
maximum range of
sequence for Genscan to analyze at once was set to 30 kb. To determine which
of these Genscan
predicted cDNA sequences encode transporters and ion channels, the encoded
polypeptides were
analyzed by querying against PFAM models for transporters and ion channels.
Potential transporters
and ion channels were also identified by homology to Incyte cDNA sequences
that had been
annotated as transporters and ion channels. These selected Genscan-predicted
sequences were then
compared by BLAST analysis to the genpept and gbpri public databases. Where
necessary, the
Genscan-predicted sequences were then edited by comparison to the top BLAST
hit from genpept to
correct errors in the sequence pxedicted by Genscan, such as extra or omitted
exons. BLAST
analysis was also used to find any Incyte cDNA or public cDNA coverage of the
Genscan-predicted
sequences, thus providing evidence for transcription. When Incyte cDNA
coverage was available,
this information was used to correct or confirm the Genscan predicted
sequence. Full length
polynucleotide sequences were obtained by assembling Genscan-predicted coding
sequences with
Incyte cDNA sequences andlor public cDNA sequences using the assembly process
described in
Example III. Alternatively, full length polynucleotide sequences were derived
entirely from edited or
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unedited Genscan-predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
"Stitched" Sequences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification program described in Example IV. Partial cDNAs assembled as
described in Example
III were mapped to genomic DNA and parsed into clusters containing related
cDNAs and Genscan
exon predictions from one or more genomic sequences. Each cluster was analyzed
using an algorithm
based on graph theory and dynamic programming to integrate cDNA and genomic
information,
generating possible splice variants that were subsequently confirmed, edited,
or extended to create a
full length sequence. Sequence intervals in which the entire length of the
interval was present on
more than one sequence in the cluster were identified, and intervals thus
identified were considered to
be equivalent by transitivity. For example, if an interval was present on a
cDNA and two genomic
sequences, then all three intervals were considered to be equivalent. This
process allows unrelated
but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals
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" Seguences
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 GenBankprimate, 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 GenB ank protein homolog.
Insertions or deletions
may occur in the chimeric protein with respect to the original GenB ank
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
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stretched sequences were examined to determine whether it contained a complete
gene.
VI. Chromosomal Mapping of TRICH Encoding Polynucleotides
The sequences which were used to assemble SEQ ID N0:28-S4 were compared with
sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
S implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ ID N0:28-S4 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for
Genome Research (WIGR), and Genethon were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ 117 NO:, to
that map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map
position of an interval, in centiMorgans, is measured relative to the terminus
of the chromosome's p-
arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
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.
In this manner, SEQ ID N0:8 was mapped to chromosome 12 within the interval
from 137.50
to 160.90 centiMorgans.
VII. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
from a particular cell type or tissue have been bound. (See, e.g., Sambrook,
supra, ch. 7; Ausubel
(1995) su ra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the computer
search can be modified to determine whether any particular match is
categorized as exact or similar.
The basis of the search is the product score, which is defined as:
CA 02410084 2002-11-20
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BLAST Score x Percent Identity
xminimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two
sequences and the
5 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
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, polynucleotide sequences encoding TRICH are analyzed with
respect to the
tissue sources from which they were derived. For example, some full length
sequences are
assembled, at least in part, with overlapping Incyte cDNA sequences (see
Example 11I). 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; hemic 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 acxoss all categories. Similarly, each human tissue is classified
into one of the following
disease/condition categories: cancer, cell line, developmental, inflammation,
neurological, trauma,
cardiovascular, pooled, and other, and the number of libraries in each
category is counted and divided
by the total number of libraries across all categories. The resulting
percentages reflect the tissue- and
disease-specific expression of cDNA encoding TRICH. cDNA sequences and cDNA
library/tissue
information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto
CA).
VIII. Extension of TRICH Encoding Polynucleotides
Full length polynucleotide sequences were also produced by extension of an
appropriate
fragment of the full length molecule using oligonucleotide primers designed
from this fragment. One
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primer was synthesized to initiate 5' extension of the known fragment, and the
other primer was
synthesized to initiate 3' extension of the known fragment. The initial
primers were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68 °C to about 72 °C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+, (NH4)2S04,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE
enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the
following parameters
for primer pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 60°C, 1 min;
Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step
6: 68°C, 5 min; Step 7: storage
at 4°C. In the alternative, the parameters for primer pair T7 and SK+
were as follows: Step 1: 94°C,
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 ~l 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 ~cl to 10 ~1 aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose gel to determine which reactions were
successful in extending the
sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Reseaxch,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agaxose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended
clones were religated using T4 lipase (New England Biolabs, Beverly MA) into
pUC 18 vector
(Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to
fill-in restriction
site overhangs, and transfected into competent E. coli cells. Transformed
cells were selected on
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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 Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the
following
parameters: Step 1: 94 ° C, 3 min; Step 2: 94 ° C, 15 sec; Step
3: 60 ° C, 1 min; Step 4: 72 ° C, 2 min; Step
5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7:
storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted with
20% dimethysulfoxide (1:2, vlv), and sequenced using DYENAMIC energy transfer
sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the
above procedure or
are used to obtain 5' regulatory sequences using the above procedure along
with oligonucleotides
designed for such extension, and an appropriate genomic library.
IX. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:28-54 are employed to screen
cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide
fragments. Oligonucleotides are designed using state-of the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 ~Ci of
~,Y 32P, adenosine triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-based
hybridization analysis of human genomic DNA digested with one of the following
endonucleases: Ase
I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
X. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
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photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, supra.), mechanical
microspotting technologies, and derivatives thereof. The substrate in each of
the aforementioned
technologies should be uniform and solid with a non-porous surface (Schena
(1999), supra).
Suggested substrates include silicon, silica, glass slides, glass chips, and
silicon wafers. Alternatively, a
procedure analogous to a dot or slot blot may also be used to arrange and link
elements to the surface
of a substrate using thermal, W, chemical, or mechanical bonding procedures. A
typical array may
be produced using available methods and machines well known to those of
ordinary skill in the art and
may contain any appropriate number of elements. (See, e.g., Schena, M. et al.
(1995) Science
270:467-470; Shalom D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and
J. Hodgson (1998)
l0 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/~1 oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 units/~1 RNase inhibitor, 500 ~M dATP, 500 ~M dGTP, 500 ~M
dTTP, 40 ~M
dCTP, 40 ~ M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amershaxn Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)+ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of O.SM sodium
hydroxide and
incubated for 20 minutes at 85° C to the stop the reaction and degrade
the RNA. Samples are purified
using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
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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 ~l SX SSC/0.2% SDS.
Microarra~i Preparation
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses
primers complementary to the vector sequences flanking the cDNA insert. Array
elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5 ~ g.
Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water, and
coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are
cured in a 110°C
oven.
Array elements are applied to the coated glass substrate using a procedure
described in US
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 high-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, Inc., 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 ~ l of sample mixture consisting of 0.2 ~ g
each of Cy3 and
Cy5 labeled cDNA synthesis products in SX SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65° C for 5 minutes and is aliquoted onto the
microarray surface and covered with
an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber
having a cavity just slightly
larger than a microscope slide. The chamber is kept at 100% humidity
internally by the addition of 140
~1 of SX 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
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Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 em 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 puzpose of identifying genes that are
differentially expressed,
the calibration is done by labeling samples of the calibrating cDNA with the
two fluorophores and
adding identical amounts of each to the hybridization mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
finear 20-colox 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).
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XI. Complementary Polynucleotides
Sequences complementary to the TRICH-encoding sequences, or any parts thereof,
are used
to detect, decrease, or inhibit expression of naturally occurring TRICH.
Although use of
oligonucleotides comprising from about 15 to 30 base pairs is described,
essentially the same
procedure is used with smaller or with larger sequence fragments. Appropriate
oligonucleotides are
designed using OLIGO 4.06 software (National Biosciences) and the coding
sequence of TRICH. To
inhibit transcription, a complementary oligonucleotide is designed from the
most unique 5' sequence
and used to prevent promoter binding to the coding sequence. To inhibit
translation, a complementary
oligonucleotide is designed to prevent ribosomal binding to the TRICH-encoding
transcript.
XII. Expression of TRICH
Expression and purification of TRICH is achieved using bacterial or virus-
based expression
systems. For expression of TRICH in bacteria, cDNA is subcloned into an
appropriate vector
containing an antibiotic resistance gene and an inducible promoter that
directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the
trp-lac (tac) hybrid
promoter and the TS or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21 (DE3).
Antibiotic resistant bacteria express TRICH upon induction with isopropyl beta-
D-
thiogalactopyranoside (IPTG). Expression of TRICH in eukaryotic cells is
achieved by infecting
insect or mammalian cell lines with recombinant Auto~ra~hica californica
nuclear polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding TRICH by either homologous recombination or
bacterial-mediated
transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect Spodoptera fru~iperda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus. (See Engelhard, E.K. et
al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)
Hum. Gene Ther.
7:1937-1945.)
In most expression systems, TRICH is synthesized as a fusion protein with,
e.g., glutathione
S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His,
permitting rapid, single-step,
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-kilodalton
enzyme from Schistosoma japonicum, enables the purification of fusion proteins
on immobilized
glutathione under conditions that maintain protein activity and antigenicity
(Amersham Pharmacia
Biotech). Following purification, the GST moiety can be proteolytically
cleaved from TRICH at
specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity purification
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using commercially available monoclonal and polyclonal anti-FLAG antibodies
(Eastman Kodak). 6-
His, a stretch of six consecutive histidine residues, enables purification on
metal-chelate resins
(QIAGEN). Methods for pxotein expression and purification are discussed in
Ausubel (1995, supra,
ch. 10 and 16). Purified TRICH obtained by these methods can be used directly
in the assays shown
in Examples XVI, XVII, and XVITI, where applicable.
XIII. Functional Assays
TRICH function is assessed by expressing the sequences encoding TRICH at
physiologically
elevated levels in mammalian cell culture systems. cDNA is subcloned into a
mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice
include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad ~'A),
both of which
contain the cytomegalovirus promoter. 5-10 ~cg of recombinant vector are
transiently transfected into
a human cell line, for example, an endothelial or hematopoietic cell line,
using either liposome
formulations or electroporation. 1-2 ~g of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker pxotein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
eDNA 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 C~tometry, 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
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analysis or microarray techniques.
XIV. 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 rabbits and to produce antibodies using standard protocols.
Alternatively, the TRICH amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions axe well
described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
KLH (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) to
increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are
immunized with the
oligopeptide-KLH 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.
XV. Purification of Naturally Occurring TRICH Using Specific Antibodies
Naturally occurring or recombinant TRICH is substantially purified by
immunoaffinity
chromatography using antibodies specific for TRICH. An immunoaffinity column
is constructed by
covalently coupling anti-TRICH antibody to an activated chromatographic resin,
such as
CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the
resin is
blocked and washed according to the manufacturer's instructions.
Media containing TRICH are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of TRICH (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/TRICH binding (e.g., a buffer of pH 2 to pH 3, or a high
concentration of a chaotrope, such
as urea or thiocyanate ion), and TRICH is collected.
XVI. Identification of Molecules Which Interact with TRICH
Molecules which interact with TRICH may include transporter substrates,
agonists or
antagonists, modulatory proteins such as G(3y proteins (Reimann, su ra) or
proteins involved in TRICH
localization or clustering such as MAGUKs (Craven, supra). TRICH, or
biologically active fragments
thereof, are labeled with lzsl Bolton-Hunter reagent. (See, e.g., Bolton A.E.
and W.M. Hunter (1973)
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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 0. 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 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, 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.
XVII. 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 eukaxyotic expression vector encoding TRICH. Eukaryotic
expression vectors are
commercially available, and the techniques to introduce them into cells are
well known to those skilled
in the art. A second plasmid which expresses any one of a number of marker
genes, such as 13-
galactosidase, is co-transformed into the cells to allow rapid identification
of those cells which have
taken up and expressed the foreign DNA. The cells are incubated for 48-72
hours after
transformation under conditions appropriate for the cell line to allow
expression and accumulation of
TRICH and 13-galactosidase.
Transformed cells expressing 13-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 cation conductance relative to control cells. The
contribution of TRICH to
CA 02410084 2002-11-20
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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 Xenopus laevis oocyte membrane using the two-electrode voltage-
clamp technique (Ishi et
al., supra; Jegla, T. and L. Salkoff (1997) J. Neurosci. 17:32-44). TRICH is
subcloned into an
appropriate Xenopus oocyte expression vector, such as pBF, and 0.5-5 ng of
mRNA is injected into
mature stage IV oocytes. Injected oocytes are incubated at 18°C for 1-5
days. Inside-out
macropatches are excised into an intracellular solution containing 116 mM K-
gluconate, 4 mM KCI,
and 10 mM Hepes (pH 7.2). The intracellular solution is supplemented with
varying concentrations of
the TRICH mediator, such as CAMP, cGMP, or Ca+2 (in the form of CaCI~, 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.
In particular the activity of TRICH-10 is measured as ration conductance in
the presence of
heat, the activity of TRICH-12 is measured as anion conductance in the
presence of GABA, the
activity of TRICH-13 is measured as Nay conductance, the activity of TRICH-21
is measured as
voltage-gated Cl- conductance, the activity of TRICH-22 is measured as Ca2+
conductance, the
activity of TRICH-24 is measured as voltage-gated Ca2+ conductance, the
activity of TRICH-26 is
measured as K+ conductance in the presence of cyclic nucleotides, and the
activity of TRICH-27 is
measured as Cl- conductance.
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 NaCI,
2.5 mM KCI, 1mM CaCl2,
1mM MgCl2, 1mM Na2HP04, 5 mM Hepes, 3.8 mM NaOH , 50~g/ml gentamycin, pH 7.8)
to allow
expression of TRICH. Oocytes are then transferred to standard uptake medium
(100mM NaCI, 2
mM KCI, 1mM CaCl2, 1mM MgCl2, 10 mM Hepes/T'ris pH 7.5). Uptake of various
substrates (e.g.,
amino acids, sugars, drugs, ions, and neurotransmitters) is initiated by
adding labeled substrate (e.g.
radiolabeled with 3H, fluorescently labeled with rhodamine, etc.) to the
oocytes. After incubating for
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. In particular, test substrates include organic
rations for TRICH-9,
carnitine and acylcarnitine for TRICH-11, galactose and other sugars for TRICH-
14, glucose for
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TRICH-15, monocarboxylate for TRICH-16, canons for TRICH-17, estramustine and
related drugs
for TRICH-18, amino acids for TRICH-19, glucose for TRICH-20, sugars for TRICH-
23, and glucose
or fructose for TRICH-25.
In the alternative, TRICH transport activity can be demonstrated through the
use of a ligand
mixing assay that is used to measure transport from early to late endosomal
compartments in X. laevis
oocytes. Ovaries are dissected from adult female X. laevis, and oocytes are
isolated.
(Mukhopadhyay A. et al. (1997) J. Cell. Biol. 136(6): 1227-1237). Oocytes are
pulsed with 2mglml
avidin for Shrs at 18~ C, washed, then incubated for 16 hrs to allow avidin to
transport to a late
compartment. The oocytes are then incubated with lmglml biotin-horseradish
peroxidase (HRP) for
30 minutes at 18 ° C to label early endocytic compartments. Varying
amounts of TRICH are injected
into the oocytes, and the oocytes axe incubated at 18 ° C. Oocytes are
collected at several time points
after TRICH injection, washed, and lysed in 1001 of phosphate-buffered saline
containing 0.3% Triton
X-100, 0.2% methylbenzethorium chloride, and 400 ~glml of BSA-biotin as a
scavenger. Finally, the
Iysates are centrifuged for 30 seconds in a microfuge, and the avidin-biotin
complexes are
immunoprecipitated using anti-avidin antibody-coated plates by incubation at 4
°C overnight, The
plates are washed at least 5 times to remove unbound proteins. Transport from
the early endosomes
to the late compartments is quantified by measuring the amount of
immunoprecipitated HRP;
increased transport due to TRICH is quantitated by comparison with control
oocytes. Potential
inhibitors of proton-dependent histidine transport such as dipeptides and
tripeptides can subsequently
be tested in the expression system described above (Yamashita, T. et al.
(1997) J. Cell. Biol. l 36(6):
1227-1237).
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 32P 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.
XVIII. 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 XVII.
Alternatively, ion
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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
Ca2+ indicator Fluo-4 AM, sodium-sensitive dyes such as SBFI and sodium green,
or the CI- 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). DiBACø equilibrates between the extracellular solution and
cellular sites
l0 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 methods and systems of
the invention
will be apparent to those skilled in the art without departing from the scope
and spirit of the invention.
Although the invention has been described in connection with certain
embodiments, it should be
understood that the invention as claimed should not be unduly limited to such
specific embodiments.
Indeed, various modifications of the described modes for carrying out the
invention which are obvious
to those skilled in molecular biology or related fields are intended to be
within the scope of the
following claims.
88
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CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
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CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
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132
CA 02410084 2002-11-20
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<110> INCYTE GENOMICS, INC.
THORNTON, Michael
WALIA, Narinder K.
YUE, Henry
NGUYEN, Danniel B.
LAL, Preeti
GANDHI, Ameena R.
TRIBOULEY, Catherine M.
YAO, Monique G.
RAMKUMAR, JayalaHIni
AU-YOUNG, Janice
LU, Yan
TANG, Y. Tom
AZIMZAI, Yalda
BRUNS, Christopher M.
GRIFFIN, Jennifer A.
YANG, Junming
BAUGHN, Mariah R.
SANJANWALA, Madhu S.
RAUMANN, Brigitte E.
LEE, Ernestine A.
HAFALIA, April
GREENE, Barrie D.
KHAN, Farrah A.
KEARNEY, Liam
ELLIOTT, Vicky S.
SEILHAMER, Jeffrey J.
POLICKY, Jennifer L.
BOROWSKY, Mark L.
BURFORD, Neil
DING, Li
LU, Dyung Aina M.
HILLMAN, Jennifer L.
<120> TRANSPORTERS AND ION CHANNELS
<130> PI-0103 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/208,424; 60/209,001; 60/210,588; 60/212,335; 60/213,747; 60/215,391
<151> 2000-05-26; 2000-06-01; 2000-06-08; 2000-06-16; 2000-06-22; 2000-06-29
<160> 54
<170> PERL Program .
<210> 1
<211> 842
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7475353CD1
<400> 1
Met Val Thr Val Gly Asn Tyr Cys Glu Ala Glu Gly Pro Val Gly
1 5 10 15
Pro Ala Trp Met Gln Asp Gly Leu Ser Pro Cys Phe Phe Phe Thr
20 25 30
Leu Val Pro Ser Thr Arg Met Ala Leu Gly Thr Leu Ala Leu Val
35 40 45
1/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
Leu Ala Leu Pro Cys Arg Arg Arg Glu Arg Pro Ala Gly Ala Asp
50 55 60
Ser Leu Ser Trp Gly Ala Gly Pro Arg Ile Ser Pro Tyr Val Leu
65 70 75
Gln Leu Leu Leu Ala Thr Leu Gln Ala Ala Leu Pro Leu Ala Gly
80 85 90
Leu Ala Gly Arg Val Gly Thr Ala Arg Gly Ala Pro Leu Pro Ser
95 100 105
Tyr Leu Leu Leu Ala Ser Val Leu Glu Ser Leu Ala Gly Ala Cys
110 115 120
Gly Leu Trp Leu Leu Val Val Glu Arg Ser Gln Ala Arg Gln Arg
125 130 135
Leu Ala Met Gly Ile Trp Ile Lys Phe Arg His Ser Pro Gly Leu
140 145 150
Leu Leu Leu Trp Thr Val Ala Phe Ala Ala Glu Asn Leu Ala Leu
155 160 165
Val Ser Trp Asn Ser Pro Gln Trp Trp Trp Ala Arg Ala Asp Leu
170 175 180
Gly Gln Gln Val Gln Phe Ser Leu Trp Val Leu Arg Tyr Val Val
185 190 195
Ser Gly Gly Leu Phe Val Leu Gly Leu Trp Ala Pro Gly Leu Arg
200 205 210
Pro Gln Ser Tyr Thr Leu Gln Val His Glu Glu Asp Gln Asp Val
215 220 225
Glu Arg Ser Gln Val Arg Ser Ala Ala Gln Gln Ser Thr Trp Arg
230 235 240
Asp Phe Gly Arg Lys Leu Arg Leu Leu Ser Gly Tyr Leu Trp Pro
245 250 255
Arg Gly Ser Pro Ala Leu Gln Leu Val Val Leu Ile Cys Leu Gly
260 265 270
Leu Met G1y Leu Glu Arg Ala Leu Asn Val Leu Val Pro Ile Phe
275 280 285
Tyr Arg Asn Ile Val Asn Leu Leu Thr Glu Lys Ala Pro Trp Asn
290 295 300
Ser Leu Ala Trp Thr Val Thr Ser Tyr Val Phe Leu Lys Phe Leu
305 310 315
Gln Gly Gly Gly Thr Gly Ser Thr Gly Phe Val Ser Asn Leu Arg
320 325 330
Thr Phe Leu Trp Ile Arg Val Gln Gln Phe Thr Ser Arg Arg Val
335 340 345
Glu Leu Leu Ile Phe Ser His Leu His Glu Leu Ser Leu Arg Trp
350 355 360
His Leu Gly Arg Arg Thr Gly Glu Val Leu Arg Ile Ala Asp Arg
365 370 375
Gly Thr Ser Ser Val Thr Gly Leu Leu Ser Tyr Leu Val Phe Asn
380 385 390
Val Ile Pro Thr Leu Ala Asp Ile Ile Ile Gly Ile Ile Tyr Phe
395 400 405
Ser Met Phe Phe Asn Ala Trp Phe Gly Leu Ile Val Phe Leu Cys
410 415 420
Met Ser Leu Tyr Leu Thr Leu Thr Ile Val Val Thr Glu Trp Arg
425 430 435
Thr Lys Phe Arg Arg Ala Met Asn Thr Gln Glu Asn Ala Thr Arg
440 445 450
Ala Arg Ala Val Asp Ser Leu Leu Asn Phe Glu Thr Val Lys Tyr
455 460 465
Tyr Asn Ala Glu Ser Tyr Glu Val Glu Arg Tyr Arg Glu Ala Ile
470 475 480
Ile Lys Tyr Gln Gly Leu Glu Trp Lys Ser Ser Ala Ser Leu Val
485 490 495
Leu Leu Asn Gln Thr Gln Asn Leu Val Ile Gly Leu Gly Leu Leu
500 505 510
Ala Gly Ser Leu Leu Cys Ala Tyr Phe Val Thr Glu Gln Lys Leu
2/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
515 520 525
Gln Val Gly Asp Tyr Val Leu Phe Gly Thr Tyr Ile Ile Gln Leu
530 535 540
Tyr Met Pro Leu Asn Trp Phe Gly Thr Tyr Tyr Arg Met Ile Gln
545 550 555
Thr Asn Phe Ile Asp Met Glu Asn Met Phe Asp Leu Leu Lys Glu
560 565 570
Glu Thr Glu Val Lys Asp Leu Pro Gly Ala Gly Pro Leu Arg Phe
575 580 585
Gln Lys Gly Arg Ile Glu Phe Glu Asn Val His Phe Ser Tyr Ala
590 595 600
Asp Gly Arg Glu Thr Leu Gln Asp Val Ser Phe Thr Val Met Pro
605 610 615
Gly Gln Thr Leu Ala Leu Val Gly Pro Ser Gly Ala Gly Lys Ser
620 625 630
Thr Ile Leu Arg Leu Leu Phe Arg Phe Tyr Asp Ile Ser Ser Gly
635 640 645
Cys Ile Arg Ile Asp Gly Gln Asp Ile Ser Gln Val Thr Gln Ala
650 655 660
Ser Leu Arg Ser His Ile Gly Val Val Pro Gln Asp Thr Val Leu
665 670 675
Phe Asn Asp Thr Ile Ala Asp Asn Ile Arg Tyr Gly Arg Val Thr
680 685 690
Ala Gly Asn Asp Glu Val Glu Ala Ala Ala Gln Ala Ala Gly Ile
695 700 705
His Asp Ala Ile Met Ala Phe Pro Glu Gly Tyr Arg Thr Gln Val
710 715 720
Gly Glu Arg Gly Leu Lys Leu Ser Gly Gly Glu Lys.Gln Arg Val
725 730 735
Ala Ile Ala Arg Thr Ile Leu Lys Ala Pro Gly Ile Ile Leu Leu
740 745 750
Asp Glu Ala Thr Ser Ala Leu Asp Thr Ser Asn Glu Arg Ala Ile
755 760 765
Gln Ala Ser Leu Ala Lys Val Cys Ala Asn Arg Thr Thr Ile Val
770 775 780
Val Ala His Arg Leu Ser Thr Val Val Asn Ala Asp Gln Ile Leu
785 790 795
Val Ile Lys Asp Gly Cys Ile Val Glu Arg G1y Arg His Glu Ala
800 805 810
Leu Leu Ser Arg Gly Gly Val Tyr Ala Asp Met Trp Gln Leu Gln
815 820 825
Gln Gly Gln Glu Glu Thr Ser Glu Asp Thr Lys Pro Gln Thr Met
830 835 840
Glu Arg
<210> 2
<211> 461
<212> PRT
<213> Homo sapiens
<220>
<221> misc_~eature
<223> Incyte ID No: 3107278CD1
<400> 2
Met Pro Gly Arg Ser Ile Ser Leu Ser Ser Pro Tyr Trp Trp Ile
1 5 10 15
Asn Leu Trp Tyr Leu Ile Thr Gly Cys Ile Ala Asp Trp Val Gly
20 25 30
Arg Arg Pro Val Leu Leu Phe Ser Ile Ile Phe Ile Leu Ile Phe
35 40 45
Gly Leu Thr Val Ala Leu Ser Val Asn Val Thr Met Phe Ser Thr
3/67
Tyr Arg Asn Ile Val Asn Leu Leu Thr Glu Lys Ala Pro Trp
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
50 55 60
Leu Arg Phe Phe Glu Gly Phe Cys Leu Ala Gly Ile Ile Leu Thr
65 70 75
Leu Tyr Ala Leu Arg Ile Glu Leu Cys Pro Pro Gly Lys Arg Phe
80 85 90
Met Ile Thr Met Val Ala Ser Phe Val Ala Met Ala Gly Gln Phe
95 100 105
Leu Met Pro Gly Leu Ala Ala Leu Cys Arg Asp Trp Gln Val Leu
110 115 120
Gln Ala Leu Ile Ile Cys Pro Phe Leu Leu Met Leu Leu Tyr Trp
125 130 135
Ser Ile Phe Pro Glu Ser Leu Arg Trp Leu Met Ala Thr Gln Gln
140 145 150
Phe Glu Ser Ala Lys Arg Leu Ile Leu His Phe Thr Gln Lys Asn
155 160 165
Arg Met Asn Pro Glu Gly Asp Ile Lys Gly Val Ile Pro Glu Leu
170 175 180
Glu Lys Glu Leu Ser Arg Arg Pro Lys Lys Val Cys Ile Val Lys
185 190 195
Val Val Gly Thr Arg Asn Leu Trp Lys Asn Ile Val Val Leu Cys
200 205 210
Val Asn Ser Leu Thr Gly Tyr Gly Ile His His Cys Phe Ala Arg
215 220 225
Ser Met Met Gly His Glu Val Lys Val Pro Leu Leu Glu Asn Phe
230 235 240
Tyr Ala Asp Tyr Tyr Thr Thr Ala Ser Ile Ala Leu Val Ser Cys
245 250 255
Leu Ala Met Cys Val Val Val Arg Phe Leu Gly Arg Arg Gly Gly
260 265 270
Leu Leu Leu Phe Met Ile Leu Thr Ala Leu Ala Ser Leu Leu Gln
275 280 285
Leu Gly Leu Leu Asn Leu Ile Gly Lys Tyr Ser Gln His Pro Asp
290 295 300
Ser Gly Met Ser Asp Ser Val Lys Asp Lys Phe Ser Ile Ala Phe
305 310 315
Ser Ile Val Gly Met Phe Ala Ser His Ala Val Gly Ser Leu Ser
320 325 330
Val Phe Phe Cys Ala Glu Ile Thr Pro Thr Val Ile Arg Cys Gly
335 340 345
Gly Leu Gly Leu Val Leu Ala Ser Ala Gly Phe Gly Met Leu Thr
350 355 360
Ala Pro Ile Ile Glu Leu His Asn Gln Lys Gly Tyr Phe Leu His
365 370 375
His Ile Ile Phe Ala Cys Cys Thr Leu Ile Cys Ile Ile Cys Ile
380 385 390
Leu Leu Leu Pro Glu Ser Arg Asp Gln Asn Leu Pro Glu Asn Ile
395 400 405
Ser Asn Gly Glu His Tyr Thr Arg Gln Pro Leu Leu Pro His Lys
410 415 420
Lys Gly Glu Gln Pro Leu Leu Leu Thr Asn Ala Glu Leu Lys Asp
425 430 435
Tyr Ser Gly Leu His Asp Ala Ala Ala Ala Gly Asp Thr Leu Pro
44b 445 450
Glu Gly Ala Thr Ala Asn Gly Met Lys Ala Met
455 460
<210> 3
<211> 485
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
4167
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
<223> Incyte ID No: 7473394CD1
<400> 3
Met Gln Asn Ile Thr Lys Glu Phe Gly Thr Phe Lys Ala Asn Asp
1 5 10 15
Asn Ile Asn Leu Gln Val Lys Ala Gly Glu Ile His Ala Leu Leu
20 25 30
Gly Glu Asn Gly Ala Gly Lys Ser Thr Leu Met Asn Val Leu Ser
35 40 45
Gly Leu Leu Glu Pro Thr Ser Gly Lys Ile Leu Met Arg Gly Lys
50 55 60
Glu Val Gln Ile Thr Ser Pro Thr Lys Ala Asn Gln Leu Gly Ile
65 70 75
Gly Met Val His Gln His Phe Met Leu Val Asp Ala Phe Thr Val
80 85 90
Thr Glu Asn Ile Val Leu Gly Ser Glu Pro Ser Arg Ala Gly Met
95 100 105
Leu Asp His Lys Lys Ala Arg Lys Glu Ile Gln Lys Val Ser Glu
110 115 120
Gln Tyr Gly Leu Ser Val Asn Pro Asp Ala Tyr Val Arg Asp Ile
125 130 135
Ser Val Gly Met Glu Gln Arg Val Glu Ile Leu Lys Thr Leu Tyr
140 145 150
Arg Gly Ala Asp Val Leu Ile Phe Asp Glu Pro Thr Ala Val Leu
155 160 165
Thr Pro Gln Glu Ile Asp Glu Leu Ile Val Ile Met Lys Glu Leu
170 175 180
Val Lys Glu Gly Lys Ser Ile Ile Leu Ile Thr His Lys Leu Asp
185 190 195
Glu Ile Lys Ala Val Ala Asp Arg Cys Thr Val Ile Arg Arg Gly
200 205 210
Lys Gly Ile Gly Thr Val Asn Val Lys Asp Val Thr Ser Gln Gln
215 220 225
Leu Ala Asp Met Met Val Gly Arg Ala Val Ser Phe Lys Thr Met
230 235 240
Lys Lys Glu Ala Lys Pro Gln Glu Va1 Val Leu Ser Ile Glu Asn
245 250 255
Leu Val Val Lys Glu Asn Arg Gly Leu Glu Ala Val Lys Asn Leu
260 265 270
Asn Leu G1u Val Arg Ala Gly Glu Val Leu Gly Ile Ala Gly Ile
275 280 285
Asp Gly Asn Gly Gln Ser Glu Leu Ile Gln Ala Leu Thr Gly Leu
290 295 300
Arg Lys Ala Glu Ser Gly His Ile Lys Leu Lys Gly Glu Asp Ile
305 310 315
Thr Asn Lys Lys Pro Arg Lys Ile Thr Glu His Gly Val Gly His
320 325 330
Val Pro Glu Asp Arg His Lys Tyr Gly Leu Val Leu Asp Met Thr
335 340 345
Leu Ser Glu Asn Ile Ala Leu Gln Thr Tyr His Gln Lys Pro Tyr
350 355 360
Ser Lys Asn Gly Met Leu Asn Tyr Ser Val Ile Asn Glu His Ala
365 370 375
Arg Glu Leu Ile Glu Glu Tyr Asp Val Arg Thr Thr Asn Glu Leu
380 385 390
Val Pro Ala Lys Ala Leu Ser Gly Gly Asn Gln Gln Lys Ala Ile
395 400 405
Ile Ala Arg Ile Val Asp Arg Asp Pro Asp Leu Leu Ile Val Ala
410 415 420
Asn Pro Thr Arg Gly Leu Asp Val Gly Ala Ile Glu Phe Ile His
425 430 435
Lys Arg Leu Ile Glu Gln Arg Asp Lys Tyr Lys Ala Val Leu Leu
440 445 450
5/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
Ile Ser Phe Glu Leu Glu Glu Ile Leu Asn Val Ser Asp Arg Ile
455 460 465
Ala Val Ile His Glu Gly Glu Ile Val Gly Ile Val Asp Pro Lys
470 475 480
Glu Thr Ser Glu Asn
485
<210> 4
<211> 301
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473900CD1
<400> 4
Met Lys Ser Gly Pro Gly Ile Gln Ala Ala Ile Asp Leu Thr Ala
1 5 10 15
Gly Ala Ala Gly Gly Thr Ala Cys Val Leu Thr Gly Gln Pro Phe
20 25 30
Asp Thr Ile Lys Val Lys Met Gln Thr Phe Pro Asp Leu Tyr Lys
35 40 45
Gly Leu Thr Asp Cys Phe Leu Lys Thr Tyr Ala Gln Val Gly Leu
50 55 60
Arg Gly Phe Tyr Lys Gly Thr Gly Pro Ala Leu Met Ala Tyr Val
65 70 75
Ala Glu Asn Ser Val Leu Phe Met Cys Tyr Gly Phe Cys Gln Gln
80 85 90
Phe Val Arg Lys Val Ala Gly Met Asp Lys Gln Ala Lys Leu Ser
95 100 105
Asp Leu Gln Thr Ala Ala Ala Gly Ser Phe Ala Ser Ala Phe Ala
110 115 120
Ala Leu Ala Leu Cys Pro Thr Glu Leu Val Lys Cys Arg Leu Gln
125 130 135
Thr Met Tyr Glu Met Glu Met Ser Gly Lys Ile Ala Lys Ser His
140 145 150
Asn Thr Ile Trp Ser Val Val Lys Gly Ile Leu Lys Lys Asp Gly
155 160 165
Pro Leu Gly Phe Tyr His Gly Leu Ser Ser Thr Leu Leu Gln Glu
170 175 180
Val Pro Gly Tyr Phe Phe Phe Phe Gly Gly Tyr Glu Leu Ser Arg
185 190 195
Ser Phe Phe Ala Ser Gly Arg Ser Lys Asp Glu Leu Gly Pro Val
200 205 210
His Leu Met Leu Ser Gly Gly Val Ala Gly Ile Cys Leu Trp Leu
215 220 225
Val Val Phe Pro Val Asp Cys Ile Lys Ser Arg Ile Gln Val Leu
230 235 240
Ser Met Tyr Gly Lys Gln Ala Gly Phe Ile Gly Thr Leu Leu Ser
245 250 255
Val Val Arg Asn Glu Gly Ile Val Ala Leu Tyr Ser Gly Leu Lys
260 265 270
Ala Thr Met Ile Arg Ala Ile Pro Ala Asn Gly Ala Leu Phe Val
275 280 285
Ala Tyr Glu Tyr Ser Arg Lys Met Met Met Lys Gln Leu Glu Ala
290 295 300
Tyr
<210> 5
<211> 304
<212> PRT
6/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7475045CD1
<400> 5
Met Glu Thr Val Pro Pro Ala Val Asp Leu Val Leu Gly Ala Ser
1 5 10 15
Ala Cys Cys Leu Ala Cys Val Phe Thr Asn Pro Leu Glu Val Val
20 25 30
Lys Thr Arg Leu Gln Leu Gln Gly Glu Leu Gln Ala Arg Gly Thr
35 40 45
Tyr Pro Arg Pro Tyr His Gly Phe Ile Ala Ser Val Ala Ala Val
50 55 60
Ala Arg Ala Asp Gly Leu Trp Gly Leu Gln Lys Gly Leu Ala Ala
65 70 75
Gly Leu Leu Tyr Gln Gly Leu Met Asn Gly Val Arg Phe Tyr Cys
80 85 90
Tyr Ser Leu Ala Cys Gln Ala Gly Leu Thr Gln Gln Pro Gly Gly
95 100 105
Thr Val Val Ala Gly Ala Val Ala Gly Ala Leu Gly Ala Phe Val
110 115 120
Gly Ser Pro Ala Tyr Leu Ile Lys Thr Gln Leu Gln Ala Gln Thr
125 130 135
Val Ala Ala Val Ala Val Gly His Gln His Asn His Gln Thr Val
140 145 150
Leu Gly Ala Leu Glu Thr Ile Trp Arg Gln Gln Gly Leu Leu Gly
155 160 165
Leu Trp Gln Gly Val Gly Gly Ala Va1 Pro Arg Val Met Val Gly
170 175 180
Ser Ala Ala Gln Leu Ala Thr Phe Ala Ser Ala Lys Ala Trp Val
185 190 195
Gln Lys Gln Gln Trp Leu Pro Glu Asp Ser Trp Leu Val Ala Leu
200 205 210
Ala Gly Gly Met Ile Ser Ser Tle A1a Val Val Val Val Met Thr
215 220 225
Pro Phe Asp Val Val Ser Thr Arg Leu Tyr Asn Gln Pro Val Asp
230 235 240
Thr Ala Gly Arg Gly Gln Leu Tyr Gly Gly Leu Thr Asp Cys Met
245 250 255
Val Lys Ile Trp Arg Gln Glu G1y Pro Leu Ala Leu Tyr Lys Gly
260 265 270
Leu Gly Pro Ala Tyr Leu Arg Leu Gly Pro His Thr Ile Leu Ser
275 280 285
Met Leu Phe Trp Asp Glu Leu Arg Lys Leu Ala Gly Arg Ala Gln
290 295 300
His Lys Gly Thr
<210> 6
<211> 278
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7475611CD1
<400> 6
Met Ser Ala Lys Val Leu Leu Ser Thr Glu His Leu Tyr Ala Thr
1 5 10 15
His Pro Gly Arg Pro Met Val Leu Thr Asp Val Asn Val Ser Phe
7/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
20 25 30
Arg Ala Gly Val Arg Val Ala Ile Leu Gly Ala Asn Gly Ser Gly
35 40 45
Lys Thr Thr Leu Met Arg Cys Leu Ser Gly Ser Leu Lys Pro Ala
50 55 60
Lys Gly His Val Lys Arg Gly Asp Ile Val Val Ser Tyr Gly Arg
65 70 75
Ala Gln Leu Arg Glu His Arg Arg Ala Val Gln Leu Val Leu Gln
80 85 90
Asp Pro Asp Asp Gln Leu Phe Ser Ala Asp Val Ser Gln Asp Val
95 100 105
Ser Phe Gly Pro Met Asn Met Gly Leu Lys Val Asp Glu Val Arg
110 115 120
Asp Arg Val Ser Glu Ser Leu Glu Leu Leu Gly Ala Ser His Leu
125 130 135
Ala Glu Arg Ala Thr Tyr Gln Leu Ser Tyr Gly Glu Arg Lys Arg
140 145 150
Val Ala Val Ala Gly Ala Val Ala Met Arg Pro Asp Leu Leu Leu
155 160 165
Leu Asp Glu Pro Thr Ala Gly Leu Asp Pro Val Gly Val Thr Gln
170 175 180
Met Leu Glu Ala Leu Asp Arg Leu Arg Asp His Gly Thr Thr Val
185 190 195
Ala Met Ala Thr His Asp Val Asp Leu Ala Leu Ala Trp Ala Gln
200 205 210
Glu Ala Leu Val Val Val Asp Gly Gln Val His Gln Gly Pro Ile
215 220 225
Gly Glu Leu Leu Ala Asp Ala Asp Thr Val Gly Arg Ala His Leu
230 235 240
His Leu Pro Trp Pro Leu Glu Leu Ala Arg Arg Leu Gly Val Arg
245 250 255
Asp Leu Pro Arg Thr Met Asp Asp Val Val Ala Met Leu Ser Asp
260 265 270
Asn Pro Ser Pro Ala Pro Ser Asn
275
<210> 7
<211> 673
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7475617CD1
<400> 7
Met Glu Glu Leu A1a Thr Glu Lys Glu Ala Glu Glu Ser His Arg
1 5 10 15
Gln Asp Ser Val Ser Leu Leu Thr Phe Ile Leu Leu Leu Thr Leu
20 25 30
Thr Ile Leu Thr Ile Trp Leu Phe Lys His Arg Arg Val Arg Phe
35 40 45
Leu His Glu Thr Gly Leu Ala Met Ile Tyr Gly Leu Ile Val Gly
50 55 60
Val Ile Leu Arg Tyr Gly Thr Pro Ala Thr Ser Gly Arg Asp Lys
65 70 75
Ser Leu Ser Cys Thr Gln Glu Asp Arg Ala Phe Ser Thr Leu Leu
80 85 90
Val Asn Val Ser Gly Lys Phe Phe Glu Tyr Thr Leu Lys Gly Glu
95 100 105
I1e Ser Pro Gly Lys Ile Asn Ser Va1 Glu Gln Asn Asp Met Leu
11.0 115 120
Arg Lys Val Thr Phe Asp Pro Glu Val Phe Phe Asn Ile Leu Leu
8/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
125 130 135
Pro Pro Ile Ile Phe His Ala Gly Tyr Ser Leu Lys Lys Arg His
140 145 150
Phe Phe Arg Asn Leu Gly Ser Ile Leu Ala Tyr A1a Phe Leu Gly
155 160 165
Thr Ala Val Ser Cys Phe Ile Ile Gly Asn Leu Met Tyr Gly Val
170 175 180
Val Lys Leu Met Lys Ile Met Gly Gln Leu Ser Asp Lys Phe Tyr
185 190 195
Tyr Thr Asp Cys Leu Phe Phe Gly Ala Ile Ile Ser Ala Thr Asp
200 205 210
pro Val Thr Val Leu Ala Ile Phe Asn Glu Leu His Ala Asp Val
215 220 225
Asp Leu Tyr Ala Leu Leu Phe Gly Glu Ser Val Leu Asn Asp Ala
230 235 240
Val Ala Ile Val Leu Ser Ser Ser Ile Val Ala Tyr Gln Pro Ala
245 250 255
Gly Leu Asn Thr His Ala Phe Asp Ala Ala Ala Phe Phe Lys Ser
260 265 270
Val Gly Ile Phe Leu Gly Ile Phe Ser Gly Ser Phe Thr Met Gly
275 280 285
Ala Val Thr Gly Val Val Thr Ala Leu Val Thr Lys Phe Thr Lys
290 295 300
Leu His Cys Phe Pro Leu Leu Glu Thr Ala Leu Phe Phe Leu Met
305 310 315
Ser Trp Ser Thr Phe Leu Leu Ala Glu Ala Cys Gly Phe Thr Gly
320 325 330
Val Val Ala Val Leu Phe Cys Gly Ile Thr Gln Ala His Tyr Thr
335 340 345
Tyr Asn Asn Leu Ser Val Glu Ser Arg Ser Arg Thr Lys Gln Leu
350 355 360
Phe Glu Val Leu His Phe Leu Ala Glu Asn Phe Ile Phe Ser Tyr
365 370 375
Met Gly Leu Ala Leu Phe Thr Phe Gln Lys His Val Phe Ser Pro
380 385 390
Ile Phe Ile Ile Gly Ala Phe Val Ala Ile Phe Leu Gly Arg Ala
395 400 405
Ala His Ile Tyr Pro Leu Ser Phe Phe Leu Asn Leu Gly Arg Arg
410 415 420
His Lys Ile Gly Trp Asn Phe Gln His Met Met Met Phe Ser Gly
425 430 435
Leu Arg Gly Ala Met Ala Phe Ala Leu Ala Ile Arg Asp Thr Ala
440 445 450
Ser Tyr Ala Arg Gln Met Met Phe Thr Thr Thr Leu Leu Ile Val
455 460 465
Phe Phe Thr Val Trp Ile Ile Gly Gly Gly Thr Thr Pro Met Leu
470 475 480
Ser Trp Leu Asn Ile Arg Val Gly Val Glu Glu Pro Ser Glu Glu
485 490 495
Asp Gln Asn Glu His His Trp Gln Tyr Phe Arg Val Gly Val Asp
500 505 510
Pro Asp Gln Asp Pro Pro Pro Asn Asn Asp Ser Phe Gln Val Leu
515 520 525
Gln Gly Asp Gly Pro Asp Ser Ala Arg Gly Asn Arg Thr Lys Gln
530 535 540
Glu Ser Ala Trp Ile Phe Arg Leu Trp Tyr Ser Phe Asp His Asn
545 550 555
Tyr Leu Lys Pro Ile Leu Thr His Ser Gly Pro Pro Leu Thr Thr
560 565 570
Thr Leu Pro Ala Trp Cys Gly Leu Leu Ala Arg Cys Leu Thr Ser
575 580 585
Pro Gln Val Tyr Asp Asn Gln Glu Pro Leu Arg Glu Glu Asp Ser
590 595 600
9/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
Asp Phe Ile Leu Thr Glu Gly Asp Leu Thr Leu Thr Tyr Gly Asp
605 610 615
Ser Thr Val Thr Ala Asn Gly Ser Ser Ser Ser His Thr Ala Ser
620 625 630
Thr Ser Leu Glu Gly Ser Arg Arg Thr Lys Ser Ser Ser Glu Glu
635 640 645
Val Leu Glu Arg Asp Leu Gly Met Gly Asp Gln Lys Va1 Ser Ser
650 655 660
Arg Gly Thr Arg Leu Val Phe Pro Leu Glu Asp Asn Ala
665 670
<210> 8
<211> 576
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473314CD1
<400> 8
Met Glu Gly Ser Gly Gly Gly Ala Gly Glu Arg Ala Pro Leu Leu
1 5 . 10 15
Gly Ala Arg Arg Ala Ala Ala Ala Ala Ala Ala Gly Ala Phe Ala
20 25 30
Gly Arg Arg Ala Ala Cys Gly Ala Val Leu Leu Thr Glu Leu Leu
35 40 45
Glu Arg Ala Ala Phe Tyr Gly Ile Thr Ser Asn Leu Val Leu Phe
50 55 60
Leu Asn Gly Ala Pro Phe Cys Trp Glu Gly Ala Gln Ala Ser Glu
65 70 75
Ala Leu Leu Leu Phe Met Gly Leu Thr Tyr Leu Gly Ser Pro Phe
80 85 90
Gly Gly Trp Leu Ala Asp Ala Arg Leu Gly Arg Ala Arg Ala Ile
95 100 105
Leu Leu Ser Leu Ala Leu Tyr Leu Leu Gly Met Leu A1a Phe Pro
110 115 120
Leu Leu Ala Ala Pro Ala Thr Arg Ala Ala Leu Cys Gly Ser Ala
125 130 135
Arg Leu Leu Asn Cys Thr Ala Pro Gly Pro Asp Ala Ala Ala Arg
140 145 150
Cys Cys Ser Pro Ala Thr Phe Ala Gly Leu Val Leu Val Gly Leu
155 160 165
Gly Val Ala Thr Val Lys Ala Asn Ile Thr Pro Phe Gly Ala Asp
170 . 175 180
Gln Val Lys Asp Arg Gly Pro Glu Ala Thr Arg Arg Phe Phe Asn
185 190 195
Trp Phe Tyr Trp Ser Ile Asn Leu Gly Ala Ile Leu Ser Leu Gly
200 205 210
Gly Ile Ala Tyr I1e Gln Gln Asn Val Ser Phe Val Thr Gly Tyr
215 220 225
Ala Ile Pro Thr Val Cys Val Gly Leu Ala Phe Val Ala Phe Leu
230 235 240
Cys Gly Gln Ser Val Phe Ile Thr Lys Pro Pro Asp Gly Ser Ala
245 250 255
Phe Thr Asp Met Phe Lys Ile Leu Thr Tyr Ser Cys Cys Ser Gln
260 265 270
Lys Arg Ser Gly Glu Arg Gln Ser Asn Gly Glu Gly Ile Gly Val
275 280 285
Phe Gln Gln Ser Ser Lys Gln Ser Leu Phe Asp Ser Cys Lys Met
290 295 300
Ser His Gly Gly Pro Phe Thr Glu Glu Lys Val Glu Asp Val Lys
305 310 315
10/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
Ala Leu Val Lys Ile Val Pro Val Phe Leu Ala Leu Ile Pro Tyr
320 325 330
Trp Thr Val Tyr Phe Gln Met Gln Thr Thr Tyr Val Leu Gln Ser
335 340 345
Leu His Leu Arg Ile Pro Glu Ile Ser Asn Ile Thr Thr Thr Pro
350 355 360
His Thr Leu Pro Ala Ala Trp Leu Thr Met Phe Asp Ala Val Leu
365 370 375
Ile Leu Leu Leu Ile Pro Leu Lys Asp Lys Leu Val Asp Pro Ile
380 385 390
Leu Arg Arg His Gly Leu Leu Pro Ser Ser Leu Lys Arg Ile Ala
395 400 405
Val Gly Met Phe Phe Val Met Cys Ser Ala Phe Ala Ala Gly Ile
410 415 420
Leu Glu Ser Lys Arg Leu Asn Leu Val Lys Glu Lys Thr Ile Asn
425 430 435
Gln Thr Ile Gly Asn Val Val Tyr His Ala Ala Asp Leu Ser Leu
440 445 450
Trp Trp Gln Val Pro Gln Tyr Leu Leu Ile Gly Ile Ser Glu Ile
455 460 465
Phe Ala Ser Ile Ala Gly Leu Glu Phe Ala Tyr Ser Ala Ala Pro
470 475 480
Lys Ser Met Gln Ser Ala Ile Met Gly Leu Phe Phe Phe Phe Ser
485 490 495
Gly Val Gly Ser Phe Val Gly Ser Gly Leu Leu Ala Leu Val Ser
500 505 5l0
Ile Lys Ala I12 Gly Trp Met Ser Ser His Thr Asp Phe Gly Asn
5l5 520 525
Ile Asn Gly Cys Tyr Leu Asn Tyr Tyr Phe Phe Leu Leu Ala Ala
530 535 540
Ile Gln Gly Ala Thr Leu Leu Leu Phe Leu Ile Ile Ser Val Lys
545 550 555
Tyr Asp His His Arg Asp His Gln Arg Ser Arg Ala Asn Gly Val
560 565 570
Pro Thr Ser Arg Arg Ala
575
<210> 9
<211> 550
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 70356714CD1
<400> 9
Met Ala Phe Ser Lys Leu Leu Glu Gln Ala Gly Gly Val Gly Leu
1 5 10 15
Phe Gln Thr Leu Gln Val Leu Thr Phe Ile Leu Pro Cys Leu Met
20 25 30
Ile Pro Ser Gln Met Leu Leu Glu Asn Phe Ser Ala Ala Ile Pro
35 40 45
Gly His Arg Cys Trp Thr His Met Leu Asp Asn Gly Ser Ala Val
50 55 , 60
Ser Thr Asn Met Thr Pro Lys Ala Leu Leu Thr Ile Ser Ile Pro
65 70 75
Pro Gly Pro Asn Gln Gly Pro His Gln Cys Arg Arg Phe Arg Gln
80 85 90
Pro Gln Trp Gln Leu Leu Asp Pro Asn Ala Thr Ala Thr Ser Trp
95 100 105
Ser Glu Ala Asp Thr Glu Pro Cys Val Asp Gly Trp Val Tyr Asp
110 115 120
11/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
Arg Ser Val Phe Thr Ser Thr Ile Val Ala Lys Trp Asp Leu Val
125 130 135
Cys Ser Ser Gln Gly Leu Lys Pro Leu Ser Gln Ser Ile Phe Met
140 145 150
Ser Gly Ile Leu Va1 Gly Ser Phe Ile Trp Gly Leu Leu Ser Tyr
155 160 165
Arg Phe Gly Arg Lys Pro Met Leu Ser Trp Cys Cys Leu Gln Leu
170 175 180
Ala Val Ala Gly Thr Ser Thr Ile Phe Ala Pro Thr Phe Val Ile
185 190 195
Tyr Cys Gly Leu Arg Phe Val Ala Ala Phe Gly Met Ala Gly Ile
200 205 210
Phe Leu Ser Ser Leu Thr Leu Met Val Glu Trp Thr Thr Thr Ser
215 220 225
Arg Arg Ala Val Thr Met Thr Val Val Gly Cys Ala Phe Ser Ala
230 235 240
Gly Gln Ala Ala Leu Gly Gly Leu Ala Phe Ala Leu Arg Asp Trp
245 250 255
Arg Thr Leu Gln Leu Ala Ala Ser Val Pro Phe Phe Ala Ile Ser
260 265 270
Leu I1e Ser Trp Trp Leu Pro Glu Ser A1a Arg Trp Leu Ile Ile
275 280 285
Lys Gly Lys Pro Asp Gln Ala Leu Gln Glu Leu Arg Lys Val Ala
290 295 300
Arg Ile Asn Gly His Lys Glu Ala Lys Asn Leu Thr Ile Glu Val
305 310 315
Leu Met Ser Ser Val Lys Glu Glu Val Ala Ser Ala Lys Glu Pro
320 325 330
Arg Ser Val Leu Asp Leu Phe Cys Val Pro Val Leu Arg Trp Arg
335 340 345
Ser Cys Ala Met Leu Val Val Asn Phe Ser Leu Leu Ile Ser Tyr
350 355 360
Tyr Gly Leu Val Phe Asp Leu Gln Ser Leu Gly Arg Asp Ile Phe
365 370 375
Leu Leu Gln Ala Leu Phe Gly Ala Val Asp Phe Leu Gly Arg Ala
380 385 390
Thr Thr Ala Leu Leu Leu Ser Phe Leu Gly Arg Arg Thr Ile Gln
395 400 405
Ala Gly Ser Gln Ala Met Ala Gly Leu A1a Ile Leu Ala Asn Met
410 415 420
Leu Val Pro Gln Asp Leu Gln Thr Leu Arg Val Val Phe Ala Val
425 430 435
Leu Gly Lys Gly Cys Phe Gly Ile Ser Leu Thr Cys Leu Thr Tle
440 445 450
Tyr Lys Ala Glu Leu Phe Pro Thr Pro Val Arg Met Thr Ala Asp
455 460 465
Gly Ile Leu His Thr Val Gly Arg Leu G1y Ala Met Met Gly Pro
470 475 480
Leu Ile Leu Met Ser Arg Gln Ala Leu Pro Leu Leu Pro Pro Leu
485 490 495
Leu Tyr G1y Val Ile Ser Ile Ala Ser Ser Leu Val Val Leu Phe
500 505 510
Phe Leu Pro Glu Thr G1n Gly Leu Pro Leu Pro Asp Thr Ile Gln
515 520 525
Asp Leu Glu Ser Gln Lys Ser Thr Ala Ala Gln Gly Asn Arg Gln
530 535 540
Glu Ala Val Thr Val Glu Ser Thr Ser Leu
545 550
<210> 10
<211> 559
<212> PRT
<213> Homo Sapiens
12167
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
<220>
<221> misc_feature
<223> Incyte ID No: 7611491CD1
<400> 10
Met Arg Arg Gln Asp Ser Arg Gly Asn Thr Val Leu His Ala Leu
1 5 10 15
Val Ala Ile Ala Asp Asn Thr Arg Glu Asn Thr Lys Phe Val Thr
20 ' 25 30
Lys Met Tyr Asp Leu Leu Leu Leu Lys Cys Ala Arg Leu Phe Pro
35 40 45
Asp Ser Asn Leu Glu Ala Val Leu Asn Asn Asp Gly Leu Ser Pro
50 55 60
Leu Met Met Met Ala Ala Lys Thr Gly Lys Ile Gly Ile Phe Gln
65 70 75
His Ile Ile Arg Arg Glu Val Thr Asp Glu Asp Thr Arg His Leu
80 85 90
Ser Arg Lys Phe Lys Asp Trp Ala Tyr Gly Pro Val Tyr Ser Ser
95 100 ~ 105
Leu Tyr Asp Leu Ser Ser Leu Asp Thr Cys Gly Glu Glu Ala Ser
110 115 120
Val Leu Glu Ile Leu Val Tyr Asn Ser Lys Ile Glu Asn Arg His
125 130 135
Glu Met Leu Ala Val Glu Pro Ile Asn Glu Leu Leu Arg Asp Lys
140 145 150
Trp Arg Lys Phe Gly Ala Val Ser Phe Tyr Ile Asn Val Val Ser
155 160 165
Tyr Leu Cys Ala Met Val Ile Phe Thr Leu Thr Ala Tyr Tyr Gln
170 175 180
Pro Leu Glu Gly Thr Pro Pro Tyr Pro Tyr Arg Thr Thr Val Asp
185 190 195
Tyr Leu Arg Leu Ala Gly Glu Val Ile Thr Leu Phe Thr Gly Val
200 205 210
Leu Phe Phe Phe Thr Asn Ile Lys Asp Leu Phe Met Lys Lys Cys
215 220 225
Pro Gly Val Asn Ser Leu Phe Ile Asp Gly Ser Phe Gln Leu Leu
230 235 240
Tyr Phe Ile Tyr Ser Val Leu Val Ile Val Ser Ala Ala Leu Tyr
245 250 255
Leu Ala Gly Ile Glu Ala Tyr Leu Ala Val Met Val Phe Ala Leu
260 265 270
Val Leu Gly Trp Met Asn Ala Leu Tyr Phe Thr Arg Gly Leu Lys
275 280 285
Leu Thr Gly Thr Tyr Ser Ile Met Ile Gln Lys Ile Leu Phe Lys
290 295 300
Asp Leu Phe Arg Phe Leu Leu Val Tyr Leu Leu Phe Met Ile Gly
305 310 315
Tyr Ala Ser Ala Leu Val Ser Leu Leu Asn Pro Cys Ala Asn Met
320 325 330
Lys Val Cys Asn Glu Asp Gln Thr Asn Cys Thr Val Pro Thr Tyr
335 340 345
Pro Ser Cys Arg Asp Ser Glu Thr Phe Ser Thr Phe Leu Leu Asp
350 355 360
Leu Phe Lys Leu Thr Ile Gly Met Gly Asp Leu Glu Met Leu Ser
365 370 375
Ser Thr Lys Tyr Pro Val Val Phe Ile Ile Leu Leu Val Thr Tyr
380 385 390
Ile Ile Leu Thr Phe Val Leu Leu Leu Asn Met Leu I1e Ala Leu
395 400 405
Met Gly Glu Thr Val Gly Gln Val Ser Lys Glu Ser Lys His Ile
410 415 420
Trp Lys Leu Gln Trp Ala Thr Thr Ile Leu Asp Ile Glu Arg Ser
425 430 435
13!67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
Phe Pro Val Phe Leu Arg Lys Ala Phe Arg Ser Gly Glu Met Val
440 445 450
Thr Val Gly Lys Ser Ser Asp Gly Thr Pro Asp Arg Arg Trp Cys
455 460 465
Phe Arg Val Asp Glu Val Asn Trp Ser His Trp Asn Gln Asn Leu
470 475 480
Gly Ile Ile Asn Glu Asp Pro Gly Lys Asn Glu Thr Tyr Gln Tyr
485 490 495
Tyr Gly Phe Ser His Thr Val Gly Arg Leu Arg Arg Asp Arg Trp
500 505 510
Ser Ser Val Val Pro Arg Val Val Glu Leu Asn Lys Asn Ser Asn
515 520 525
Pro Asp Glu Val Val Val Pro Leu Asp Ser Thr Gly Asn Pro Arg
530 535 540
Cys Asp Gly His Gln Gln Gly Tyr Pro Arg Lys Trp Arg Thr Asp
545 550 555
Asp Ala Pro Leu
<210> 11
<211> 181
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 171968CD1
<400> 11
Met Phe His His Gln Gln Ala Tyr Cys Leu Ala Pro Phe Asp Leu
1 5 10 15
Ile Lys Val Arg Leu Gln Asn Gln Thr Glu Pro Arg Ala Gln Pro
20 25 30
Gly Ser Pro Pro Pro Arg Tyr Gln Gly Pro Val His Cys Ala Ala
35 40 45
Ser Ile Phe Arg Glu Glu Gly Pro Arg Gly Leu Phe Arg Gly Ala
50 55 60
Trp Ala Leu Thr Leu Arg Asp Thr Pro Thr Val Gly Ile Tyr Phe
65 70 75
Ile Thr Tyr Glu Gly Leu Cys Arg Gln Tyr Thr Pro Glu Gly Gln
80 85 90
Asn Pro Ser Ser Ala Thr Va1 Leu Val Ala Gly Gly Phe Ala Gly
95 100 105
Ile Ala Ser Trp Val Ala Ala Thr Pro Leu Asp Val Ile Lys Ser
110 115 120
Arg Met Gln Met Asp Gly Leu Arg Arg Arg Val Tyr Gln Gly Met
125 130 135
Leu Asp Cys Met Val Ser Ser Ile Arg Gln Glu Gly Leu Gly Val
140 145 150
Phe Phe Arg Gly Val Thr Ile Asn Ser Ala Arg Ala Phe Pro Val
155 160 165
Asn Ala Val Thr Phe Leu Ser Tyr Glu Tyr Leu Leu Arg Trp Trp
170 175 180
Gly
<210> 12
<211> 124
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
14/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
<223> Incyte ID No: 257274CD1
<400> 12
Met Cys Ser Gly Leu Leu Glu Leu Leu Leu Pro Ile Trp Leu Ser
1 5 10 15
Trp Thr Leu Gly Thr Arg Gly Ser G1u Pro Arg Ser Val Asn Asp
20 25 30
Pro Gly Asn Met Ser Phe Val Lys Glu Thr Va1 Asp Lys Leu Leu
35 40 45
Lys Gly Tyr Asp Ile Arg Leu Arg Pro Asp Phe Gly Gly Pro Pro
50 55 60
Val Cys Val Gly Met Asn Ile Asp Ile Ala Ser Ile Asp Met Val
65 70 75
Ser Glu Val Asn Met Arg Phe Trp Leu Gln Glu Arg Gly Thr Lys
80 85 90
Thr Val Val Cys Ala Phe Gln Gly Cys Leu Cys Gly Phe Ser Lys
95 100 105
Ala Ala Ser Trp Thr Gly Arg Pro G1y Pro Gly Thr Ala Ser Leu
110 115 120
Cys Pro Arg Cys
<210> 13
<211> 2009
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6355991CD1
<400> 13
Met Glu Gln Thr Val Leu Val Pro Pro Gly Pro Asp Ser Phe Asn
1 5 10 15
Phe Phe Thr Arg Glu Ser Leu A1a Ala Ile Glu Arg Arg Ile Ala
20 25 30
Glu Glu Lys Ala Lys Asn Pro Lys Pro Asp Lys Lys Asp Asp Asp
35 40 45
Glu Asn Gly Pro Lys Pro Asn Ser Asp Leu Glu Ala Gly Lys Asn
50 55 60
Leu Pro Phe Ile Tyr Gly Asp Ile Pro Pro Glu Met Val Ser Glu
65 70 75
Pro Leu Glu Asp Leu Asp Pro Tyr Tyr Ile Asn Lys Gln Thr Phe
80 85 90
Ile Val Leu Asn Lys Gly Lys Ala Ile Phe Arg Phe Ser Ala Thr
95 100 105
Ser Ala Leu Tyr Ile Leu Thr Pro Phe Asn Pro Leu Arg Lys Ile
110 115 120
Ala Ile Lys Ile Leu Val His Ser Leu Phe Ser Met Leu Ile Met
125 130 135
Cys Thr Ile Leu Thr Asn Cys Val Phe Met Thr Met Ser Asn Pro
140 145 150
Pro Asp Trp Thr Lys Asn Val Glu Tyr Thr Phe Thr Gly Ile Tyr
155 160 165
Thr Phe Glu Ser Leu Ile Lys Ile Ile Ala Arg Gly Phe Cys Leu
170 175 180
Glu Asp Phe Thr Phe Leu Arg Asp Pro Trp Asn Trp Leu Asp Phe
185 190 195
Thr Val Ile Thr Phe Ala Tyr Val Thr Glu Phe Val Asp Leu Gly
200 205 210
Asn Val Ser Ala Leu Arg Thr Phe Arg Val Leu Arg Ala Leu Lys
215 220 225
Thr Ile Ser Val Ile Pro Gly Leu Lys Thr Ile Val Gly Ala Leu
15167
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
230 235 240
Ile Gln Ser Val Lys Lys Leu Ser Asp Val Met Ile Leu Thr Val
245 250 255
Phe Cys Leu Ser Val Phe Ala Leu Ile Gly Leu Gln Leu Phe Met
260 265 270
Gly Asn Leu Arg Asn Lys Cys Ile Gln Trp Pro Pro Thr Asn Ala
275 280 285
Ser Leu Glu Glu His Ser Ile flu Lys Asn Ile Thr Val Asn Tyr
290 295 300
Asn Gly Thr Leu Ile Asn Glu Thr Val Phe Glu Phe Asp Trp Lys
305 310 315
Ser Tyr Ile Gln Asp Ser Gly Tyr His Tyr Phe Leu Glu Gly Phe
320 325 330
Leu Asp Ala Leu Leu Cys Gly Asn Ser Ser Asp Ala Gly Gln Cys
335 , 340 345
Pro Glu Gly Tyr Met Cys Val Lys Ala Gly Arg Asn Pro Asn Tyr
350 355 360
Gly Tyr Thr Ser Phe Asp Thr Phe Ser Trp Ala Phe Leu Ser Leu
365 370 375
Phe Arg Leu Met Thr Gln Asp Phe Trp Glu Asn Leu Tyr Gln Leu
380 385 390
Thr Leu Arg Ala A1a Gly Lys Thr Tyr Met Ile Phe Phe Val Leu
395 400 405
Va1 Ile Phe Leu Gly Ser Phe Tyr Leu Ile Asn Leu Ile Leu Ala
410 415 420
Val Val Ala Met Ala Tyr Glu Glu Gln Asn Gln Ala Thr Leu Glu
425 430 435
Glu Ala Glu Gln Lys Glu Ala G1u Phe Gln Gln Met Ile Glu Gln
440 445 450
Leu Lys Lys Gln Gln Glu Ala Ala Gln Gln A1a Ala Thr Ala Thr
455 460 465
Ala Ser Glu His Ser Arg G1u Pro Ser Ala Ala Gly Arg Leu Ser
470 475 480
Asp Ser Ser Ser Glu Ala Ser Lys Leu Ser Ser Lys Ser Ala Lys
485 490 495
Glu Arg Arg Asn Arg Arg Lys Lys Arg Lys Gln Lys Glu Gln Ser
500 505 510
Gly Gly Glu Glu Lys Asp Glu Asp Glu Phe Gln Lys Ser Glu Ser
515 520 525
Glu Asp Ser Ile Arg Arg Lys Gly Phe Arg Phe Ser Ile Glu Gly
530 535 540
Asn Arg Leu Thr Tyr Glu Lys Arg Tyr Ser Ser Pro His Gln Ser
545 550 555
Leu Leu Ser Ile Arg Gly Ser Leu Phe Ser Pro Arg Arg Asn Ser
560 565 570
Arg Thr Ser Leu Phe Ser Phe Arg Gly Arg Ala Lys Asp Val Gly
575 580 585
Ser Glu Asn Asp Phe Ala Asp Asp Glu His Ser Thr Phe Glu Asp
590 595 600
Asn Glu Ser Arg Arg Asp Ser Leu Phe Val Pro Arg Arg His Gly
605 610 615
Glu Arg Arg Asn Ser Asn Leu Ser Gln Thr Ser Arg Ser Ser Arg
620 625 630
Met Leu Ala Val Phe Pro Ala Asn Gly Lys Met His Ser Thr Val
635 640 645
Asp Cys Asn Gly Val Val Ser Leu Val Gly Gly Pro Ser Val Pro
650 655 660
Thr Ser Pro Val Gly Gln Leu Leu Pro Glu Val Ile Ile Asp Lys
665 670 675
Pro Ala Thr Asp Asp Asn Gly Thr Thr Thr Glu Thr Glu Met Arg
680 ~ 685 690
Lys Arg Arg Ser Ser Ser Phe His Val Ser Met Asp Phe Leu Glu
695 700 705
16/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
Asp Pro Ser Gln Arg Gln Arg Ala Met Ser Ile Ala Ser Ile Leu
710 715 720
Thr Asn Thr Val Glu Glu Leu Glu Glu Ser Arg G1n Lys Cys,Pro
725 730 735
Pro Cys Trp Tyr Lys Phe Ser Asn Ile Phe Leu Ile Trp Asp Cys
740 745 750
Ser Pro Tyr Trp Leu Lys Val Lys His Val Val Asn Leu Val Val
755 760 765
Met Asp Pro Phe Val Asp Leu Ala Tle Thr Ile Cys Ile Val Leu
770 775 780
Asn Thr Leu Phe Met Ala Met Glu His Tyr Pro Met Thr Asp His
785 790 ' 795
Phe Asn Asn Val Leu Thr Val Gly Asn Leu Val Phe Thr Gly Ile
800 805 810
Phe Thr Ala Glu Met Phe Leu Lys Tle Ile Ala Met Asp Pro Tyr
815 820 825
Tyr Tyr Phe Gln Glu Gly Trp Asn Ile Phe Asp Gly Phe Ile Val
830 835 840
Thr Leu Ser Leu Val Glu Leu Gly Leu Ala Asn Val Glu Gly Leu
845 850 855
Ser Val Leu Arg Ser Phe Arg Leu Leu Arg Val Phe Lys Leu Ala
860 865 870
Lys Ser Trp Pro Thr Leu Asn Met Leu Ile Lys Ile Ile Gly Asn
875 880 885
Ser Gly Gly Ala Leu Gly Asn Leu Thr Leu Val Leu Ala Ile Ile
890 895 900
Val Phe Ile Phe Ala Val Val Gly Met Gln Leu Phe Gly Lys Ser
905 910 915
Tyr Lys Asp Cys Val Cys Lys Ile Ala Ser Asp Cys Gln Leu Pro
920 925 930
Arg Trp His Met Asn Asp Phe Phe His Ser Phe Leu Ile Val Phe
935 940 945
Arg Val Leu Cys Gly Glu Trp Ile Glu Thr Met Trp Asp Cys Met
950 955 960
Glu Val Ala Gly Gln Ala Met Cys Leu Thr Val Phe Met Met Val
965 970 975
Met Val Ile Gly Asn Leu Val Val Leu Asn Leu Phe Leu Ala Leu
980 985 990
Leu Leu Ser Ser Phe Ser Ala Asp Asn Leu Ala Ala Thr Asp Asp
995 1000 1005
Asp Asn Glu Met Asn Asn Leu Gln Ile Ala Val Asp Arg Met His
1010 1015 1020
Lys Gly Val Ala Tyr Val Lys Arg Lys Ile Tyr Glu Phe Ile Gln
1025 1030 1035
Gln Ser Phe Ile Arg Lys Gln Lys Ile Leu Asp Glu Ile Lys Pro
1040 1045 1050
Leu Asp Asp Leu Asn Asn Lys Lys Asp Ser Cys Met Ser Asn His
1055 1060 1065
Thr Ala Glu Ile Gly Lys Asp Leu Asp Tyr Leu Lys Asp Val Asn
1070 1075 1080
Gly Thr Thr Ser Gly Ile Gly Thr Gly Ser Ser Val Glu Lys Tyr
1085 1090 1095
Ile Ile Asp Glu Ser Asp Tyr Met Ser Phe Ile Asn Asn Pro Ser
1100 1105 1110
Leu Thr Val Thr Val Pro Ile Ala Val Gly Glu Ser Asp Phe Glu
1115 1120 1125
Asn Leu Asn Thr Glu Asp Phe Ser Ser Glu Ser Asp Leu Glu Glu
1130 1135 1140
Ser Lys Glu Lys Leu Asn Glu Ser Ser Ser Ser Ser Glu Gly Ser
1145 1150 1155
Thr Val Asp Ile Gly Ala Pro Val Glu Glu Gln Pro Val Val Glu
1160 1165 1170
Pro Glu Glu Thr Leu Glu Pro Glu Ala Cys Phe Thr Glu Gly Cys
17/67
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1175 1180 1185
Val Gln Arg Phe Lys Cys Cys Gln Ile Asn Va1 Glu Glu Gly Arg
1190 1195 1200
Gly Lys Gln Trp Trp Asn Leu Arg Arg Thr Cys Phe Arg Ile Val
2205 1210 1215
Glu His Asn Trp Phe Glu Thr Phe Ile Val Phe Met Ile Leu Leu
1220 1225 1230
Ser Ser Gly.Ala Leu Ala Phe Glu Asp Ile Tyr Ile Asp Gln Arg
1235 1240 1245
Lys Thr Ile Lys Thr Met Leu Glu Tyr Ala Asp Lys Val Phe Thr
1250 1255 1260
Tyr Ile Phe Ile Leu Glu Met Leu Leu Lys Trp Val Ala Tyr Gly
1265 1270 1275
Tyr Gln Thr Tyr Phe Thr Asn Ala Trp Cys Trp Leu Asp Phe Leu
1280 1285 1290
Ile Val Asp Val Ser Leu Val Ser Leu Thr Ala Asn Ala Leu Gly
1295 1300 1305
Tyr Ser Glu Leu Gly Ala Ile Lys Ser Leu Arg Thr Leu Arg Ala
1310 1315 1320
Leu Arg Pro Leu Arg Ala Leu Ser Arg Phe Glu Gly Met Arg Val
1325 1330 1335
Val Val Asn Ala Leu Leu Gly Ala Ile Pro Ser Ile Met Asn Val
1340 1345 1350
Leu Leu Val Cys Leu Ile Phe Trp Leu Ile Phe Ser Ile Met Gly
1355 1360 1365
Val Asn Leu Phe Ala Gly Lys Phe Tyr His Cys Ile Asn Thr Thr
1370 1375 1380
Thr Gly Asp Arg Phe Asp Ile Glu Asp Val Asn Asn His Thr Asp
1385 1390 1395
Cys Leu Lys Leu Ile Glu Arg Asn Glu Thr Ala Arg Trp Lys Asn
1400 1405 1410
Val Lys Val Asn Phe Asp Asn Val Gly Phe Gly Tyr Leu Ser Leu
1415 1420 1425
Leu Gln Val Ala Thr Phe Lys Gly Trp Met Asp Ile Met Tyr Ala
1430 1435 1440
Ala Val Asp Ser Arg Asn Val Glu Leu Gln Pro Lys Tyr Glu Glu
1445 1450 1455
Ser Leu Tyr Met Tyr Leu Tyr Phe Val Ile Phe Ile Ile Phe Gly
1460 1465 1470
Ser Phe Phe Thr Leu Asn Leu Phe Ile Gly Val Ile Ile Asp Asn
1475 1480 1485
Phe Asn Gln Gln Lys Lys Lys Phe Gly Gly Gln Asp Ile Phe Met
1490 1495 1500
Thr Glu Glu Gln Lys Lys Tyr Tyr Asn Ala Met Lys Lys Leu Gly
1505 1510 1515
Ser Lys Lys Pro Gln Lys Pro Ile Pro Arg Pro Gly Asn Lys Phe
1520 1525 1530
Gln Gly Met Val Phe Asp Phe Val Thr Arg Gln Val Phe Asp Ile
1535 1540 1545
Ser Ile Met Ile Leu Ile Cys Leu Asn Met Val Thr Met Met Val
1550 1555 1560
Glu Thr Asp Asp Gln Ser Glu Tyr Val Thr Thr Ile Leu Ser Arg
1565 1570 1575
Ile Asn Leu Val Phe Ile Val Leu Phe Thr Gly Glu Cys Val Leu
1580 1585 1590
Lys Leu Ile Ser Leu Arg His Tyr Tyr Phe Thr Ile Gly Trp Asn
1595 1600 1605
Ile Phe Asp Phe Val Val Val Ile Leu Ser Ile VaI Gly Met Phe
1610 1615 1620
Leu Ala Glu Leu Ile Glu Lys Tyr Phe Val Ser Pro Thr Leu Phe
1625 1630 1635
Arg Val Ile Arg Leu Ala Arg Ile Gly Arg Ile Leu Arg Leu Ile
1640 1645 1650
18/67
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Lys Gly Ala Lys Gly Ile Arg Thr Leu Leu Phe Ala Leu Met Met
1655 1660 1665
Ser Leu Pro Ala Leu Phe Asn Ile G1y Leu Leu Leu Phe Leu Val
1670 1675 1680
Met Phe I1e Tyr Ala Ile Phe Gly Met Ser Asn Phe Ala Tyr Val
1685 1690 1695
Lys Arg Glu Val Gly Ile Asp Asp Met Phe Asn Phe Glu Thr Phe
1700 1705 1710
Gly Asn Ser Met Ile Cys Leu Phe Gln Ile Thr Thr Ser Ala Gly
1715 1720 1725
Trp Asp Gly Leu Leu Ala Pro Ile Leu Asn Ser Lys Pro Pro Asp
1730 1735 1740
Cys Asp Pro Asn Lys Val Asn Pro Gly Ser Ser Val Lys Gly Asp
1745 1750 1755
Cys Gly Asn Pro Ser Val Gly Ile Phe Phe Phe Val Ser Tyr Ile
1760 1765 1770
Ile Ile Ser Phe Leu Val Val Val Asn Met Tyr Ile Ala Val Ile
1775 1780 1785
Leu Glu Asn Phe Ser Val Ala Thr Glu Glu Ser Ala Glu Pro Leu
2790 1795 1800
Ser Glu Asp Asp Phe Glu Met Phe Tyr Glu Val Trp Glu Lys Phe
1805 1810 1815
Asp Pro Asp Ala Thr Gln Phe Met Glu Phe Glu Lys Leu Ser Gln
1820 2825 1830
Phe Ala Ala Ala Leu Glu Pro Pro Leu Asn Leu Pro Gln Pro Asn
1835 1840 1845
Lys Leu Gln Leu Ile Ala Met Asp Leu Pro Met Val Ser Gly Asp
1850 1855 1860
Arg Ile His Cys Leu Asp Ile Leu Phe Ala Phe Thr Lys Arg Val
1865 1870 1875
Leu Gly Glu Ser Gly Glu Met Asp Ala Leu Arg Ile Gln Met Glu
1880 1885 1890
G1u Arg Phe Met Ala Ser Asn Pro Ser Lys Val Ser Tyr Gln Pro
1895 1900 1905
Ile Thr Thr Thr Leu Lys Arg Lys Gln Glu Glu Val Ser Ala Val
1910 1915 1920
Ile Ile Gln Arg Ala Tyr Arg Arg His Leu Leu Lys Arg Thr Val
1925 1930 1935
Lys Gln Ala Ser Phe Thr Tyr Asn Lys Asn Lys Ile Lys Gly Gly
1940 1945 1950
Ala Asn Leu Leu Ile Lys Glu Asp Met Ile Ile Asp Arg Ile Asn
1955 1960 1965
Glu Asn Ser Ile Thr Glu Lys Thr Asp Leu Thr Met Ser Thr Ala
1970 1975 1980
Ala Cys Pro Pro Ser Tyr Asp Arg Val Thr Lys Pro Ile Val Glu
1985 1990 1995
Lys His Glu Gln Glu Gly Lys Asp Glu Lys Ala Lys Gly Lys
2000 2005
<210> 14
<211> 538
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 70035348CD1
<400> 14
Met Val Val Glu Thr Glu Gly Ser Leu Leu Asn
Pro Asn Pro Gln
1 5 10 15
Lys Gly Ala Val Thr Glu Gly Gly Ser Arg His
Thr Glu Ser Pro
20 25 30
19/67
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Pro Trp Ala Arg Gly Cys Gly Met Phe Thr Phe Leu Ser Ser Val
35 40 45
Thr Ala Ala Val Ser Gly Leu Leu Val Gly Tyr Glu Leu Gly Ile
50 55 60
Ile Ser Gly Ala Leu Leu Gln Ile Lys Thr Leu Leu Ala Leu Ser
65 70 75
Cys His Glu Gln Glu Met Val Val Ser Ser Leu Val Ile Gly Ala
80 85 90
Leu Leu Ala Ser Leu Thr Gly Gly Val Leu Ile Asp Arg Tyr Gly
95 100 105
Arg Arg Thr Ala Ile Ile Leu Ser Ser Cys Leu Leu Gly Leu Gly
110 115 120
Ser Leu Val Leu Ile Leu Ser Leu Ser Tyr Thr Val Leu Ile Val
125 130 135
Gly Arg Ile Ala Ile Gly Val Ser Ile Ser Leu Ser Ser Ile Ala
140 145 150
Thr Cys Val Tyr Ile Ala Glu Ile Ala Pro Gln His Arg Arg Gly
155 . 160 165
Leu Leu Val Ser Leu Asn Glu Leu Met Ile Val Ile Gly Ile Leu
170 175 180
. Ser Ala Tyr Ile Ser Asn Tyr Ala Phe Ala Asn Val Phe His Gly
185 190 195
Trp Lys Tyr Met Phe Gly Leu Val Ile Pro Leu Gly Val Leu Gln
200 205 210
Ala Ile Ala Met Tyr Phe Leu Pro Pro Ser Pro Arg Phe Leu Val
215 220 225
Met Lys Gly Gln Glu Gly Ala Ala Ser Lys Val Leu Gly Arg Leu
230 235 240
Arg Ala Leu Ser Asp Thr Thr Glu Glu Leu Thr Val Ile Lys Ser
245 250 255
Ser Leu Lys Asp Glu Tyr Gln Tyr Ser Phe Trp Asp Leu Phe Arg
260 265 270
Ser Lys Asp Asn Met Arg Thr Arg Ile Met Ile Gly Leu Thr Leu
275 280 285
Va1 Phe Phe Val Gln Ile Thr Gly Gln Pro Asn Ile Leu Phe Tyr
290 295 300
Ala Ser Thr Val Leu Lys Ser Val Gly Phe Gln Ser Asn Glu Ala
305 310 315
Ala Ser Leu Ala Ser Thr Gly Val Gly Val Val Lys Val Ile Ser
320 325 330
Thr Ile Pro Ala Thr Leu Leu Val Asp His Val Gly Ser Lys Thr
335 340 345
Phe Leu Cys Ile Gly Ser Ser Val Met Ala Ala Ser Leu Val Thr
350 355 360
Met Gly Ile Val Asn Leu Asn Ile His Met Asn Phe Thr His Ile
365 370 375
Cys Arg Ser His Asn Ser Ile Asn Gln Ser Leu Asp Glu Ser Val
380 385 390
Ile Tyr Gly Pro Gly Asn Leu Ser Thr Asn Asn Asn Thr Leu Arg
395 400 405
Asp His Phe Lys Gly Ile Ser Ser His Ser Arg Ser Ser Leu Met
410 415 420
Pro Leu Arg Asn Asp Val Asp Lys Arg Gly Glu Thr Thr Ser Ala
425 430 435
Ser Leu Leu Asn Ala Gly Leu Ser His Thr Glu Tyr Gln Ile Val
440 445 450
Thr Asp Pro Gly Asp Val Pro Ala Phe Leu Lys Trp Leu Ser Leu
455 460 465
Ala Ser Leu Leu Val Tyr Val Ala Ala Phe Ser Ile Gly Leu Gly
470 475 480
Pro Arg Asp Val Ile Phe Ile Gly Gln Ser Thr Asn Leu Pro Ser
485 490 495
Ala Pro Glu Gly Asp Thr Ile Ser Ile Ser Lys Thr Ile Tyr Tyr
20/67
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500 505 510
Ala Ala Tyr Asn Lys Ala Ile Ile Gln Thr Ala Leu Glu Arg Gln
515 520 525
Pro Arg Ala Lys Thr Val Ser Ala Phe Ser His Lys Thr
530 535
<210> 15
<211> 742
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472539CD1
<400> 15
Met Glu Tyr Gln Ala Ser Glu Val Ile Gly Gln Arg Gln Ser Ser
1 5 10 15
Ala Thr Lys Pro Gly Arg Ser Gly Lys G1u Ser Val Thr Glu Pro
20 25 30
Trp Ala Arg Val Pro Gly Ala Leu Gly Val Ala Ala Arg Gln Met
35 40 45
His Pro Lys Ser Ile Ile Thr Phe Arg Glu Ile Asn Gly Glu Tyr
50 55 60
Thr Gly Ala Val Asp Phe Pro Arg Leu Gly Val Arg Ala Ser Glu
65 70 75
Glu Thr Ala Leu Arg Glu Leu Lys Met Ser Lys Glu Leu Ala Ala
80 85 90
Met Gly Pro Gly Ala Ser Gly Asp Gly Val Arg Thr Glu Thr Ala
95 100 105
Pro His Ile Ala Leu Asp Ser Arg Val Gly Leu His Ala Tyr Asp
110 115 120
Ile Ser Val Val Val Ile Tyr Phe Val Phe Val Ile Ala Val Gly
125 130 135
Ile Trp Ser Ser Ile Arg Ala Ser Arg Gly Thr Ile Gly Gly Tyr
140 145 150
Phe Leu Ala Gly Ser Trp Ser Ile Ser Asp Val Gln Gln Cys Gly
155 160 165
Gln Trp Leu Val His Arg Pro Gly Trp Asp Arg Gly Cys Arg Arg
170 175 180
Pro Cys Arg Arg Trp Leu Arg Val Glu Leu Leu Leu Ala Leu Gly
185 190 195
Trp Val Phe Val Pro Val Tyr Ile Ala Ala Gly Val Val Thr Met
200 205 210
Pro Gln Tyr Leu Lys Lys Arg Phe Gly Gly Gln Arg Ile Gln Val
215 220 225
Tyr Met Ser Val Leu Ser Leu Ile Leu Tyr Ile Phe Thr Lys Ile
230 235 240
Ser Thr Asp Ile Phe Ser Gly Ala Leu Phe Ile Gln Met Ala Leu
245 250 255
Gly Trp Asn Leu Tyr Leu Ser Thr Gly Ile Leu Leu Val Val Thr
260 265 270
Ala Val Tyr Thr Ile Ala Gly Gly Leu Met Ala Val Ile Tyr Thr
275 280 285
Asp Ala Leu Gln Thr Val Ile Met Val Gly Gly Ala Leu Val Leu
290 295 300
Met Phe Leu Gly Phe Gln Asp Val Gly Trp Tyr Pro Gly Leu Glu
305 310 315
Gln Arg Tyr Arg Gln Ala Ile Pro Asn Val Thr Val Pro Asn Thr
320 325 330
Thr Cys His Leu Pro Arg Pro Asp Ala Phe His Ile Leu Arg Asp
335 340 345
Pro Val Ser Gly Asp Ile Pro Trp Pro Gly Leu Ile Phe Gly Leu
21 /67
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350 355 360
Thr Val Leu Ala Thr Trp Cys Trp Cys Thr Asp Gln Val Ile Val
365 370 375
G1n Arg Ser Leu Ser Ala Lys Ser Leu Ser His Ala Lys Gly Gly
380 385 390
Ser Val Leu G1y Gly Tyr Leu Lys Ile Leu Pro Met Phe Phe Tle
395 400 405
Val Met Pro Gly Met Ile Ser Arg Ala Leu Phe Pro Asp Glu Val
410 415 420
Gly Cys Val Asp Pro Asp Val Cys Gln Arg Ile Cys Gly Ala Arg
425 430 435
Val Gly Cys Ser Asn Ile Ala Tyr Pro Lys Leu Val Met Ala Leu
440 445 450
Met Pro Val Gly Leu Arg Gly Leu Met Ile Ala Val Ile Met Ala
455 460 465
Ala Leu Met Ser Ser Leu Thr Ser Ile Phe Asn Ser Ser Ser Thr
470 475 480
Leu Phe Thr Ile Asp Val Trp Gln Arg Phe Arg Arg Lys Ser Thr
485 490 495
Glu Gln Glu Leu Met Val Val Gly Arg Val Phe Val Val Phe Leu
500 505 510
Val Val Ile Ser Ile Leu Trp Ile Pro Ile Ile Gln Ser Ser Asn
5l5 520 525
Ser Gly Gln Leu Phe Asp Tyr Ile Gln Ala Val Thr Ser Tyr Leu
530 535 540
Ala Pro Pro Ile Thr Ala Leu Phe Leu Leu Ala Ile Phe Cys Lys
545 550 555
Arg Val Thr Glu Pro Gly Ala Phe Trp Gly Leu Val Phe Gly Leu
560 565 570
Gly Val Gly Leu Leu Arg Met Ile Leu Glu Phe Ser Tyr Pro Ala
575 580 585
Pro Ala Cys Gly Glu Val Asp Arg Arg Pro Ala Val Leu Lys Asp
590 595 600
Phe His Tyr Leu Tyr Phe Ala I1e Leu Leu Cys Gly Leu Thr Ala
605 610 615
Tle Val Ile Val Ile Leu Thr Arg Leu Thr Trp Trp Thr Arg Asn
620 625 630
Cys Pro Leu Ser Glu Leu Glu Lys Glu Ala His Glu Ser Thr Pro
635 640 645
Glu Ile Ser Glu Arg Pro Ala Gly Glu Cys Pro Ala Gly Gly Gly
650 655 660
Ala Ala Glu Asn Ser Ser Leu Gly Gln Glu Gln Pro Glu Ala Pro
665 670 675
Ser Arg Ser Trp Gly Lys Leu Leu Trp Ser Trp Phe Cys Gly Leu
680 685 690
Ser Gly Thr Pro Glu Gln Ala Leu Ser Pro Ala Glu Lys Ala Ala
695 700 705
Leu Glu Gln Lys Leu Thr Ser Ile Glu Glu Glu Pro Leu Trp Arg
710 715 720
His Val Cys Asn Ile Asn Ala Val Leu Leu Leu Ala Ile Asn Ile
725 730 735
Phe Leu Trp Gly Tyr Phe Ala
740
<210> 16
<211> 426
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 817477CD1
22/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
<400> 16
Met Ala Arg Arg Thr Glu Pro Pro Asp Gly Gly Trp Gly Trp Val
1 5 10 15
Val Val Leu Ser Ala Phe Phe Gln Ser Ala Leu Val Phe Gly Val
20 25 30
Leu Arg Ser Phe Gly Val Phe Phe Val Glu Phe Val Ala Ala Phe
35 40 45
Glu Glu Gln Ala Ala Arg Val Ser Trp Ile Ala Ser Ile Gly Tle
50 55 60
Ala Val Gln Gln Phe Gly Ser Pro Val Gly Ser Ala Leu Ser Thr
65 70 75
Lys Phe Gly Pro Arg Pro Val Val Met Thr Gly Gly Ile Leu Ala
80 85 90
Ala Leu Gly Met Leu Leu Ala Ser Phe Ala Thr Ser Leu Thr His
95 100 105
Leu Tyr Leu Ser Ile Gly Leu Leu Ser Gly Ser Gly Trp Ala Leu
110 115 120
Thr Phe Ala Pro Thr Leu Ala Cys Leu Ser Cys Tyr Phe Ser Arg
125 130 135
Arg Arg Ser Leu Ala Thr Gly Leu Ala Leu Thr Gly Val Gly Leu
140 145 150
Ser Ser Phe Thr Phe Ala Pro Phe Phe Gln Trp Leu Leu Ser His
155 160. 165
Tyr Ala Trp Arg Gly Ser Leu Leu Leu Val Ser Ala Leu Ser Leu
170 175 180
His Leu Val Ala Cys Gly Ala Leu Leu Arg Pro Pro Ser Leu Ala
185 190 195
Glu Asp Pro Ala Val Gly Gly Pro Arg Ala Gln Leu Thr Ser Leu
200 205 210
Leu His His Gly Pro Phe Leu Arg Tyr Thr Val Ala Leu Thr Leu
215 220 225
:Lle Asn Thr Gly Tyr Phe Ile Pro Tyr Leu His Leu Val Ala His
230 235 240
Leu Gln Asp Leu Asp Trp Asp Pro Leu Pro Ala Ala Phe Leu Leu
245 250 255
Ser Val Val Ala Ile Ser Asp Leu Val Gly Arg Val Val Ser Gly
260 265 270
Trp Leu Gly Asp Ala Val Pro Gly Pro Val Thr Arg Leu Leu Met
275 280 285
Leu Trp Thr Thr Leu Thr Gly Val Ser Leu Ala Leu Phe Pro Val
290 295 300
Ala Gln Ala Pro Thr Ala Leu Val Ala Leu Ala Val Ala Tyr Gly
305 310 315
Phe Thr Ser Gly Ala Leu Ala Pro Leu Ala Phe Ser Val Leu Pro
320 325 330
Glu Leu Ile Gly Thr Arg Arg Ile Tyr Cys Gly Leu Gly Leu Leu
335 340 345
Gln Met Ile Glu Ser Ile G1y Gly Leu Leu Gly Pro Pro Leu Ser
350 355 360
Gly Tyr Leu Arg Asp Val Thr Gly Asn Tyr Thr Ala Ser Phe Val
365 370 375
Val Ala Gly Ala Phe Leu Leu Ser Gly Ser Gly Ile Leu Leu Thr
380 385 390
Leu Pro His Phe Phe Cys Phe Ser Thr Thr Thr Ser Gly Pro Gln
395 400 405
Asp Leu Val Thr Glu Ala Leu Asp Thr Lys Val Pro Leu Pro Lys
410 415 420
Glu Gly Leu Glu Glu Asp
425
<210> 17
<211> 1197
<212> PRT
23/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
<213> Homo sapiens
<220>
<221> misC_feature
<223> Incyte ID No: 1442166CD1
<400> 17
Met Ala Ala Ala Ala Ala Val Gly Asn Ala Val Pro Cys Gly Ala
1 5 10 l5
Arg Pro Cys Gly Val Arg Pro Asp Gly G1n Pro Lys Pro Gly Pro
20 25 30
Gln Pro Arg Ala Leu Leu Ala Ala Gly Pro Ala Leu Ile A1a Asn
35 40 45
Gly Asp Glu Leu Val Ala Ala Val Trp Pro Tyr Arg Arg Leu Ala
50 55 ' 60
Leu Leu Arg Arg Leu Thr Val Leu Pro Phe Ala Gly Leu Leu Tyr
65 70 75
Pro Ala Trp Leu Gly Ala Ala Ala Ala Gly Cys Trp Gly Trp Gly
80 85 90
Ser Ser Trp Val Gln Ile Pro Glu Ala Ala Leu Leu Val Leu Ala
95 100 105
Thr Ile Cys Leu Ala His Ala Leu Thr Va1 Leu Ser Gly His Trp
110 115 120
Ser Val His Ala His Cys Ala Leu Thr Cys Thr Pro Glu Tyr Asp
125 130 135
Pro Ser Lys Ala Thr Phe Val Lys Val Val Pro Thr Pro Asn Asn
140 145 150
Gly Ser Thr Glu Leu Val Ala Leu His Arg Asn Glu Gly Glu Asp
155 160 165
Gly Leu Glu Val Leu Ser Phe Glu Phe Gln Lys Ile Lys Tyr Ser
170 175 180
Tyr Asp AIa Leu Glu Lys Lys Gln Phe Leu Pro Val Ala Phe Pro
185 190 195
Val Gly Asn Ala Phe Ser Tyr Tyr Gln Ser Asn Arg Gly Phe Gln
200 205 210
Glu Asp Ser Glu Ile Arg Ala Ala Glu Lys Lys Phe Gly Ser Asn
215 220 225
Lys Ala Glu Met Val Val Pro Asp Phe Ser G1u Leu Phe Lys Glu
230 235 240
Arg Ala Thr Ala Pro Phe Phe Val Phe Gln Val Phe Cys Val Gly
245 250 255
Leu Trp Cys Leu Asp Glu Tyr Trp Tyr Tyr Ser Val Phe Thr Leu
260 265 270
Ser Met Leu Val Ala Phe Glu Ala Ser Leu Val Gln Gln Gln Met
275 280 285
Arg Asn Met Ser Glu Ile Arg Lys Met Gly Asn Lys Pro His Met
290 295 300
Ile Gln Val Tyr Arg Ser Arg Lys Trp Arg Pro Ile Ala Ser Asp
305 310 315
Glu Ile Val Pro Gly Asp~Ile Val Ser Ile Gly Arg Ser Pro Gln
320 325 330
Glu Asn Leu Val Pro Cys Asp Val Leu Leu Leu Arg Gly Arg Cys
335 340 345
Ile 'Jal Asp Glu Ala Met Leu Thr Gly Glu Ser Val Pro Gln Met
350 355 360
Lys Glu Pro Ile Glu Asp Leu Ser Pro Asp Arg Val Leu Asp Leu
365 370 375
Gln Ala Asp Ser Arg Leu His Val Ile Phe Gly Gly Thr Lys Val
380 385 390
Val Gln His Ile Pro Pro Gln Lys Ala Thr Thr Gly Leu Lys Pro
395 400 405
Val Asp Ser Gly Cys Val Ala Tyr Val Leu Arg Thr Gly Phe Asn
410 415 420
24/67
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Thr Ser Gln Gly Lys Leu Leu Arg Thr Ile Leu Phe Gly Val Lys
425 430 435
Arg Val Thr Ala Asn Asn Leu G1u Thr Phe Ile Phe Ile Leu Phe
440 445 450
Leu Leu Val Phe Ala Ile Ala Ala Ala Ala Tyr Val Trp Ile Glu
455 460 465
Gly Thr Lys Asp Pro Ser Arg Asn Arg Tyr Lys Leu Phe Leu Glu
470 475 480
Cys Thr Leu Ile Leu Thr Ser Val Val Pro Pro Glu Leu Pro Ile
485 490 495
Glu Leu Ser Leu Ala Val Asn Thr Ser Leu Ile Ala Leu Ala Lys
500 505 510
Leu Tyr Met Tyr Cys Thr Glu Pro Phe Arg Ile Pro Phe Ala Gly
515 520 525
Lys Val Glu Val Cys Cys Phe Asp Lys Thf Gly Thr Leu Thr Ser
530 535 540
Asp Ser Leu Val Val Arg Gly Val A1a Gly Leu Arg Asp Gly Lys
545 550 555
Glu Val Thr Pro Val Ser Ser Ile Pro Val Glu Thr His Arg Ala
560 565 570
Leu Ala Ser Cys His Ser Leu Met Gln Leu Asp Asp Gly Thr Leu
575 580 585
Val Gly Asp Pro Leu Glu Lys Ala Met Leu Thr Ala Val Asp Trp
590 595 600
Thr Leu Thr Lys Asp Glu Lys Val Phe Pro Arg Ser Ile Lys Thr
605 610 615
Gln Gly Leu Lys Ile His Gln Arg Phe His Phe Ala Ser Ala Leu
620 625 630
Lys Arg Met Ser Val Leu Ala Ser Tyr Glu Lys Leu Gly Ser Thr
635 640 645
Asp Leu Cys Tyr Ile Ala Ala Val Lys Gly Ala Pro Glu Thr Leu
650 655 660
His Ser Met Phe Ser Gln Cys Pro Pro Asp Tyr His His Ile His
665 670 675
Thr Glu Ile Ser Arg Glu Gly Ala Arg Val Leu Ala Leu Gly Tyr
680 685 690
Lys Glu Leu Gly His Leu Thr His Gln Gln Ala Arg Glu Val Lys
695 700 705
Arg Glu Ala Leu Glu Cys Ser Leu Lys Phe Val Gly Phe Ile Val
710 715 720
Val Ser Cys Pro Leu Lys Ala Asp Ser Lys Ala Val Ile Arg G1u
725 730 735
Ile Gln Asn Ala Ser His Arg Val Val Met Ile Thr Gly Asp Asn
740 745 750
Pro Leu Thr Ala Cys His Val Ala Gln Glu Leu His Phe Ile Glu
755 760 765
Lys Ala His Thr Leu Ile Leu Gln Pro Pro Ser Glu Lys Gly Arg
770 775 780
Gln Cys Glu Trp Arg Ser Ile Asp Gly Sex Ile Val Leu Pro Leu
785 790 795
Ala Arg Gly Ser Pro Lys Ala Leu Ala Leu Glu Tyr Ala Leu Cys
800 805 810
Leu Thr G1y Asp Gly Leu Ala His Leu Gln Ala Thr Asp Pro Gln
815 820 825
Gln Leu Leu Arg Leu Ile Pro His Val Gln Val Phe Ala Arg Val
830 835 840
Ala Pro Lys Gln Lys Glu Phe Val Ile Thr Ser Leu Lys Glu Leu
845 850 855
Gly Tyr Val Thr Leu Met Cys Gly Asp Gly Thr Asn Asp Val Gly
860 865 870
Ala Leu Lys His Ala Asp Val Gly Val Ala Leu Leu Ala Asn Ala
875 880 885
Pro Glu Arg Val Val Glu Arg Arg Arg Arg Pro Arg Asp Ser Pro
25/67
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890 895 900
Thr Leu Ser Asn Ser Gly Ile Arg Ala Thr Ser Arg Thr Ala Lys
905 910 915
Gln Arg Ser Gly Leu Pro Pro Ser Glu Glu Gln Pro Thr Ser Gln
920 925 930
Arg Asp Arg Leu Ser Gln Val Leu Arg Asp Leu Glu Asp Glu Ser
935 940 945
Thr Pro Ile Val Lys Leu Gly Asp Ala Ser Ile Ala Ala Pro Phe
950 955 960
Thr Ser Lys Leu Ser Ser Ile Gln Cys Ile Cys His Val Ile Lys
965 970 975
Gln Gly Arg Cys Thr Leu Val Thr Thr Leu Gln Met Phe Lys Ile
980 985 990
Leu Ala Leu Asn Ala Leu Ile Leu Ala Tyr Ser Gln Ser Val Leu
995 1000 1005
Tyr Leu Glu Gly Val Lys Phe Ser Asp Phe Gln Ala Thr Leu Gln
1010 1015 1020
Gly Leu Leu Leu Ala Gly Cys Phe Leu Phe Ile Ser Arg Ser Lys
1025 1030 1035
Pro Leu Lys Thr Leu Ser Arg Glu Arg Pro Leu Pro Asn Ile Phe
1040 1045 1050
Asn Leu Tyr Thr Ile Leu Thr Val Met Leu Gln Phe Phe Val His
1055 1060 2065
Phe Leu Ser Leu Val Tyr Leu Tyr Arg Glu Ala Gln Ala Arg Ser
1070 1075 1080
Pro Glu Lys Gln Glu Gln Phe Val Asp Leu Tyr Lys Glu Phe Glu
1085 1090 1095
Pro Ser Leu Val Asn Ser Thr Val Tyr Ile Met Ala Met Ala Met
1100 1105 1110
Gln Met Ala Thr Phe Ala Ile Asn Tyr Lys Gly Pro Pro Phe Met
1225 1120 2125
Glu Ser Leu Pro Glu Asn Lys Pro Leu Val Trp Ser Leu Ala Val
1230 1135 1140
Ser Leu Leu A1a Ile I1e Gly Leu Leu Leu Gly Ser Ser Pro Asp
1145 1150 1155
Phe Asn Ser Gln Phe Gly Leu Val Asp Ile Pro Val Glu Val Leu
1160 1165 1270
Leu Leu Asp Phe Cys Leu Ala Leu Leu Ala Asp Arg Val Leu Gln
2175 1180 1185
Phe Phe Leu Gly Thr Pro Lys Leu Lys Val Pro Ser
1190 1195
<210> 18
<212> 2771
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2311751CD1
<400> 18 '
Met Met Glu Arg Ala Ile Ile Asp Thr Phe Val Gly His Asp Val
1 5 10 15
Val Glu Pro Gly Ser Tyr Val Gln Met Phe Pro Tyr Pro Cys Tyr
20 25 30
Thr Arg Asp Asp Phe Leu Phe Val Ile Glu His Met Met Pro Leu
35 40 45
Cys Met Val Ile Ser Trp Val Tyr Ser Val Ala Met Thr Ile Gln
50 55 60
His Ile Val Ala Glu Lys Glu His Arg Leu Lys Glu Val Met Lys
65 70 75
Thr Met Gly Leu Asn Asn Ala Val His Trp Val Ala Trp Phe Ile
26/67
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80 85 90
Thr Gly Phe Val Gln Leu Ser Ile Ser Val Thr Ala Leu Thr Ala
95 100 105
Ile Leu Lys Tyr Gly Gln Val Leu Met His Ser His Val Val Ile
110 115 120
Ile Trp Leu Phe Leu Ala Val Tyr Ala Val Ala Thr I1e Met Phe
125 130 135
Cys Phe Leu Val Ser Val Leu Tyr Ser Lys Ala Lys Leu Ala Ser
140 145 150
Ala Cys Gly Gly Ile Ile Tyr Phe Leu Ser Tyr Val Pro Tyr Met
155 160 165
Tyr Val Ala Ile Arg Glu Glu Val Ala His Asp Lys Ile Thr Ala
170 175 180
Phe Glu Lys Cys Ile Ala Ser Leu Met Ser Thr Thr Ala Phe Gly
185 190 195
Leu Gly Ser Lys Tyr Phe Ala Leu Tyr Glu Val Ala Gly Val Gly
200 205 210
Ile Gln Trp His Thr Phe Ser Gln Ser Pro Val Glu Gly Asp Asp
215 220 225
Phe Asn Leu Leu Leu Ala Val Thr Met Leu Met Val Asp Ala Val
230 ~ 235 240
Val Tyr Gly Ile Leu Thr Trp Tyr Ile Glu Ala Val His Pro Gly
245 250 255
Met Tyr Gly Leu Pro Arg Pro Trp Tyr Phe Pro Leu Gln Lys Ser
260 265 270
Tyr Trp Leu Gly Ser Gly Arg Thr Glu Ala Trp Glu Trp Ser Trp
275 280 285
Pro Trp Ala Arg Thr Pro Arg Leu Ser Val Met Glu Glu Asp Gln
290 295 300
Ala Cys Ala Met Glu Ser Arg.Arg Phe Glu Glu Thr Arg Gly Met
305 310 315
Glu Glu Glu Pro Thr His Leu Pro Leu Val Val Cys Val Asp Lys
320 325 330
Leu Thr Lys Val Tyr Lys Asp Asp Lys Lys Leu Ala Leu Asn Lys
335 340 345
Leu Ser Leu Asn Leu Tyr Glu Asn Gln Val Va1 Ser Phe Leu Gly
350 355 360
His Asn Gly Ala Gly Lys Thr Thr Thr Met Ser Ile Leu Thr Gly
365 370 375
Leu Phe Pro Pro Thr Ser Gly Ser Ala Thr Ile Tyr Gly His Asp
380 385 390
Ile Arg Thr Glu Met Asp Glu Ile Arg Lys Asn Leu Gly Met Cys
395 400 405
Pro Gln His Asn Val Leu Phe Asp Arg Leu Thr Val Glu Glu His
410 415 420
Leu Trp Phe Tyr Ser Arg Leu Lys Ser Met Ala Gln Glu Glu Ile
425 430 435
Arg Arg Glu Met Asp Lys Met Ile Glu Asp Leu Glu Leu Ser Asn
440 445 450
Lys Arg His Ser Leu Val Gln Thr Leu Ser Gly Gly Met Lys Arg
455 460 465
Lys Leu Ser Val Ala Ile Ala Phe Val Gly Gly Ser Arg Ala Ile
470 475 480
Ile Leu Asp Glu Pro Thr Ala Gly Val Asp Pro Tyr Ala Arg Arg
485 490 495
Ala Ile Trp Asp Leu Ile Leu Lys Tyr Lys Pro GIy Arg Thr Ile
500 505 510
Leu Leu Ser Thr His His Met Asp Glu Ala Asp Leu Leu Gly Asp
515 520 525
Arg Ile A1a Ile Ile Ser His Gly Lys Leu Lys Cys Cys Gly Ser
530 535 540
Pro Leu Phe Leu Lys Gly Thr Tyr Gly Asp Gly Tyr Arg Leu Thr
545 550 555
27/67
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Leu Val Lys Arg Pro Ala Glu Pro Gly Gly Pro Gln Glu Pro G1y
560 565 570
Leu Ala Ser Ser Pro Pro Gly Arg Ala Pro Leu Ser Ser Cys Ser
575 580 585
Glu Leu Gln Val Ser Gln Phe Ile Arg Lys His Val Ala Ser Cys
590 595 600
Leu Leu Val Ser Asp Thr Ser Thr Glu Leu Ser Tyr Ile Leu Pro
605 610 615
Ser Glu Ala Ala Lys Lys Gly Ala Phe Glu Arg Leu Phe Gln His
620 625 630
Leu Glu Arg Ser Leu Asp Ala Leu His Leu Ser Ser Phe Gly Leu
635 640 645
Met Asp Thr Thr Leu Glu Glu Val Phe Leu Lys Val Ser Glu Glu
650 655 660
Asp Gln Ser Leu Glu Asn Ser Glu Ala Asp Val Lys Glu Ser Arg
665 670 675
Lys Asp Val Leu Pro Gly Ala Glu Gly Pro Ala Ser Gly Glu Gly
680 685 690
His Ala Gly Asn Leu Ala Arg Cys Ser Glu Leu Thr Gln Ser Gln
695 700 705
Ala Ser Leu Gln Ser Ala Ser Ser Val Gly Ser Ala Arg Gly Asp
710 715 720
Glu Gly Ala Gly Tyr Thr Asp Val Tyr Gly Asp Tyr Arg Pro Leu w
725 730 735
Phe Asp Asn Pro Gln Asp Pro Asp Asn Val Ser Leu Gln Glu Val
740 745 750
Glu Ala G1u Ala Leu Ser Arg Val Gly Gln Gly Ser Arg Lys Leu
755 ~ 760 765
Asp Gly Gly Trp Leu Lys Val Arg Gln Phe His Gly Leu Leu Val
770 775 780
Lys Arg Phe His Cys Ala Arg Arg Asn Ser Lys Ala Leu Phe Ser
785 790 795
Gln Ile Leu Leu Pro Ala Phe Phe Val Cys Val Ala Met Thr Val
800 805 810
Ala Leu Ser Val Pro Glu Ile Gly Asp Leu Pro Pro Leu Val Leu
815 820 825
Ser Pro Ser Gln Tyr His Asn Tyr Thr Gln Pro Arg Gly Asn Phe
830 835 840
Ile Pro Tyr Ala Asn Glu G1u Arg Arg Glu Tyr Arg Leu Arg Leu
845 850 855
Ser Pro Asp Ala Ser Pro Gln Gln Leu Val Ser Thr Phe Arg Leu
860 865 870
Pro Ser Gly Val Gly Ala Thr Cys Val Leu Lys Ser Pro Ala Asn
875 880 885
Gly Ser Leu Gly Pro Thr Leu Asn Leu Ser Ser Gly Glu Ser Arg
890 895 900
Leu Leu Ala Ala Arg Phe Phe Asp Ser Met Cys Leu Glu Ser Phe
905 910 915
Thr Gln Gly Leu Pro Leu Ser Asn Phe Val Pro Pro Pro Pro Ser
920 925 930
Pro Ala Pro Ser Asp Ser Pro Ala Ser Pro Asp Glu Asp Leu Gln
935 940 945
Ala Trp Asn Val Ser Leu Pro Pro Thr Ala Gly Pro Glu Met Trp
950 955 960
Thr Ser Ala Pro Ser Leu Pro Arg Leu Val Arg Glu Pro Val Arg
965 970 975
Cys Thr Cys Ser Ala Gln Gly Thr Gly Phe Ser Cys Pro Ser Ser
980 985 990
VaI Gly GIy His Pro Pro Gln Met Arg Val Val Thr Gly Asp Ile
995 1000 1005
Leu Thr Asp Ile Thr Gly His Asn Val Ser Glu Tyr Leu Leu Phe
1010 1015 1020
Thr Ser Asp Arg Phe Arg Leu His Arg Tyr Gly Ala Ile Thr Phe
28/67
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1.025 1030 1035
Gly Asn Val Leu Lys Ser Ile Pro Ala Ser Phe Gly Thr Arg A1a
1040 1045 1050
Pro Pro Met Val Arg Lys Ile Ala Val Arg Arg A1a Ala Gln Val
1055 1060 1065
Phe Tyr Asn Asn Lys Gly Tyr His Ser Met Pro Thr Tyr Leu Asn
1070 1075 1080
Ser Leu Asn Asn Ala Ile Leu Arg Ala Asn Leu Pro Lys Ser Lys
1085 1090 1095
Gly Asn Pro Ala Ala Tyr Gly Ile Thr Val Thr Asn His Pro Met
1100 1105 1110
Asn Lys Thr Ser Ala Ser Leu Ser Leu Asp Tyr Leu Leu Gln Gly
1115 1120 1125
Thr Asp Val Val Ile Ala Ile Phe Ile Ile Val Ala Met Sex Phe
1130 1135 1140
Val Pro Ala Ser Phe Val Val Phe Leu Val A1a Glu Lys Ser Thr
1145 1150 1155
Lys Ala Lys His Leu Gln Phe Val Ser Gly Cys Asn Pro Ile Ile
1160 1165 1170
Tyr Trp Leu Ala Asn Tyr Val Trp Asp Met Leu Asn Tyr Leu Val
1175 1180 1185
Pro Ala Thr Cys Cys Val Ile Ile Leu Phe Val Phe Asp Leu Pro
1190 1195 1200
Ala Tyr Thr Ser Pro Thr Asn Phe Pro Ala Val Leu Ser Leu Phe
1205 1210 1215
Leu Leu Tyr Gly Trp Ser Ile Thr Pro Tle Met Tyr Pro Ala Ser
1220 1225 1230
Phe Trp Phe Glu Val Pro Ser Ser Ala Tyr Val Phe Leu Ile Val
1235 1240 1245
Ile Asn Leu Phe Ile Gly Ile Thr A1a Thr Val Ala Thr Phe Leu
1250 1255 .1260
Leu Gln Leu Phe Glu His Asp Lys Asp Leu Lys Val Val Asn Ser
1265 1270 1275
Tyr Leu Lys Ser Cys Phe Leu Ile Phe Pro Asn Tyr Asn Leu Gly
1280 1285 1290
His Gly Leu Met Glu Met Ala Tyr Asn Glu Tyr Ile Asn Glu Tyr
1295 1300 1305
Tyr Ala Lys Ile Gly Gln Phe Asp Lys Met Lys Ser Pro Phe Glu
1310 1315 1320
Trp Asp Ile Val Thr Arg Gly Leu Val A1a Met Ala Val Glu Gly
1325 1330 1335
Val Val Gly Phe Leu Leu Thr Ile Met Cys Gln Tyr Asn Phe Leu
1340 1345 1350
Arg Arg Pro Gln Arg Met Pro Val Ser Thr Lys Pro Val Glu Asp
1355 1360 1365
Asp Val Asp Val Ala Ser Glu Arg Gln Arg Val Leu Arg Gly Asp
1370 1375 1380
Ala Asp Asn Asp Met Val Lys Ile Glu Asn Leu Thr Lys Val Tyr
1385 1390 1395
Lys Ser Arg Lys Ile Gly Arg Ile Leu Ala Val Asp Arg Leu Cys
1400 1405 1410
Leu Gly Val Arg Pro Gly Glu Cys Phe G1y Leu Leu Gly Val Asn
1415 1420 1425
Gly Ala Gly Lys Thr Ser Thr Phe Lys Met Leu Thr Gly Asp Glu
1430 1435 1440
Ser Thr Thr Gly Gly Glu Ala Phe Val Asn Gly His Ser Val Leu
1445 1450 1455
Lys Glu Leu Leu Gln Val Gln Gln Ser Leu Gly Tyr Cys Pro Gln
1460 1465 1470
Cys Asp Ala Leu Phe Asp Glu Leu Thr Ala Arg Glu His Leu Gln
1475 1480 1485
Leu Tyr Thr Arg Leu Arg Gly Ile Ser Trp Lys Asp Glu Ala Arg
1490 1495 1500
29/67
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Val Val Lys Trp Ala Leu Glu Lys Leu Glu Leu Thr Lys Tyr Ala
1505 1510 1515
Asp Lys Pro Ala Gly Thr Tyr Ser Gly Gly Asn Lys Arg Lys Leu
1520 1525 1530
Ser Thr Ala Ile Ala Leu Ile Gly Tyr Pro Ala Phe Ile Phe Leu
1535 2540 1545
Asp Glu Pro Thr Thr Gly Met Asp Pro Lys Ala Arg Arg Phe Leu
1550 1555 1560
Trp Asn Leu Ile Leu Asp Leu Ile Lys Thr Gly Arg Ser Val Val
1565 1570 1575
Leu Thr Ser His Ser Met Glu Glu Cys Glu Ala Leu Cys Thr Arg
1580 1585 1590
Leu Ala Ile Met Val Asn Gly Arg Leu Arg Cys Leu Gly Ser Ile
2595 1600 2605
Gln His Leu Lys Asn Arg Phe Gly Asp Gly Tyr Met Ile Thr Val
1610 1615 1620
Arg Thr Lys Ser Ser Gln Ser Val Lys Asp Val Val Arg Phe Phe
1625 1630 1635
Asn Arg Asn Phe Pro Glu Ala Met Leu Lys Glu Arg His His Thr
1640 1645 1650
Lys Val Gln Tyr Gln Leu Lys Ser Glu His Ile Ser Leu Ala Gln
1655 1660 1665
Val Phe Ser Lys Met Glu Gln Val Ser Gly Val Leu Gly Ile Glu
1670 1675 1680
Asp Tyr Ser Val Ser Gln Thr Thr Leu Asp Asn Val Phe Val Asn
1685 1690 1695
Phe Ala Lys Lys Gln Ser Asp Asn Leu Glu Gln Gln Glu Thr Glu
1700 1705 1710
Pro Pro Ser Ala Leu Gln Ser Pro Leu Gly Cys Leu Leu Ser Leu
1715 1720 1725
Leu Arg Pro Arg Ser Ala Pro Thr Glu Leu Arg Ala Leu Val Ala
1730 1735 1740
Asp Glu Pro Glu Asp Leu Asp Thr Glu Asp Glu Gly Leu Ile Ser
1745 ,1750 1755
Phe Glu Glu Glu Arg AIa Gln Leu Ser Phe Asn Thr Asp Thr Leu
1760 1765 1770
Cys
<210> 19
<211> 474
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472537CD1
<400> 19
Met Phe Ser Leu Ser Tyr Leu Cys Val Cys Val Phe Ser Gln Phe
1 5 10 15
Ala Asn Glu Asp Thr Glu Ser Gln Lys Phe Leu Thr Asn Gly Phe
20 25 30
Leu Gly Lys Lys Lys Leu Ala Asp Pro Phe Phe Phe Lys His Pro
35 40 45
Gly Thr Thr Ser Phe Gly Met Ser Ser Phe Asn Leu Ser Asn Ala
50 55 60
Ile Met G1y Ser Gly Ile Leu Gly Leu Ser Tyr Ala Met Ala Asn
65 70 75
Thr Gly Ile Ile Leu Phe Met Phe Met Leu Leu Ala Val Ala Ile
80 85 90
Leu Ser Leu Tyr Ser Val His Leu Leu Leu Lys Thr Ser Leu Ile
95 100 105
30/67
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Val Gly Ser Leu Ile Tyr Glu Lys Leu Gly Glu Lys Ala Phe Gly
110 115 120
Trp Pro Gly Lys Ile Gly Ala Phe Val Ser Ile Thr Met Gln Asn
125 130 135
Ile Gly Ala Met Ser Ser Tyr Leu Phe Ile Ile Lys Tyr Glu Leu
140 145 150
Pro Glu Val Ile Arg Ala Phe Met Gly Leu Glu Glu Thr Ser Arg
155 160 165
Glu Trp Tyr Leu Asn Gly Asn Tyr Leu I1e Ile Phe Val Ser Val
170 175 180
Gly Ile Ile Leu Pro Leu Ser Leu Leu Lys Asn Leu Gly Tyr Leu
185 190 195
G1y Tyr Thr Ser Gly Phe Ser Leu Thr Cys Met Val Phe Phe Val
200 205 210
Ser Val Val Ile Tyr Lys Lys Phe Gln Ile Pro Cys Pro Leu Pro
215 220 225
Glu Asn Gln Ala Lys Gly Ser Leu His Asp Ser Gly Val Glu Tyr
230 235 240
Glu Ala His Ser Asp Asp Lys Cys Glu Pro Lys Tyr Phe Val Phe
245 250 255
Asn Ser Gln Thr Ala Tyr Ala Ile Pro Ile Leu Val Phe Ala Phe
260 265 270
Val Cys His Pro Glu Val Leu Pro Ile Tyr Ser Glu Leu Lys Asp
275 280 285
Arg Ser Arg Arg Lys Met Gln Thr Val Ser Asn Ile Ser Ile Thr
290 295 300
Gly Met Leu Val Met Tyr Leu Leu Ala Ala Leu Phe Gly Tyr Leu
305 310 315
Thr Phe Tyr Gly Arg Val Glu Asp Glu Leu Leu His Ala Tyr Ser
320 325 330
Lys Val Tyr Thr Leu Asp Ile Pro Leu Leu Met Val Arg Leu Ala
335 340 345
Val Leu Val Ala Val Thr Leu Thr Val Pro Ile Val Leu Phe Pro
350 355 360
Val Arg Thr Ser Val Ile Thr Leu Leu Phe Pro Lys Arg Pro Phe
365 370 375
Ser Trp Ile Arg His Phe Leu Ile Ala Ala Va1 Leu Ile Ala Leu
380 385 390
Asn Asn Val Leu Val Ile Leu Val Pro Thr Ile Lys Tyr Ile Phe
395 400 405
Gly Phe Ile Gly Ala Ser Ser Ala Thr Met Leu Ile Phe Ile Leu
410 415 420
Pro Ala Val Phe Tyr Leu Lys Leu Val Lys Lys Glu Thr Phe Arg
425 430 435
Ser Pro Pro Glu Leu Gln Ala Leu Ile Phe Leu Val Val Gly Ile
440 445 450
Phe Phe Met I1e Gly Ser Met Ala Leu Ile Ile Ile Asp Trp Ile
455 460 465
Tyr Asp Pro Pro Asn Ser Lys His His
470
<210> 20
<211> 752
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472546CD1
<400> 20
Met Glu Tyr Gln Ala Ser Glu Val Ile Gly Gln Arg Gln Ser Ser
1 5 10 15
31/67
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Ala Thr Lys Pro Gly Arg Ser GIy Lys Glu Ser Val Thr Glu Pro
20 25 30
Trp Ala Arg Val Pro Gly Ala Leu Gly Val Ala Ala Arg Gln Met
35 40 45
His Pro Lys Ser Ile Ile Thr Phe Arg Glu Ile Asn Gly Glu Tyr
50 55 60
Thr Gly Ala Val Asp Phe Pro Arg Leu Gly Val Arg Ala Ser G1u
65 70 75
Glu Thr Ala Leu Arg Glu Leu Lys Met Ser Lys Glu Leu Ala Ala
80 85 90
Met Gly Pro Gly Ala Ser Gly Asp Gly Val Arg Thr Glu Thr Ala
95 100 105
Pro His Ile Ala Leu Asp Ser Arg VaI Gly Leu His Ala Tyr Asp
110 115 120
Ile Ser Val Val Val Ile Tyr Phe Val Phe Val Ile Ala Val Gly
125 ~ 130 135
Ile Trp Ser Ser Ile Arg Ala Ser Arg Gly Thr Ile Gly Gly Tyr
140 145 l50
Phe Leu Ala Gly Arg Ser Met Ser Trp Trp Pro Ile Gly Ala Ser
155 160 165
Leu Met Ser Ser Asn Val Gly Ser Gly Leu Phe Ile Gly Leu Ala
170 175 180
Gly Thr Gly Ala Ala Gly Gly Leu Ala Val Gly Gly Phe Glu Trp
185 290 195
Asn Ala Thr Trp Leu Leu Leu Ala Leu Gly Trp Val Phe Val Pro
200 205 210
Val Tyr Ile Ala Ala Gly Val Val Thr Met Pro Gln Tyr Leu Lys
215 220 225
Lys Arg Phe Gly Gly Gln Arg Ile Gln Val Tyr Met Ser Val Leu
230 235 240
Ser Leu Ile Leu Tyr Ile Phe Thr Lys Ile Ser Thr Asp Ile Phe
245 250 255
Ser Gly Ala Leu Phe Ile Gln Met Ala Leu Gly Trp'Asn Leu Tyr
260 265 270
Leu Ser Thr Gly Ile Leu Leu Val Val Thr Ala Val Tyr Thr Ile
275 280 285
Ala Gly Gly Leu Met Ala Val Ile Tyr Thr Asp Ala Leu GIn Thr
290 295 300
Val Ile Met Val Gly Gly Ala Leu Val Leu Met Phe Leu Gly Phe
305 310 315
Gln Asp Val Gly Trp Tyr Pro Gly Leu Glu Gln Arg Tyr Arg Gln
320 325 330
Ala Ile Pro Asn Val Thr Val Pro Asn Thr Thr Cys His Leu Pro
335 340 345
Arg Pro Asp Ala Phe His Ile Leu Arg Asp Pro Val Ser Gly Asp
350 355 360
Ile Pro Trp Pro GIy Leu Ile Phe Gly Leu Thr Val Leu Ala Thr
365 370 375
Trp Cys Trp Cys Thr Asp Gln Val Ile Val Gln Arg Ser Leu Ser
380 385 390
Ala Lys Ser Leu Ser His Ala Lys Gly Gly Ser Val Leu Gly Gly
395 400 405
Tyr Leu Lys Ile Leu Pro Met Phe Phe Ile Val Met Pro Gly Met
410 415 420
Ile Ser Arg Ala Leu Phe Pro Asp Glu Val Gly Cys Val Asp Pro
425 430 435
Asp Val Cys Gln Arg Ile Cys Gly Ala Arg Val Gly Cys Ser Asn
440 445 450
Ile Ala Tyr Pro Lys Leu Val Met Ala Leu Met Pro Val Gly Leu
455 460 465
Arg Gly Leu Met Ile Ala Val Ile Met Ala Ala Leu Met Ser Ser
470 475 480
Leu Thr Ser Ile Phe Asn Ser Ser Ser Thr Leu Phe Thr Ile Asp
32/67
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485 490 495
Val Trp Gln Arg Phe Arg Arg Lys Ser Thr Glu Gln Glu Leu Met
500 505 510
Val Val Gly Arg Val Phe Val Val Phe Leu Val Val Ile Ser Ile
515 520 525
Leu Trp Ile Pro Ile Ile Gln Ser Ser Asn Ser Gly Gln Leu Phe
530 535 540
Asp Tyr Ile Gln Ala Val Thr Ser Tyr Leu Ala Pro Pro Ile Thr
545 550 555
Ala Leu Phe Leu Leu Ala Ile Phe Cys Lys Arg Val Thr Glu Pro
560 565 570
Gly Ala Phe Trp Gly Leu Val Phe Gly Leu Gly Val Gly Leu Leu
575 580 585
Arg Met Ile Leu Glu Phe Ser Tyr Pro Ala Pro Ala Cys Gly Glu
590 595 600
Val Asp Arg Arg Pro Ala Val Leu Lys Asp Phe His Tyr Leu Tyr
605 610 615
Phe Ala Ile Leu Leu Cys Gly Leu Thr Ala Ile Val Tle Val Ile
620 625 630
Leu Thr Arg Leu Thr Trp Trp Thr Arg Asn Cys Pro Leu Ser Glu
635 640 645
Leu Glu Lys Glu Ala His G1u Ser Thr Pro Glu Ile Ser Glu Arg
650 655 660
Pro Ala Gly Glu Cys Pro Ala Gly Gly Gly Ala~Ala Glu Asn Ser
665 670 675
Ser Leu Gly Gln Glu Gln Pro Glu Ala Pro Ser Arg Ser Trp Gly
680 685 690
Lys Leu Leu Trp Ser Trp Phe Cys Gly Leu Ser Gly Thr Pro Glu
695 700 705
Gln Ala Leu Ser Pro Ala Glu Lys Ala Ala Leu Glu Gln Lys Leu
710 715 720
Thr Ser Ile Glu Glu Glu Pro Leu Trp Arg His Val Cys Asn Ile
725 730 735
Asn Ala Val Leu Leu Leu Ala Ile Asn Ile Phe Leu Trp Gly Tyr
740 745 750
Phe Ala
<210> 21
<211> 654
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7474202CD1
<400> 21
Met Glu Glu Leu Val Gly Leu Arg Glu Gly Phe Ser Gly Asp Pro
1 5 10 15
Val Thr Leu Gln Glu Leu Trp Gly Pro Cys Pro His Ile Arg Arg
20 25 30
Ala Ile Gln GIy Gly Leu Glu Trp Leu Lys Gln Lys Val Phe Arg
35 40 45
Leu Gly Glu Asp Trp Tyr Phe Leu Met Thr Leu Gly Val Leu Met
50 55 60
Ala Leu Val Ser Tyr Ala Met Asn Phe Ala Ile Gly Cys Val Val
65 70 75
Arg Gly Phe Ser Gln Ser Ile Thr Pro Ser Ser Gly Gly Ser Gly
80 85 90
Ile Pro Glu Leu Lys Thr Met Leu Ala Gly Val Ile Leu Glu Asp
95 100 205
Tyr Leu Asp Ile Lys Asn Phe Gly Ala Lys Val Val Gly Leu Ser
33/67
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110 115 120
Cys Thr Leu Ala Thr Gly Ser Thr Leu Phe Leu Gly Lys Val Gly
125 130 135
Pro Phe Val His Leu Ser Val Met Ile Ala Ala Tyr Leu Gly Arg
140 145 150
Val Arg Thr Thr Thr Ile Gly Glu Pro Glu Asn Lys Ser Lys Gln
155 160 165
Asn Glu Met Leu Val Ala Ala Ala Ala Val Gly Val Ala Thr Val
170 175 180
Phe Ala A1a Pro Phe Ser Gly Val Leu Phe Ser Ile Glu Val Met
185 190 195
Ser Ser His Phe Ser Val Arg Asp Tyr Trp Arg Gly Phe Phe Ala
200 205 210
Ala Thr Cys Gly Ala Phe Ile Phe Arg Leu Leu Ala Val Phe Asn
215 220 225
Ser Glu Gln Glu Thr Ile Thr Ser Leu Tyr Lys Thr Ser Phe Arg
230 235 240
Val Asp Val Pro Phe Asp Leu Pro Glu I1e Phe Phe Phe Val Ala
245 250 255
Leu Gly Gly Ile Cys Gly Val Leu Ser Cys Ala Tyr Leu Phe Cys
260 265 270
Gln Arg Thr Phe Leu Ser Phe Ile Lys Thr Asn Arg Tyr Ser Ser
275 280 285
Lys Leu Leu Ala Thr Ser Lys Pro Val Tyr Ser Ala Leu Ala Thr
290 295 300
Leu Leu Leu Ala Ser Ile Thr Tyr Pro Pro Gly Val Gly His Phe
305 310 315
Leu Ala Ser Arg Leu Ser Met Lys Gln His Leu Asp Ser Leu Phe
320 325 330
Asp Asn His Ser Trp Ala Leu Met Thr Gln Asn Ser Ser Pro.Pro
335 340 345
Trp Pro Glu Glu Leu Asp Pro Gln His Leu Trp Trp Glu Trp Tyr
350 355 360
His Pro Arg Phe Thr Ile Phe G1y Thr Leu Ala Phe Phe Leu Val
365 370 375
Met Lys Phe Trp Met Leu Ile Leu Ala Thr Thr Ile Pro Met Pro
380 385 390
Ala Gly Tyr Phe Met Pro Ile Phe Ile Leu Gly Ala Ala Ile Gly
395 400 405
Arg Leu Leu Gly Glu Ala Leu Ala Val Ala Phe Pro Glu Gly Ile
410 415 420
Val Thr Gly Gly Val Thr Asn Pro Ile Met Pro Gly Gly Tyr Ala
425 430 435
Leu Ala Gly Ala A1a Ala Phe Ser Gly Ala Val Thr His Thr Ile
440 445 450
Ser Thr Ala Leu Leu Ala Phe Glu Leu Thr Gly Gln Ile Val His
455 460 465
Ala Leu Pro Val Leu Met Ala Val Leu Ala Ala Asn Ala Ile Ala
470 475 480
Gln Ser Cys Gln Pro Ser Phe Tyr Asp Gly Thr Ile Ile Val Lys
485 490 495
Lys Leu Pro Tyr Leu Pro Arg Ile Leu Gly Arg Asn Ile Gly Ser
500 505 510
His His Val Arg Val Glu His Phe Met Asn His Ser Ile Thr Thr
515 520 525
Leu A1a Lys Asp Thr Pro Leu Glu Glu Val Val Lys Val Val Thr
530 535 540
Ser Thr Asp Val Thr Glu Tyr Pro Leu Val Glu Ser Thr Glu Ser
545 550 555
Gln Ile Leu Val Gly Ile Val Gln Arg Ala Gln Leu Val Gln Ala
560 565 570
Leu Gln Ala Glu Pro Pro Ser Arg Ala Pro Gly His Gln Cys Leu
575 580 585
34/67
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Gln Asp Ile Leu Ala Arg Gly Cys Pro Thr Glu Pro Val Thr Leu
590 595 600
Thr Leu Phe Ser Glu Thr Thr Leu His Gln Ala Gln Asn Leu Phe
605 610 615
Lys Leu Leu Asn Leu Gln Ser Leu Phe Val Thr Ser Arg Gly Arg
620 625 630
Ala Val Gly Cys Val Ser Trp Val Glu Met Lys Lys Ala Ile Ser
635 640 645
Asn Leu Thr Asn Pro Pro Ala Pro Lys
650
<210> 22
<211> 886
<212> PRT
<213> Homo Sapiens
<220>
<221> misC_feature
<223> Incyte ID No: 7476280CD1
<400> 22
Met Asp Pro T1e Thr Pro Asn Trp Thr Glu Ile Val Asn Arg Lys
1 5 10 15
Leu Ser Phe Pro Pro Pro Leu Leu Asp Ala Ile Gln Glu Gly Arg
20 25 30
Leu Gly Phe Val Gln Gln Leu Leu Glu Ser Glu Val Glu Ala Ala
35 40 45
Ser Ser Gly Pro Gly Trp Pro Leu Trp Asn Val Glu Glu Ala Glu
50 55 60
Asp Arg Cys Trp Arg Glu Ala Leu Asn Leu Ala Ile Arg Leu Gly
65 70 75
His Glu Ala Leu Thr Asp Val Leu Leu Ala Ser Val Lys Phe Asp
80 85 90
Phe Arg Gln Ile His Glu Ala Leu Leu Val Ala Val Asp Thr Asn
95 100 105
Gln Ala Val Val Arg Arg Leu Pro Ala Arg Leu Glu Arg Glu Lys
110 115 120
Gly Arg Lys Val Asp Thr Arg Ser Phe Ser Leu Ala Phe Phe Asp
125 130 135
Ser Ser Ile Asp Gly Ser Arg Phe Ala Pro Gly Val Thr Pro Leu
140 145 150
Pro Gln Ala Cys Gln Lys Asp Leu Tyr Glu Ile Ala Gln Leu Leu
155 160 165
Met Glu Gln Gly His Thr Ile Ala Arg Pro His Pro Val Ser Cys
170 175 180
Ala Cys Leu Glu Cys Ser Asn Ala Arg Arg Tyr Asp Leu Leu Lys
185 190 195
Leu Ser Leu Ser Arg Ile Asn Thr Tyr Leu Gly Ile Ala Ser Arg
200 205 210
Ala His Leu Ser Leu AIa Ser Glu Asp Ala Met Leu Ala Ala Phe
215 220 225
Gln Leu Ser Arg Glu Leu Arg Arg Leu Ala Arg Lys Glu Pro Glu
230 235 240
Phe Lys Pro Glu Tyr Ile Ala Leu Glu Ser Leu Ser Gln Asp Tyr
245 250 255
Gly Phe Gln Leu Leu Gly Met Cys Trp Asn Gln Ser Glu Val Thr
260 265 270
Ala Val Leu Asn Asp Leu Ala Glu Asp Ser Glu Thr Glu Pro Glu
275 280 285
Ala Glu Gly Leu Gly Leu Ala Phe Glu Glu Gly Ile Pro Asn Leu
290 295 300
Val Arg Leu Arg Leu Ala Val Asn Tyr Asn Gln Lys Arg Phe Val
305 310 315
35167
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Ala His Leu Ile Cys Gln Gln Val Leu Ser Ser Ile Trp Cys Gly
320 325 330
Asn Leu Ala Gly Trp Arg Gly Ser Thr Thr Ser Trp Lys Leu Phe
335 340 345
Ala Thr Phe Leu Ile Phe Leu Thr Met Pro Phe Leu Cys Leu Gly
350 355 360
Tyr Trp Leu Thr Pro Lys Ser Gln Leu Gly His Leu Leu Lys Ile
365 370 375
Pro Val Leu Lys Phe Leu Leu His Ser Ala Ser Tyr Leu Trp Phe
380 385 390
Leu Ile Phe Leu Leu Gly Glu Ser Leu Val Met Glu Thr Gln Leu
395 400 405
Ser Thr Phe Arg Gly Arg Ser Gln Ser Val Trp Glu Thr Ser Leu
410 415 420
His Met Ile Cys Val Thr Gly Phe Leu Trp Phe Glu Cys Lys Glu
425 430 435
Val Trp Ile Glu Gly Leu Arg Ser Tyr Leu Leu Asp Trp Trp Asn
440 445 450
Phe Leu Asp Met Val Val Leu Ser Leu Tyr Leu Ala Ala Phe Ala
455 460 465
Leu Arg Leu Leu Leu Ala Gly Leu Ala Pro Met His Cys Arg Asp
470 475 480
Ala Ser Gln Ala Ala Ala Cys His Tyr Phe Thr Met Ala Glu Arg
485 490 495
Ser Glu Trp His Thr G1u Asp Pro Gln Phe Leu Ala Glu Val Leu
500 505 5l0
Phe Thr Ala Thr Ser Met Leu Ser Phe Thr Arg Leu Ala Tyr Ile
515 520 525
Leu Pro Ala His Glu Ser Leu Gly Thr Leu Gln Ile Ser Ile Gly
530 535 540
Lys Met Ile Glu Asp Met Ile Arg Phe Met Phe Ile Leu Met Ile
545 550 555
Ile Leu Thr Ala Phe Leu Cys Gly Leu Asn Asn Ile Tyr Val Pro
560 565 570
Tyr Gln Lys Thr Glu Trp Leu Gly Lys Ser Phe Asn Glu Thr Phe
575 580 585
Gln Phe Leu Phe Trp Thr Met Phe Gly Met Glu Glu His Ser Val
590 595 600
Val Asp Val Pro Gln Phe Leu Val Pro Glu Phe Ala Gly Arg Ala
605 610 615
Leu Tyr Gly Ile Phe Thr Ile Ile Met Val Ile Val Leu Leu Asn
620 625 630
Met Leu Ile Ala Met Ile Thr Asn Ser Phe Gln Lys Ile Glu Asp
635 640 645
Asp Ala Asp Val Glu Trp Thr Phe Ala Arg Ser Lys Leu Tyr Leu
650 655 660
Phe Tyr Phe Arg Glu Gly Leu Thr Leu Pro Val Pro Phe Asn Ile
665 670 675
Leu Pro Ser Ser Lys Ala Val Phe Tyr Leu Leu Arg Arg Ile Cys
680 685 690
Gln Phe Ile Cys Cys Cys Cys Ser Cys Cys Lys Thr Lys Lys Pro
695 700 705
Asp Tyr Pro Pro Ile Pro Thr Phe Val Asn Pro Arg Ala Gly Ala
710 . 715 720
Val Pro Gly Glu Gly Glu Arg Gly Ser Tyr Arg Leu His Val Ile
725 730 735
Lys Ala Leu Val Gln Arg Tyr Thr Glu Thr Ala Arg Arg Glu Phe
740 745 750
Glu Glu Thr Arg Arg Lys Asp Leu Gly Asn Arg Leu Thr Glu Leu
755 760 765
Thr Lys Thr Ile Ser Arg Leu Gln Ser Glu Val Ala Gly Val Arg
770 775 780
Arg Thr Leu Ala Glu Gly Gly Thr Pro Arg Pro Pro Asp Gly Ala
36/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
785 790 795
Ser Val Leu Ser His Tyr Ile Thr Gln Val His Asn Ser Phe Gln
800 805 810
Asn Leu G1y Pro Pro Ile Pro Glu Thr Pro Glu Leu Thr Gly Pro
815 820 825
Gly Ile Val Arg Thr Gln Glu Ser Ser Gly Thr Gly Leu Gln Asp
830 835 840
Thr Gly Gly Val Arg Thr Leu Ala Ser Gly GIu Ser Gly Pro Cys
845 850 855
Ser Pro Ala His Val Leu Val His Arg Glu Gln Glu Ala Glu Gly
860 865 870
Ala Gly Asp Leu Pro Gln Gly Glu Asp Ser Gly Thr Glu Arg Arg
875 880 885
Ser
<210> 23
<211> 512
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1713377CD1
<400> 23
Met Ala Gly Gly Met Ser Ala Glu Cys Pro Glu Pro Gly Pro Gly
1 5 10 15
Gly Leu Gln Gly Gln Ser Pro Gly Pro Gly Arg Gln Cys Pro Pro
20 25 30
Pro Ile Thr Pro Thr Ser Trp Ser Leu Pro Pro Trp Arg Ala Tyr
35 40 45
Val Ala Ala Ala Val Leu Cys Tyr Ile Asn Leu Leu Asn Tyr Met
50 55 60
Asn Trp Phe Ile Ile Ala Gly Val Leu Leu Asp Ile G1n Glu Val
65 70 75
Phe Gln Ile Ser Asp Asn His Ala Gly Leu Leu Gln Thr Val Phe
80 85 90
Val Ser Cys Leu Leu Leu Ser Ala Pro Val Phe Gly Tyr Leu Gly
95 100 105
Asp Arg His Ser Arg Lys Ala Thr Met Ser Phe Gly Ile Leu Leu
110 115 120
Trp Ser Gly Ala Gly Leu Ser Ser Ser Phe Ile Ser Pro Arg Tyr
125 130 135
Ser Trp Leu Phe Phe Leu Ser Arg Gly Ile Val Gly Thr Gly Ser
140 145 250
Ala Ser Tyr Ser Thr Ile Ala Pro Thr Val Leu Gly Asp Leu Phe
155 160 165
Val Arg Asp Gln Arg Thr Arg Val Leu Ala Val Phe Tyr Ile Phe
170 175 180
Ile Pro Val Gly Ser Gly Leu Gly Tyr Val Leu Gly Ser Ala Val
185 190 195
Thr Met Leu Thr Gly Asn Trp Arg Trp Ala Leu Arg Val Met Pro
200 205 210
Cys Leu Glu Ala Val Ala Leu Ile Leu Leu Ile Leu Leu Val Pro
215 220 225
Asp Pro Pro Arg Gly Ala Ala Glu Thr Gln Gly Glu Gly Ala Val
230 235 240
Gly Gly Phe Arg Ser Ser Trp Cys Glu Asp Val Arg Tyr Leu Gly
245 250 255
Lys Asn Trp Ser Phe Val Trp Ser Thr Leu Gly Val Thr Ala Met
260 265 270
Ala Phe Val Thr Gly Ala Leu Gly Phe Trp Ala Pro Lys Phe Leu
37/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
275 280 285
Leu Glu Ala Arg Val Val His Gly Leu Gln Pro Pro Cys.Phe Gln
290 295 300
Glu Pro Cys Ser Asn Pro Asp Ser Leu Ile Phe Gly Ala Leu Thr
305 310 315
Ile Met Thr Gly Va1 Ile Gly Val Ile Leu Gly Ala Glu Ala Ser
320 325 330
Arg Arg Tyr Lys Lys Val Ile Pro Gly Ala Glu Pro Leu Ile Cys
335 340 345
Ala Ser Ser Leu Leu Ala Thr Ala Pro Cys Leu Tyr Leu Ala Leu
350 355 360
Val Leu Ala Pro Thr Thr Leu Leu Ala Ser Tyr Val Phe Leu G1y
365 370 375
Leu Gly Glu Leu Leu Leu Ser Cys Asn Trp Ala Val Val Ala Asp
380 385 390
Ile Leu Leu Ser Val Val Val Pro Arg Cys Arg Gly Thr Ala Glu
395 400 405
Ala Leu Gln Ile Thr Val Gly His Ile Leu Gly Asp A1a Gly Ser
410 415 420
Pro Tyr Leu Thr Gly Leu Ile Ser Ser Val Leu Arg Ala Arg Arg
425 430 435
Pro Asp Ser Tyr Leu Gln Arg Phe Arg Ser Leu Gln Gln Ser Phe
440 445 450
Leu Cys Cys Ala Phe Val Ile Ala Leu Gly Gly Gly Cys Phe Leu
455 460 465
Leu Thr Ala Leu Tyr Leu Glu Arg Asp Glu Thr Arg Ala Trp Gln
470 475 480
Pro Val Thr Gly Thr Pro Asp Ser Asn Asp Val Asp Ser Asn Asp
485 490 495
Leu Glu Arg Gln Gly Leu Leu Ser Gly Ala Gly Ala Ser Thr Glu
500 505 510
Glu Pro
<210> 24
<211> 475 '
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5842557CD1
<400> 24
Met Ile Pro Ala Tyr Ser Lys Asn Arg Ala Tyr Ala Ile Phe Phe
1 5 10 15
Ile Val Phe Thr Val Ile Gly Ser Leu Phe Leu Met Asn Leu Leu
20 25 30
Thr Ala Ile Ile Tyr Ser Gln Phe Arg Gly Tyr Leu Met Lys Ser
35 4 40 45
Leu Gln Thr Ser Leu Phe Arg Arg Arg Leu Gly Thr Arg Ala Ala
50 55 60
Phe Glu Val Leu Ser Ser Met Val Gly Glu Gly Gly Ala Phe Pro
65 70 75
Gln Ala Val Gly Val Lys Pro Gln Asn Leu Leu Gln Val Leu Gln
80 85 90
Lys Val Gln Leu Asp Ser Ser His Lys Gln Ala Met Met Glu Lys
95 100 105
Val Arg Ser Tyr Asp Ser Val Leu Leu Ser Ala Glu Glu Phe. Gln
110 115 120
Lys Leu Phe Asn Glu Leu Asp Arg Ser Val Val Lys Glu His Pro
125 130 135
Pro Arg Pro Glu Tyr Gln Ser Pro Phe Leu Gln Ser Ala Gln Phe
38167
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
140 145 150
Leu Phe Gly His Tyr Tyr Phe Asp Tyr Leu Gly Asn Leu Ile Ala
155 160 165
Leu Ala Asn Leu Val Ser Ile Cys Val Phe Leu Val Leu Asp Ala
170 175 180
Asp Val Leu Pro Ala Glu Arg Asp Asp Phe Ile Leu Gly Ile Leu
185 190 195
Asn Cys Val Phe Ile Val Tyr Tyr Leu Leu Glu Met Leu Leu Lys
200 205 210
Val Phe Ala Leu Gly Leu Arg Gly Tyr Leu Ser Tyr Pro Ser Asn
215 220 225
Val Phe Asp Gly Leu Leu Thr Val Va1 Leu Leu Val Leu Glu Ile
230 235 240
Ser Thr Leu Ala Val Tyr Arg Leu Pro His Pro Gly Trp Arg Pro
245 250 255
Glu Met Val Gly Leu Leu Ser Leu Trp Asp Met Thr Arg Met Leu
260 265 270
Asn Met Leu Ile Val Phe Arg Phe Leu Arg Ile Ile Pro Ser Met
275 280 285
Lys Pro Met Ala Val Val Ala Ser Thr Val Leu Gly Leu Val Gln
290 295 300
Asn Met Arg Ala Phe Gly Gly Ile Leu Val Val Val Tyr Tyr Val
305 310 315
Phe Ala Ile Ile Gly Ile Asn Leu Phe Arg Gly Val Ile Val Ala
320 325 330
Leu Pro Gly Asn Ser Ser Leu Ala Pro Ala Asn Gly Ser Ala Pro
335 340 345
Cys Gly Ser Phe Glu Gln Leu Glu Tyr Trp Ala Asn Asn Phe Asp
350 355 360
Asp Phe Ala Ala Ala Leu Val Thr Leu Trp Asn Leu Met Val Val
365 370 375
Asn Asn Trp Gln Val Phe Leu Asp Ala Tyr Arg Arg Tyr Ser Gly
380 385 390
Pro Trp Ser Lys Ile Tyr Phe Val Leu Trp Trp Leu Val Ser Ser
395 400 405
Val Ile Trp Val Asn Leu Phe Leu Ala Leu Ile Leu Glu Asn Phe
410 415 420
Leu His Lys Trp Asp Pro Arg Ser His Leu Gln Pro Leu Ala Gly
425 430 435
Thr Pro Glu Ala Thr Tyr Gln Met Thr Val Glu Leu Leu Phe Arg
440 445 450
Asp Ile Leu Glu Glu Pro Glu Glu Asp Glu Leu Thr Glu Arg Leu
455 460 465
Ser Gln His Pro His Leu Trp Leu Cys Arg
470 475
<210> 25
<211> 537
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7476643CD1
<400> 25
Met Ala Arg Lys Gln Asn Arg Asn Ser Lys Glu Leu Gly Leu Val
1 5 l0 15
Pro Leu Thr Asp Asp Thr Ser His Ala Arg Pro Pro Gly Pro Gly
20 25 30
Arg Ala Leu Leu Glu Cys Asp His Leu Arg Ser Gly Val Pro Gly
35 40 45
Gly Arg Arg Arg Lys Asp Trp Ser Cys Ser Leu Leu Val Ala Ser
39/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
50 55 60
Leu Ala Gly Ala Phe Gly Ser Ser Phe Leu Tyr Gly Tyr Asn Leu
65 70 75
Ser Val Val Asn Ala Pro Thr Pro Tyr Ile Lys Ala Phe Tyr Asn
80 85 90
Glu Ser Trp Glu Arg Arg His Gly Arg Pro Ile Asp Pro Asp Thr
95 100 105
Leu Thr Leu Leu Trp Ser Val Thr Val Ser Ile Phe Ala I1e Gly
110 115 120
Gly Leu Val Gly Thr Leu Ile Val Lys Met Ile Gly Lys Val Leu
125 130 135
Gly Arg Lys His Thr Leu Leu Ala Asn Asn Gly Phe Ala Ile Ser
140 145 150
Ala Ala Leu Leu Met Ala Cys Ser Leu Gln Ala Gly Ala Phe Glu
155 160 265
Met Leu Ile Val Gly Arg Phe Ile Met Gly Ile Asp G1y Gly Val
170 175 180
Ala Leu Ser Val Leu Pro Met Tyr Leu Ser Glu Ile Ser Pro Lys
185 190 195
Glu Ile Arg Gly Ser Leu Gly Gln Val Thr Ala Ile Phe Ile Cys
200 205 210
Ile Gly Val Phe Thr Gly Gln Leu Leu Gly Leu Pro Glu Leu Leu
215 220 ~ 225
Gly Lys Glu Ser Thr Trp Pro Tyr Leu Phe Gly Val Ile Val Val
230 235 240
Pro Ala Val Val Gln Leu Leu Ser Leu Pro Phe Leu Pro Asp Ser
245 250 255
Pro Arg Tyr Leu Leu Leu Glu Lys His Asn Glu Ala Arg Ala Val
260 265 270
Lys Ala Phe Gln Thr Phe Leu Gly Lys Ala Asp Val Ser Gln Glu
275 280 285
Val Glu Glu Val Leu Ala Glu Ser Arg Val Gln Arg Ser Ile Arg
290 295 300
Leu Val Ser Val Leu Glu Leu Leu Arg Ala Pro Tyr Val Arg Trp
305 310 315
Gln Val Val Thr Val Ile Val Thr Met Ala Cys Tyr Gln Leu Cys
320 325 330
Gly Leu Asn A1a Ile Trp Phe Tyr Thr Asn Ser Ile Phe Gly Lys
335 340 345
Ala Gly Ile Pro Leu Ala Lys Ile Pro Tyr Val Thr Leu Ser Thr
350 355 360
Gly Gly Ile Glu Thr Leu Ala Ala Val Phe Ser Gly Leu Val Ile
365 370 375
Glu His Leu Gly Arg Arg Pro Leu Leu Ile Gly Gly Phe Gly Leu
380 385 390
Met Gly Leu Phe Phe Gly Thr Leu Thr Ile Thr Leu Thr Leu Gln
395 400 405
Asp His Ala Pro Trp Val Pro Tyr Leu Ser Ile Val Gly Ile Leu
410 415 420
Ala Ile Ile Ala Ser Phe Cys Ser Gly Pro Gly Gly Ile Pro Phe
425 430 435
Ile Leu Thr Gly Glu Phe Phe Gln Gln Ser Gln Arg Pro Ala Ala
440 445 450
Phe Ile Ile Ala Gly Thr Val Asn Trp Leu Ser Asn Phe Ala Val
455 460 465
Gly Leu Leu Phe Pro Phe Ile Gln Lys Ser Leu Asp Thr Tyr Cys
470 475 480
Phe Leu Val Phe Ala Thr Ile Cys Ile Thr Gly Ala Ile Tyr Leu
485 490 495
Tyr Phe Val Leu Pro Glu Thr Lys Asn Arg Thr Tyr Ala Glu Ile
500 505 510
Ser Gln Ala Phe Ser Lys Arg Asn Lys Ala Tyr Pro Pro Glu Glu
515 520 525
40/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
Lys Ile Asp Ser Ala Val Thr Asp Ala Gln Arg Asn
530 535
<210> 26
<211> 905
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7611651CD1
<400> 26
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 GIy 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 G1y 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
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
41/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
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 4l5 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 A1a 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 G1n 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 G1y 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 Ala Gly Gly Leu His Ser Ser Pro Arg Gln Ala
665 670 675
Pro Gly Ser Gln Asp His Gln Gly Phe Phe Leu Ser Asp Asn Gln
680 685 690
Ser Asp Ala Ala Pro Pro Leu Ser IIe Ser Asp Ala Ser GIy Leu
695 700 705
Trp Pro Glu Leu Leu Gln Glu Met Pro Pro Arg His Ser Pro Gln
7l0 715 720
Ser Pro Gln Glu Asp Pro Asp Cys Trp Pro Leu Lys Leu GIy Ser
725 730 735
Arg Leu Glu Gln Leu Gln Ala GIn Met Asn Arg Leu Glu Ser Arg
740 745 750
Val Ser Ser Asp Leu Ser Arg Ile Leu GIn Leu Leu Gln Lys Pro
755 760 765
Met Pro Gln Gly His Ala Ser Tyr Ile Leu Glu AIa Pro Ala Ser
770 775 780
Asn Asp Leu Ala Leu Val Pro Ile Ala Ser Glu Thr Thr Ser Pro
785 790 795
Gly Pro Arg Leu Pro Gln Gly Phe Leu Pro Pro Ala Gln Thr Pro
800 805 810
Ser Tyr Gly Asp Leu Asp Asp Cys Ser Pro Lys His Arg Asn Ser
815 820 825
Ser Pro Arg Met Pro His Leu Ala Val Ala Met Asp Lys Thr Leu
830 835 840
Ala Pro Ser Ser Glu Gln Glu Gln Pro Glu Gly Leu Trp Pro Pro
42/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
845 850 855
Leu Ala Ser Pro Leu His Pro Leu Glu Val Gln Gly Leu Ile Cys
860 865 870
Gly Pro Cys Phe Ser Ser Leu Pro Glu His Leu Gly Ser Val Pro
875 880 885
Lys Gln Leu Asp Phe Gln Arg His Gly Ser.Asp Pro Gly Phe Ala
890 895 900
Gly Ser Trp Gly His
905
<210> 27
<211> 686
<212> PRT
<213> Homo Sapiens
<220>
<222> misc_feature
<223> Incyte ID No: 2522075CD1
<400> 27
Met Ala Glu Ala Ala Glu Pro Glu Gly Val Ala Pro Gly Pro Gln
1 5 10 15
Gly Pro Pro Glu Val Pro Ala Pro Leu Ala Glu Arg Pro Gly Glu
20 25 30
Pro Gly Ala Ala Gly Gly Glu Ala Glu Gly Pro Glu.Gly Ser Glu
35 40 45
Gly Ala Glu Glu Ala Pro Arg Gly Ala Ala Ala Val Lys Glu Ala
50 55 60
Gly Gly G1y Gly Pro Asp Arg Gly Pro Glu Ala Glu Ala Arg Gly
65 70 75
Thr Arg Gly Ala His Gly Glu Thr Glu Ala Glu Glu Gly Ala Pro
80 85 90
Glu Gly Ala Glu Val Pro Gln Gly Gly Glu Glu Thr Ser Gly Ala
95 200 205
Gln Gln Val Glu G1y Ala Ser Pro Gly Arg Gly Ala Gln Gly Glu
110 115 220
Pro Arg Gly Glu Ala Gln Arg Glu Pro Glu Asp Ser Ala Ala Pro
125 130 135
Glu Arg Gln Glu Glu Ala Glu Gln Arg Pro Glu Val Pro Glu Gly
140 145 150
Ser Ala Ser Gly Glu Ala Gly Asp Ser Val Asp Ala Glu Gly Pro
255 260 165
Leu Gly Asp Asn Ile Glu Ala Glu Gly Pro Ala Gly Asp Ser Val
170 175 180
Glu Ala Glu Gly Arg Val Gly Asp Ser Val Asp Ala Glu Gly Pro
185 190 195
Ala Gly Asp Ser Val Asp Ala Glu G1y Pro Leu Gly Asp Asn Ile
200 205 210
Gln Ala Glu Gly Pro Ala Gly Asp Ser Val Asp Ala Glu Gly Arg
215 220 225
Val Gly Asp Ser Val Asp Ala Glu Gly Pro Ala Gly Asp Ser Val
230 235 240
Asp Ala Glu Gly Arg Val Gly Asp Ser Val Glu Ala Gly Asp Pro
245 250 255
Ala Gly Asp Gly Val Glu Ala Gly Val Pro Ala Gly Asp Ser Val
260 265 270
Glu Ala Glu Gly Pro Ala Gly Asp Ser Met Asp Ala Glu Gly Pro
275 280 285
Ala Gly Arg Ala Arg Arg Val Ser Gly Glu Pro Gln Gln Ser Gly
290 295 300
Asp Gly Ser Leu Ser Pro Gln Ala Glu Ala Ile Glu Val Ala Ala
305 310 315
Gly Glu Ser Ala Gly Arg Ser Pro Gly Glu Leu Ala Trp Asp Ala
43/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
320 325 330
Ala Glu Glu Ala Glu Val Pro Gly Val Lys Gly Ser Glu Glu Ala
335 340 345
Ala Pro Gly Asp Ala Arg Ala Asp Ala Gly Glu Asp Arg Val Gly
350 355 360
Asp Gly Pro Gln Gln Glu Pro Gly Glu Asp Glu Glu Arg Arg Glu
365 370 375
Arg Ser Pro Glu Gly Pro Arg Glu Glu Glu Ala Ala Gly Gly Glu
380 385 390
Glu Glu Ser Pro Asp Ser Ser Pro His Gly Glu Ala Ser Arg Gly
395 400 405
Ala Ala Glu Pro Glu Ala Gln Leu Ser Asn His Leu Ala Glu Glu
410 415 420
Gly Pro Ala Glu Gly Ser Gly Glu Ala Ala Arg Val Asn Gly Arg
425 430 435
Arg Glu Asp Gly Glu Ala Ser Glu Pro Arg Ala Leu Gly Gln Glu
440 445 450
His Asp Ile Thr Leu Phe Val Lys Ala Gly Tyr Asp Gly Glu Ser
455 460 465
Ile Gly Asn Cys Pro'Phe Ser Gln Arg Leu Phe Met Ile Leu Trp
470 475 480
Leu Lys Gly Val Ile Phe Asn Val Thr Thr Val Asp Leu Lys Arg
485 490 495
Lys Pro Ala Asp Leu Gln Asn Leu Ala Pro Gly Thr Asn Pro Pro
500 505 510
Phe Met Thr Phe Asp Gly Glu Val Lys Thr Asp Val Asn Lys Ile
515 520 525
Glu Glu Phe Leu Glu Glu Lys Leu Ala Pro Pro Arg Tyr Pro Lys
530 535 540
Leu Gly Thr Gln His Pro Glu Ser Asn Ser Ala Gly Asn Asp Val
545 550 555
Phe Ala Lys Phe Ser Ala Phe Ile Lys Asn Thr Lys Lys Asp Ala
560 565 570
Asn Glu Ile His Glu Lys Asn Leu Leu Lys Ala Leu Arg Lys Leu
575 580 585
Asp Asn Tyr Leu Asn Ser Pro Leu Pro Asp Glu Tle Asp Ala Tyr
590 595 600
Ser Thr Glu Asp Val Thr Val Ser Gly Arg Lys Phe Leu Gly Gly
605 610 615
Asp Glu Leu Thr Leu Ala Asp Cys Asn Leu Leu Pro Lys Leu His
620 625 630
Tle Ile Lys Ile Val Ala Lys Lys Tyr Arg Asp Phe Glu Phe Pro
635 640 645
Ser Glu Met Thr Gly Ile Trp Arg Tyr Leu Asn Asn Ala Tyr Ala
650 655 660
Arg Asp Glu Phe Thr Asn Thr Cys Pro Ala Asp Gln Glu Ile Glu
665 670 675
His Ala Tyr Ser Asp Val Ala Lys Arg Met Lys
680 685
<210> 28
<211> 2984
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7475353CB1
<400> 28
gttggcagaa gggtcccggg cccagagcca gcggggccgt gctgagacgg cgtacgtgcc 60
ctgcgtgagt gcgtggcggc ggcgcgtgcg ctaggggagt gggcggtgag gcctggtcca 120
cgtgcgtccc ttcccgggac ccccgcagct tggcgcccag cggctacgtg agccaaggca 180
44/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
cccggatgtc cgcgcccctc tccgagtgac aagtcccggc ctccggtccc gcagtgcccg 240
cagcctcggc cggcgtccac gcattgccat ggtgactgtg ggcaactact gcgaggccga 300
agggcccgtg ggtccggcct ggatgcagga tggcctgagt ccctgcttct tcttcacgct 360
cgtgccctcg acgcggatgg ctctagggac tctggccttg gtgctggctc ttccctgcag 420
acgccgggag cggcccgctg gtgctgattc gctgtcttgg ggggccggcc ctcgcatctc 480
tccctacgtg ctgcagctgc ttctggccac acttcaggcg gcgctgcccc tggccggcct 540
ggctggccgg gtgggcactg cccggggggc cccactgcca agctatctac ttctggcctc 600
cgtgctggag agtctggccg gcgcctgtgg cctgtggctg cttgtcgtgg agcggagcca 660
ggcacggcag cgtctggcaa tgggcatctg gatcaagttc aggcacagcc ctggtctcct 720
gctcctctgg actgtggcgt ttgcagctga gaacttggcc ctggtgtctt ggaacagccc 780
acagtggtgg tgggcaaggg cagacttggg ccagcaggtt cagtttagcc tgtgggtgct 840
gcggtatgtg gtctctggag ggctgtttgt cctgggtctc tgggcccctg gacttcgtcc 900
ccagtcctat acattgcagg ttcatgaaga ggaccaagat gtggaaagga gccaggttcg 960
gtcagcagcc caacagtcta cctggcgaga ttttggcagg aagctccgcc tcctgagtgg 1020
ctacctgtgg cctcgaggga gtccagctct gcagctggtg gtgctcatct gcctggggct 1080
catgggtttg gaacgggcac tcaatgtgtt ggtgcctata ttctatagga acattgtgaa 1140
cttgctgact gagaaggcac cttggaactc tctggcctgg actgttacca gttacgtctt 1200
cctcaagttc ctccaggggg gtggcactgg cagtacaggc ttcgtgagca acctgcgcac 1260
cttcctgtgg atccgggtgc agcagttcac gtctcggcgg gtggagctgc tcatcttctc 1320
ccacctgcac gagctctcac tgcgctggca cctggggcgc cgcacagggg aggtgctgcg 1380
gatcgcggat cggggcacat ccagtgtcac 'agggctgctc agctacctgg tgttcaatgt 1440
catccccacg ctggccgaca tcatcattgg catcatctac ttcagcatgt tcttcaacgc 1500
ctggtttggc ctcattgtgt tcctgtgcat gagtctttac ctcacc,ctga ccattgtggt 1560
cactgagtgg agaaccaagt ttcgtcgtgc tatgaacaca caggagaacg ctacccgggc 1620
acgagcagtg gactctctgc taaacttcga gacggtgaag tattacaacg ccgagagtta 1680
cgaagtggaa cgctatcgag aggccatcat caaatatcag ggtttggagt ggaagtcgag 1740
cgcttcactg gttttactaa atcagaccca gaacctggtg attgggctcg ggctcctcgc 1800
cggctccctg ctttgcgcat actttgtcac tgagcagaag ctacaggttg gggactatgt 1860
gctctttggc acctacatta tccagctgta catgcccctc aactggtttg gcacctacta 1920
caggatgatc cagaccaact tcattgacat ggagaacatg tttgacttgc tgaaagagga 1980
gacagaagtg aaggaccttc ctggagcagg gccccttcgc tttcagaagg gccgtattga 2040
gtttgagaac gtgcacttca gctatgccga tgggcgggag actctgcagg acgtgtcttt 2100
cactgtgatg cctggacaga cacttgccct ggtgggccca tctggggcag ggaagagcac 2160
aattttgcgc ctgctgtttc gcttctacga catcagctct ggctgcatcc gaatagatgg 2220
gcaggacatt tcacaggtga cccaggcctc tctccggtct cacattggag ttgtgcccca 2280
agacactgtc ctctttaatg acaccatcgc cgacaatatc cgttacggcc gtgtcacagc 2340
tgggaatgat gaggtggagg ctgctgctca ggctgcaggc atccatgatg ccattatggc 2400
tttccctgaa gggtacagga cacaggtggg cgagcgggga ctgaagctga gcggcgggga 2460
gaagcagcgc gtcgccattg cccgcaccat cctcaaggct ccgggcatca ttctgctgga 2520
tgaggcaacg tcagcgctgg atacatctaa tgagagggcc atccaggctt ctctggccaa 2580
agtctgtgcc aaccgcacca ccatcgtagt ggcacacagg ctctcaactg tggtcaatgc 2640
tgaccagatc ctcgtcatca aggatggctg catcgtggag aggggacgac acgaggctct 2700
gttgtcccga ggtggggtgt atgctgacat gtggcagctg cagcagggac aggaagaaac 2760
ctctgaagac actaagcctc agaccatgga acggtgacaa aagtttggcc acttccctct 2820
caaagactaa cccagaaggg aataagatgt gtctcctttc cctggcttat ttcatcctgg 2880
tcttggggta tggtgctagc tatggtaagg gaaagggacc tttccgaaaa acatcttttg 2940
gggaaataaa aatgtggact gtgaaaaaaa aaaaaaaaaa aaaa 2984
<210> 29
<211> 1846
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3107278CB1
<400> 29
aatactatca gtcttccctg cgtacggtcg gaactattcc ccttcgccac tgccccctgg 60
gaggctgcgg gcaacggagc aacagcagcg gcgcggacgg aggcgaacac cacccctcat 120
cccctccgga caagggggac aacgcctcca actgtgactg ccgcgcatgg gactacggca 180
tccgcgccgg cctcgtceag aacgtggtca gcaagtggga tctgtgtgtg ataatgcctg 240
gaaggtccat atcgctaagt tctccttact ggtggattaa tctttggtac ctaataactg 300
45/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
gatgcattgc tgactgggtc ggccggcggc ctgtgctgct gttttccatc atcttcattc 360
tgatctttgg actgactgtg gcactgtcag tgaatgtgac aatgttcagc acactcaggt 420
tctttgaagg attttgcctg gctggaatca ttctcacctt gtatgcttta cgaatagagc 480
tgtgcccccc tggaaaacgg ttcatgatta cgatggtggc gagcttcgtg gccatggcgg 540
gccagttcct catgcctggg ctagccgccc tgtgccggga ttggcaggtg ctgcaggccc 600
tCatCatCtg CCCCttCCtg CtCatgCtgC tCtaCtggtC gatattcccc gagtccctcc 660
ggtggctaat ggccacccag cagtttgagt ctgcaaagag gctgatcctc cacttcacac 720
agaagaatcg catgaaccct gagggcgaca tcaagggtgt gataccagag ctggagaaag 780
agctttcccg gaggcccaag aaggtctgca tcgtgaaggt ggtggggaca cggaacctgt 840
ggaagaacat tgtggtcctg tgtgtgaact cgctgacggg gtacgggatc caccactgct 900
ttgccaggag catgatgggc cacgaggtga aggtgccgct cctggagaac ttctatgctg 960
actactatac cacggccagc atcgcgctgg tgtcctgcct ggccatgtgc gtggtggtcc 1020
gattcctcgg gcgcagggga gggctgctgc tcttcatgat cctcaccgcc ctggcctcgc 1080
tcctgcagct cggcctcctc aacctgattg gaaagtacag ccagcaccca gactcaggga 1140
tgagtgacag cgtcaaggac aaattttcca tcgcgttttc catcgtgggc atgtttgcct 1200
cccatgcggt ggggagcctc agcgtgttct tctgtgcgga gatcaccccg acggtgataa 1260
ggtgtggcgg gctggggctg gtgctggcca gcgcgggctt cggcatgctg acggcaccca 1320
tcatcgagct gcacaaccag aaaggctact tcctgcacca catcatcttt gcctgctgca 1380
cgctcatctg catcatctgc atcctcctgc tgcccgagag cagggaccag aacctgcctg 1440
agaacatttc taacggggag cactacacgc gccagccgct gctgccgcac aagaaggggg 1500
agcagccact gctgctcacc aacgccgagc tcaaggacta ctcgggcctc cacgatgccg 2560
cagccgcggg tgacacactg cccgagggtg ccacggccaa cggcatgaag gccatgtagc 1620
ccggcctgcg gaacccgggg ctccagggtc tggggcagct tgggcacagg tttacagacc 1680
agggaccgaa cacgcagcca ggggtgggaa agatgacatc agccaagctg agcctctcaa 1740
ctggtgtggg gaaatcctgt ctttccaaaa gtecaaggag cgcgggtcgg aggagacaaa 1800
ctctttggaa ataacccttt caagactttc ttttctgccg ttaaaa 1846
<210> 30
<211> 1458
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473394CB1
<400> 30
atgca'gaata ttaccaaaga atttggaaca ttcaaggcaa atgacaacat caatttacaa 60
gtaaaggcag gagagattca tgcgttgctt ggagaaaacg gtgctggcaa atctacattg 120
atgaacgtgc tttccggatt attagagccg acatcaggga aaattttgat gcgtgggaaa 180
gaagtacaga tcacaagccc gacaaaagcc aatcaattag ggattgggat ggtccatcag 240
cactttatgc ttgttgatgc ctttactgta acagaaaaca tcgtgttggg aagcgaacct 300
agtcgtgcag ggatgcttga ccataaaaaa gcgcgaaaag agatccaaaa agtttctgaa 360
caatatggat tatcagtcaa cccggatgct tatgttcgtg atatttcagt tgggatggaa 420
caacgggtag aaattttaaa aacactttac cgaggagcag atgtactgat ttttgatgag 480
ccgacagctg tattgacccc tcaggaaatt gatgaattaa tcgtgatcat gaaggaatta 540
gtcaaagaag gcaagtcaat cattttgatt acgcataagt tagatgaaat caaagcagta 600
gctgaccgtt gtacagttat ccgccgtgga aaaggaatcg gtacagtcaa cgttaaagac 660
gttacctcac agcaattagc tgatatgatg gtcggaagag cggtttcatt caaaacgatg 720
aaaaaagaag cgaagcctca agaagtcgtt ttgtctattg aaaatctagt ggtaaaagaa 780
aatcgtggat tagaagccgt gaaaaacctg aacttagagg ttcgtgctgg cgaagtactt 840
ggtatcgctg gaatcgatgg aaacgggcag tcggagttga tccaagcttt gactggtttg 900
cgaaaggcag aaagcggaca tatcaagcta aaaggggaag acatcaccaa taaaaaacct 960
cgaaagatca ctgaacatgg tgtaggacat gtgccagaag accgtcataa atacgggttg 1020
gtcctagata tgacattgtc tgaaaacatt gccctgcaaa cgtatcatca aaaaccttac 1080
agtaaaaacg gtatgctgaa ttattcagtg ataaatgaac atgccagaga attgatcgaa 1140
gaatatgatg ttcgaacaac gaatgaactt gttcctgcaa aagctttatc aggcggaaat 1200
cagcaaaaag caatcatcgc tcggatagtc gaccgagatc ctgatctgtt gatcgttgca 1260
aatccaactc gtgggctgga tgtaggtgcg atcgaattta ttcataaacg tctgatcgaa 1320
caaagggaca aatacaaagc agtgttattg attagtttcg aattagaaga aattttaaat 1380
gtttcggatc gtattgctgt tatccatgaa ggagaaatcg tcgggatcgt tgatccgaaa 1440
gaaacatctg aaaattaa 1458
46/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
<210> 31
<211> 1234
<212> DNA
<213> Homo Sapiens
<220>
<222> misc_feature
<223> Incyte ID No: 7473900CB1
<400> 31
atgaagtccg gtcctggcat ccaagccgcc atcgacctca cagcgggggc cgcagggggg 60
acagcgtgtg tactgactgg gcagcccttc gacacaataa aagtgaagat gcagacgttc 120
cctgacctgt acaagggcct caccgactgc ttcctgaaga catacgccca agtgggtctc 180
cggggcttct acaagggcac cggcccggca cttatggcct acgtcgccga aaactcggtc 240
ctcttcatgt gctacgggtt ctgccagcag tttgtcagga aagtggctgg aatggacaag 300
caggcaaagc tgagtgatct ccagactgca gccgcggggt ccttcgcctc tgcatttgct 360
gcactggctc tctgccccac tgagcttgtg aagtgccggc tacagaccat gtatgaaatg 420
gagatgtcag ggaagatagc aaaaagccat aatacaattt ggtctgtcgt gaagggtatc 480
cttaaaaagg atggcccctt gggcttctac catggactct cgagtactct acttcaagaa 540
gtaccgggtt atttcttttt ctttggtggc tatgaactga gccgatcgtt ttttgcgtca 600
gggagatcaa aagatgaact aggccctg~c catttgatgt taagtggtgg agttgctgga 660
atttgcctgt ggcttgtcgt gttcccagtg gattgtatta aatccagaat tcaagttctt 720
tccatgtatg ggaaacaggc aggatttatt ggtaccctct taagtgttgt gagaaatgaa 780
ggaatagtag ccttatattc tggactgaaa gctactatga ttcgagcaat ccctgccaat 840
ggggcactgt ttgtggccta cgaatacagc aggaagatga tgatgaaaca gttggaagca 900
tactgaagtg tcttggtgaa cctggatccg agtccatgag tttgaggact acagttcatc 960
acagggttca gcagagtaca agaccactgt ctaattttga cttcatggga attttggttt 1020
tatcttccct tcttctaccc taaatcttaa ctttatggaa gggcctctat tttacatcat 1080
ataatttctg cccataattg tattgaaata ggaaagttgc tgctcttgca cttgctggaa 1140
tgtacagggt gggctggttg gccctatgta cctaatctga aaaactaaat atcgttctgt 1200
cagggccttt gcataaagcc atttgtgtgt acat 1234
<210> 32
<211> 1255
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7475045CB1
<400> 32
gtgacctttc cccccagatc ccaggtccag gcccgccctc ggctggcagg tgtgggcaca 60
gaggcagctg ggattggtcg cagctggcgg aggcgcgtcc caggctccgg cagaccgctg 120
gaacagttga gccagagcag gtggactgct gagatagacc agggacacca ggcagccaca 180
ggcctgtcag accaggaccc ttaccctcta gacatggcct cggtcccctg caaaccccag 240
ccccgtagcc ctgcgaggtt acagacagcc taaacgccac caccacaggg cctgtgccgt 300
gcccctgacc cgggcacaga aggccactgg cccggaggcc atggagacgg tgcccccagc 360
agtggacctg gtgctgggtg CttCtgCCtg ctgcctggcc tgtgtCttCa CCaaCCCCCt 42O
ggaggtggtg aagacgcggc tgcagctgca gggggagctg caggcccggg gcacctaccc 480
acggccctac catggcttca tagcctctgt cgctgctgtg gcccgagcag acgggctgtg 540
gggcctgcag aaggggctgg ctgccggcct tctgtaccaa ggcctcatga atggcgttcg 600
tttctactgc tacagcctgg cgtgccaggc tggcctcacg cagcaaccag gtggcaccgt 660
ggttgcggga gccgtggcgg gggcactggg agccttcgtg gggagccctg cttacctgat 720
caaaacgcag ctgcaagctc agacagtggc cgcagtggcc gtgggacacc agcacaatca 780
ccagactgtc ctgggtgcct tggagaccat ctggcggcag caagggctct tggggctgtg 840
gcagggcgtt ggtggggctg tgccccgagt catggtgggc tcagctgccc agctggccac 900
cttcgcctct gccaaggcct gggtacagaa gcaacagtgg ctccctgagg acagctggct 960
ggtggccctg gctgggggca tgatcagcag catagccgtg gttgtcgtca tgactccctt 1020
cgatgtggtc agcacgcggc tatacaatca gccggtggac acagctggca ggggccagct 1080
ctatgggggc ctcaccgact gcatggtgaa gatctggcgg caggagggcc ccctggcact 1140
ctacaagggc ctgggccccg cctacctgcg cctgggcccc cacaccatcc tcagcatgct 1200
cttctgggac gagcttcgga aactggctgg gcgggcccag cacaagggca cctag 1255
47/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
<210> 33
<212> 957
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7475611CB1
<220>
<221> unsure
<222> 1
<223> a, t, c, g, or other
<400> 33
ngccgcgctg gccaccttgc ccatcaaacg caccggttcg gtgcgataca agatcatcgt 60
cgtcatcgtc atcgctgtcc tgtgggtgat cagttggacg acgaccggaa ggattttcag 120
atgagtgcca aggtcctgct gtcgaccgag cacctgtacg ccacccaccc gggccgtcct 180
atggtactga ccgacgttaa tgtctccttt cgcgccgggg ttcgcgtagc gatcctggga 240
gctaatggat ccggtaagac gaccctcatg cgctgcctgt ccggttccct caaacccgcc 300
aagggtcacg tcaagagggg cgacatcgtt gtcagctacg ggcgcgctca acttcgtgag 360
caccgtcgag ccgtccagct tgtgctgcaa gaccctgacg accagctctt tagcgccgat 420
gtcagccagg atgtctcctt cggccccatg aatatgggcc tcaaagttga cgaggtgcgt 480
gaccgggtct ccgagtccct agaactgctc ggggccagtc atctggctga gcgtgccacg 540
tatcaactgt cctatggtga gcgcaagagg gtcgcggttg ccggtgccgt ggccatgcgc 600
ccggatctgc tgctccttga tgagcccacc gccggacttg acccggttgg agtcacccag 660
atgttggagg ccctggatcg gctgcgcgat catggaacaa cggtggcgat ggctacccac 720
gacgtcgacc tggCtctggc gtgggcgcag gaggcccttg tcgttgtcga cggtcaggtg 780
caccaaggac cgatcggcga gttacttgcc gatgccgaca ccgtgggacg ggcacacctg 840
CdCCttCCgt ggCCCCtCga gctcgcccgg cgcctcggtg ttcgggacct tcccaggacg 900
atggacgacg tcgtggcgat gctgtccgac aatccctcgc cagctccctc gaattga 957
<210> 34
<211> 2407
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7475617CB1
<400> 34
gcggccgcgg cctcggcctc ctcctctggg gcggcggcgg aggacagcag cgccatggag 60
gagctcgcta ctgagaagga ggcggaggag agccaccggc aagacagcgt gagcctgctc 120
accttcatcc tgctgctcac gctcaccatc ctcaccatct ggctcttcaa gcaccgccgg 180
gtgcgctttc tgcacgagac cgggctggcc atgatctatg ggctcatcgt tggggtgatc 240
ctgaggtatg gtacccctgc taccagtggc cgtgacaaat cactcagctg cactcaggaa 300
gacagggcct tcagtacctt attagtgaat gtcagcggaa agttcttcga atacactctg 360
aaaggagaaa tcagtcctgg caagatcaac agcgtagagc agaatgatat gctacggaag 420
gtaacattcg atccagaagt atttttcaac attcttctgc ctccaattat ttttcatgct 480
ggatacagct taaagaagag acactttttc agaaatcttg gatctatact ggcctatgcc 540
ttcttgggga ctgctgtttc atgcttcatt attggaaatc tcatgtatgg tgtggtgaag 600
ctcatgaaga ttatgggaca gctctcagat aaattttact acacagattg tctctttttt 660
ggagcaatca tctctgccac tgacccagtg actgtgctgg cgatatttaa tgaattgcat 720
gcagacgtgg atctttacgc acttcttttt ggagagagcg tcctaaatga tgctgttgcc 780
attgtactgt cctcgtctat tgttgcctac cagccagcgg gactgaacac tcacgccttt 840
gatgctgctg ccttttttaa gtcagttggc atttttctag gtatatttag tggctctttt 900
accatgggag ctgtgactgg tgttgtgact gctctagtga ctaagtttac caaactgcac 960
tgcttccccc tgctggagac ggcgctgttc ttcctcatgt cctggagcac gtttctcttg 1020
gcagaagcct gcggatttac aggtgttgta gctgtccttt tctgtggaat cacacaagct 1080
cattacacct acaacaatct gtcggtggaa tcaagaagtc gaaccaagca gctctttgag 1140
gtgttacatt tcctggcaga gaacttcatc ttctcctaca tgggcctggc actgtttacc 1200
ttccagaagc acgttttcag ccccattttc atcatcggag cttttgttgc catcttcctg 1260
48/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
ggcagagccg cgcacatcta cccgctctcc ttcttcctca acttgggcag aaggcataag 1320
attggctgga attttcaaca catgatgatg ttttcaggcc tcaggggagc aatggcattt 1380
gcgttggcca tccgtgacac ggcatcctat gctcgccaga tgatgttcac gaccaccctt 1440
ctcattgtgt tcttcactgt ctggatcatt ggaggaggca cgacacccat gttgtcatgg 1500
cttaacatca gagttggcgt cgaggagccc tccgaagagg accagaatga acaccactgg 1560
cagtacttca gagttggtgt tgaccccgat caagacccac cacccaacaa cgacagcttt 1620
caagtcttac aaggggacgg cccagattct gccagaggaa accggacaaa acaggagagc 1680
gcatggatat tcaggctgtg gtacagcttt gatcacaatt atctgaagcc catcctcaca 1740
cacagtggtc ccccactaac caccacgctc cccgcctggt gtggcttact agctcgatgt 1800
ctgaccagtc cccaggtgta cgataaccaa gagccactga gagaggaaga ctctgatttc 1860
atcctgaccg aaggcgacct gacattgacc tacggggaca gcacagtgac tgcaaatggc 1920
tcctcaagtt cgcacaccgc ctccacgagt ctggagggca gccggagaac gaagagcagc 1980
tcggaggaag tgctggagcg agacctggga atgggagacc agaaggtttc gagccggggc 2040
acccgcctag tgtttcccct ggaagataat gcttgacttt ccccccaagc cctggcgcga 2100
tggggtaggc tcccgatggg actgaagatt tgaaaataca tccacgaaca tttcaacatg 2160
gaacgaagaa ttatagttcc ttctctggct atatccataa agaacgtagt tatgaaatgt 2220
ttaaaaccaa aggcaaatag tgttatactc ttattttttg attaatctga ggaaaggagg 2280
tatttagaaa ctgatgatgg tatcactgca aaaagcattc aacttttttt tttttggtgg 2340
agatggtgtt ccactctgtc acccaggctg gagtgcggtg ccttggtctg ggcccactgc 2400
aacctct 2407
<210> 35
<221> 2767
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473314CB1
<400> 35
atggagggct ctgggggcgg tgcgggcgag cgggcgccgc tgctgggcgc gcggcgggcg 60
gcggcggccg cggcggctgg ggcgttcgcg ggccggcgcg cggcgtgcgg ggccgtgctg 120
ctgacggagc tgctggagcg cgccgctttc tacggcatca cgtccaacct ggtgctattc 180
ctgaacgggg cgccgttctg ctgggagggc gcgcaggcca gcgaggcgct gctgctcttc 240
atgggcctca cctacctggg ctcgccgttc ggaggctggc tggccgacgc gcggctgggc 300
cgggcgcgcg ccatcctgct gagcctggcg ctctacctgc tgggcatgct ggccttcccg 360
ctgctggccg cgcccgccac gcgagccgcg ctctgcggtt ccgcgcgcct gctcaactgc 420
acggcgcctg gtcccgacgc cgccgcccgc tgctgctcac cggccacctt cgcggggctg 480
gtgctggtgg gcctgggcgt ggccaccgtc aaggccaaca tcacgccctt cggcgccgac 540
caggttaaag atcgaggtcc ggaagccact aggagatttt ttaattggtt ttattggagc 600
attaacctgg gagcgatcct gtcgttaggt ggcattgcct atattcagca gaacgtcagc 660
tttgtcactg gttatgcgat ccccactgtc tgcgtcggcc ttgcttttgt ggccttcctc 720
tgtggccaga gcgttttcat caccaagcct cctgatggca gtgccttcac cgatatgttc 780
aagatactga cgtattcctg ctgttcccag aagcgaagtg gagagcgcca gagtaatggt 840
gaaggcattg gagtctttca gcaatcttct aaacaaagtc tgtttgattc atgtaagatg 900
tctcatggtg ggccatttac agaagagaaa gtggaagatg tgaaagctct ggtcaagatt 960
gtccctgttt tcttggcttt gataccttac tggacagtgt atttccaaat gcagacaaca 1020
tatgttttac agagtcttca tttgaggatt ccagaaattt caaatattac aaccactcct 1080
cacacgctcc ctgcagcctg gctgaccatg tttgatgctg tgctcatcct cctgctcatc 1140
cctctgaagg acaaactggt cgatcccatt ttgagaagac atggcctgct cccatcctcc 1200
ctgaagagga tcgccgtggg catgttcttt gtcatgtgct cagcctttgc tgcaggaatt 1260
ttggagagta aaaggctgaa ccttgttaaa gagaaaacca ttaatcagac catcggcaac 1320
gtcgtctacc atgctgccga tctgtcgctg tggtggcagg tgccgcagta cttgctgatt 1380
gggatcagcg agatctttgc aagtatcgca ggcctggaat ttgcatactc agctgccccc 1440
aagtccatgc agagtgccat aatgggcttg ttctttttct tctctggcgt cgggtcgttc 2500
gtgggttctg gactgctggc actggtgtct atcaaagcca tcggatggat gagcagtcac 1560
acagactttg gtaatattaa cggctgctat ttgaactatt actttttcct tctggctgct 1620
attcaaggag ctaccctcct gcttttcctc attatttctg tgaaatatga ccatcatcga 1680
gaccatcagc gatcaagagc caatggcgtg cccaccagca ggagggcctg accttcctga 1740
ggccatgtgc ggtttctgag gctgacatgt cagtaactga ctggggtgca ctgagaacag 1800
gcaagacttt aaattcccat aaaatgtctg acttcactga aacttgcatg ttgcctggat 1860
tgatttcttc tttccctcta tccaaaggag cttggtaagt gccttactgc agcgtgtctc 1920
49167
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
ctggcacgct gggccctccg ggaggagagc tgcagatttc gagtatgtcg cttgtcattc 1980
aaggtctctg tgaatcctct agctgggttc ccttttttac agaaactcac aaatggagat 2040
tgcaaagtct tggggaactc cacgtgttag ttggcatccc agtttcttaa acaaatagta 2100
tcacctgctt cccatagcca tatctcactg taaaaaaaaa aattaataaa ctgttactta 2160
tatttaagaa agtgaggatt tttttttttt aaagataaaa gcatggtcag atgctgcaag 2220
gattttacat aaatgccata tttatggttt ccttcctgag aacaatcatg ctcttgccat 2280
gttctttgat ttaggctggt agtaaacaca tttcatctgc tgcttcaaaa agtacttact 2340
ttttaaacca tcaacattac ttttctttct taaggcaagg catgcataag agtcatttga 2400
gaccatgtgt cccatctcaa gccacagagc aactcacggg gtacttcaca ccttacctag 2460
tcagagtgct tatatatagc tttattttgg tacgattgag actaaagact gatcatggtt 2520
gtatgtaagg aaaacattct tttgaacaga aatagtgtaa ttaaaaataa ttgaaagtgt 2580
taaatgtgaa cttgagctgt ttgaccagtc acatttttgt attgttactg tacgtgtatc 2640
tggggcttct ccgtttgtta atactttttc tgtatttgtt gctgtatttt tggcataact 2700
ttattataaa aagcatctca aatgcgaaat ccaaaaaaaa aaaaaaaaaa gatcggccgc 2760
aagctta 2767
<210> 36
<211> 2182
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 70356714CB1
<400> 36
gcagtccgct cagccgaggc agctctgttc atggcgttct cgaagctctt ggagcaagcc 60
ggaggcgtgg gcctcttcca gaCCCtgCag gtgCtCaCCt tCatCCtCCC CtgCCtCatg 120
ataccttccc agatgctcct ggagaacttc tcagccgcca tcccaggcca ccgatgctgg 180
acacacatgc tggacaatgg ctctgcggtt tccacaaaca tgacccccaa ggcccttctg 240
aCCatCtCCa tCCCgCCagg ccccaaccag gggccccacc agtgccgccg cttccgccag 300
ccacagtggc agctcttgga ccccaatgcc acggccaCCa gctggagcga agctgacacg 360
gagccgtgtg tggacggctg ggtctatgac cgcagcgtct tcacctccac catcgtggcc 420
aagtgggacc tggtgtgcag ctcccagggc ttgaagcccc taagccagtc catcttcatg 480
tccgggatcc tggtgggctc ctttatctgg ggcctcctct cctaccggtt tgggaggaag 540
ccgatgctga gctggtgctg cctgcagttg gccgtggcgg gcaccagcac catcttcgcc 600
ccaacattcg tcatctactg cggcctgcgg ttcgtggccg cttttgggat ggccggcatc 660
tttctgagtt cactgacact gatggtggag tggaccacga ccagcaggag ggcggtcacc 720
atgacggtgg tgggatgtgc cttcagcgca ggccaggcgg cgctgggcgg cctggccttt 780
gccctgcggg actggaggac tctccagctg gcagcatcag tgcccttctt tgccatctcc 840
ctgatatcct ggtggctgcc agaatccgcc cggtggctga ttattaaggg caaaccagac 900
caagcacttc aggagctcag aaagg.tggcc aggataaatg gccacaagga ggccaagaac 960
ctgaccatag aggtgctgat gtccagcgtg aaggaggagg tggcctctgc aaaggagccg 1020
cggtcggtgc tggacctgtt ctgcgtgccc gtgctccgct ggaggagctg cgccatgctg 1080
gtggtgaatt tctctctatt gatctcctac tatgggctgg tcttcgacct gcagagcctg 1140
ggccgtgaca tcttcctcct ccaggccctc ttcggggccg tggacttcct gggccgggcc 1200
accactgccc tcttgctcag tttccttggc cgccgcacca tccaggcggg ttcccaggcc 1260
atggccggcc tcgccattct agccaacatg ctggtgccgc aagatttgca gaccctgcgt 1320
gtggtctttg ctgtgctggg aaagggatgt tttgggataa gcctaacctg cctc'accatc 1380
tacaaggctg aactctttcc aacgccagtg cggatgacag cagatggcat tctgcataca 1440
gtgggccggc tgggggctat gatgggtccc ctgatcctga tgagccgcca agccctgccc 1500
CtgCtgCCtC CtCtCCCCta tggcgttatc tccattgctt ccagcctggt tgtgctgttc 1560
ttcctcccgg agacccaggg acttccgctc cctgacacta tccaggacct ggagagccag 1620
aaatcaacag cagcccaggg caaccggcaa gaggccgtca ctgtggaaag tacctcgctc 1680
tagaaattgt gcctgcatgg agccccttta gtcaaagact cctggaaagg agttgcctct 1740
tctccaatca gagcgtggag gcgagttggg cgacttcaag ggcctggcat ggcagaggcc 1800
aggcagccgt ggccgagtgg acagcgtggc cgtctgctgt ggctgaaggc agcttccaca 1860
gctcactcct cttctccctg ccctgatcag attccccacc ttacccgggc cctacaggag 1920
CCtgtgCaga tggccatgcc caaccaataa cgagacggtt CCCCtCCCtt tCCCtgCCag 1980
gctcatgtct ttacaccttc actcagccac gccaaccaga gactgggttc caatctcacc 2040
ccaccacata cagagccctc atctgtgaaa tgagaatgat cacgtgaccc accccccagg 2100
gcaggtatca gggtgaactg atcttagcac cggccaaata aatggaacct gctgagagag 2160
ctgccagaaa aaaaaaaaaa as 2182
50/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
<210> 37
<211> 2811
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7611491CB1
<400> 37
cagacgggcc tggggcaggc atggcggatt ccagcgaagg cccccgcgcg gggcccgggg ~0
aggtggctga gctccccggg gatgagagtg gcaccccagg tggggaggct tttcctctct 120
cctccctggc caatctgttt gagggggagg atggctccct ttcgccctca ccggctgatg 180
ccagtcgccc tgctggccca ggcgatgggc gaccaaatct gcgcatgaag ttccaggcgc 240
cttccgcaag ggggtgccca accccatcga tctgctggag tccaccctat atgagtcctc 300
ggtggtgcct gggcccaaga aagcacccat ggactcactg tttgactacg gcacctatcg 360
tcaccactcc agtgacaaca agaggtggag gaagaagatc atagagaagc agccgcagag 420
ccccaaagcc cctgcccctc agccgccccc catcctcaaa gtcttcaacc ggcctatcct 480
ctttgacatc gtgtcccggg gctccactgc tgacctggac gggctgctcc cattcttgct 540
gacccacaag aaacgcctaa ctgatgagga gtttcgagag ccatctacgg ggaagacctg 600
cctgcccaag gccttgctga acctgagcaa tggccgcaac gacaccatcc ctgtgctgct 660
ggacatcgcg gagcgcaccg gcaacatgag ggagttcatt aactcgccct tccgtgacat 720
ctactatcga ggtcagacag ccctgcacat cgccattgag cgtcgctgca aacactacgt 780
ggaacttctc gtggcccagg gagctgatgt ccacgcccag gcccgtgggc gcttcttcca 840
gcccaaggat gaggggggct acttctactt tggggagctg cccctgtcgc tggctgcctg 900
caccaaccag ccccacattg tcaactacct gacggagaac ccccacaaga aggggacatg 960
cggcgccagg actcgcgagg caacacagtg ctgcatgcgc tggtggccat tgctgacaac 1020
acccgtgaga acaccaagtt tgttaccaag atgtacgacc tgctgctgct caagtgtgcc 1080
cgcctcttcc ccgacagcaa cctggaggcc gtgctcaaca acgacggcct ctcgcccctc 1140
atgatgatgg ctgccaagac gggcaagatt gggatctttc agcacatcat ccggcgggag 1200
gtgacggatg aggacacacg gcacctgtcc cgcaagttca aggactgggc ctatgggcca 1260
gtgtattcct cgctttatga cctctcctcc ctggacacgt gtggggaaga ggcctccgtg 1320
ctggagatcc tggtgtacaa cagcaagatt gagaaccgcc acgagatgct ggctgtggag 1380
cccatcaatg aactgctgcg ggacaagtgg cgcaagttcg gggccgtctc cttctacatc 1440
aacgtggtct cctacctgtg tgccatggtc atcttcactc tcaccgccta ctaccagccg 1500
ctggagggca caccgccgta cccttaccgc accacggtgg actacctgcg gctggctggc 1560
gaggtcatta cgctcttcac tggggtcctg ttcttcttca ccaacatcaa agacttgttc 1620
atgaagaaat gccctggagt gaattctctc ttcattgatg gctccttcca gctgctctac 1680
ttcatctact ctgtcctggt gatcgtctca gcagccctct acctggcagg gatcgaggcc 1740
tacctggccg tgatggtctt tgccctggtc ctgggctgga tgaatgccct ttacttcacc 1800
cgtgggctga agctgacggg gacctatagc atcatgatcc agaagattct cttcaaggac 1860
CttttCCgat tCCtgCtCgt ctacttgctc ttcatgatcg gctacgcttc agccctggtc 1920
tccctcctga acccgtgtgc caacatgaag gtgtgcaatg aggaccagac caactgcaca 1980
gtgCCClCtt aCCCCtCgtg ccgtgacagc gagaccttca gcaccttcct cctggacctg 2040
tttaagctga ccatcggcat gggcgacctg gagatgctga gcagcaccaa gtaccccgtg 2100
gtcttcatca tcctgctggt gacctacatc atcctcacct ttgtgctgct cctcaacatg 2160
ctcattgccc tcatgggcga gacagtgggc caggtctcca aggagagcaa gcacatctgg 2220
aagctgcagt gggccaccac catcctggac attgagcgct ccttccccgt attcctgagg 2280
aaggccttcc gctctgggga gatggtcacc gtgggcaaga gctcggacgg cactcctgac 2340
cgcaggtggt gcttcagggt ggatgaggtg aactggtctc actggaacca gaacttgggc 2400
atcatcaacg aggacccggg caagaatgag acctaccagt attatggctt ctcgcatacc 2460
gtgggccgcc tccgcaggga tcgctggtcc tcggtggtac cccgcgtggt ggaactgaac 2520
aagaactcga acccggacga ggtggtggtg cctctggaca gcacggggaa cccccgctgc 2580
gatggccacc agcagggtta cccccgcaag tggaggactg atgacgcccc gctctaggga 2640
ctgcagccca gccccagctt ctctgcccac tcatttctag tccagccgca tttcagcagt 2700
gccttctggg gtgtcccccc acaccctgct tttggcccag aggcgaggga ccagtggagg 2760
ttcaaggagg cccaagaacc tgtggtcccc tggctctgct tcccaacctg g 2811
<210> 38
<211> 2074
<212> DNA
<213> Homo Sapiens
S 1/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
<220>
<221> misc_feature
<223> Incyte ID No: 171968CB1
<400> 38
tggaccccga ttctcacctg gactccaaaa gctatcttga cctactggca tctctgaccc 60
aaatcttaat tgcccccatc gccctctaca tccccccagc actgaccctc accaggactc 120
cagccccaat tccatcccaa atctgtgtag catctgcttc tgccgattct aagagcccta 180
gcacctgcca agtcccccca ttacccacct tcccacactc agaagcctct ttggtgggat 240
gctaatggga aggagtcttg cctctctgga ggcaggaggg gctggccttg tgcccctccg 300
ggcctctgag aggtgggcgc aggagaacag cactcacgag gggacctcct tcaccctggg 360
aaagggtggt ttctttgcta tttcacagtc acaggctgaa tccttcactt ggccctgccc 420
accgtacagg tatgctcact gccggcttta gggaggccag aaaccaacct gctcctgcaa 480
aaagaatcca ggcttgttct gagtgcctgc tgtaggccag gcaagttggt cactgttgca 540
tgaggggcag tgcctctcac tcttgggcct gatgccaagg gaggtggcct gtcccggtcg 600
catgcagaca tcctggccat cccagccaca catgcacgtg agaggctggg tgccggcagg 660
gttcctgagg gactggaaga tgtggccccc tgcctgcctc cttcctcttg tgaatataag 720
gggccagttc ccagcccaaa gccccacccg gggccctcat gtttcatcac caacaggcct 780
actgtctggc tccttttgac ctcatcaaag tccggctaca aaaccagaca gagccaaggg 840
cccagccagg gagcccccca ccccggtacc aggggcccgt gcactgtgca gcctccatct 900
tccgggagga ggggccccgg gggctgttcc gaggagcctg ggccctgacg ctgagggaca 960
cccccacggt ggggatctac ttcatcacct atgaagggct ctgtcgccag tacacaccag 1020
aaggccagaa tcccagctca gccacggtgc tggtggcagg gggctttgca ggcattgctt 1080
cctgggtggc agccacgccc ttagacgtga tcaagtcccg gatgcagatg gatggactga 1140
gacgcagagt gtaccagggg atgctggact gcatggtgag cagcatccgg caggaaggac 1200
tgggagtctt cttccggggg gtcaccatca acagtgcccg cgcctttccc gtcaatgctg 1260
tcaccttcct cagctacgaa tatctcctcc gctggtgggg atgagccctg cggcaatgcc 1320
agcagctccc catcaggccc acggcctgga ggccagtttg agattggagg ccaggttgaa 1380
agcttgcaaa tcagtgcaag aggctcagcc cttcctaacc aaggtgcctc ccacccgcgc 1440
agatctgggc tgggcagaca cctgtgggag ccggaagcca gggggcctgt gcagcctccc 1500
tgtgtagctg gccttgactc ctttgcctcc cacatctgtg aaacagggag catgaggcac 1560
aagtgagctg gcaagtggtg ctggtgacat cccagctcct gtcctgtgcc ttcacctctt 1620
tttttttttt tttttttttt ggggagaggg ggaggtcttt ctctcttggt cccccagggg 1680
cgtgggattc gcaggggcgg gtgagactcc tcgggttcat tggaaacctt cggcgttttc 1740
CaCCtttCCg gggtctcaag cgcaattctt cctggcctta agccttccca aggtacgctg 1800
ggagacatat tagcgcggcc cggcaacaca aacccaggta taaatttttg ggtattttta 1860
aggctaagaa gacaggggtt taccccattg tcagcgccag ggttgggtcc tggagatctc 1920
tctggatctt tggggagatc cggcccgggt ggtgggcctt ctcaagagtt gcgcggggag 1980
attacacagg gctgtgagag gccccacccg ggcgccccgg ggtggcctta cactcttctt 2040
aagggagcct cggaggactc cctttttgga aagg 2074
<210> 39
<211> 1340
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 257274CB1
<400> 39
aggaacagcg ccatgtgctc cgggctcctg gagctcctgc tgcccatctg gctctcctgg 60
accctgggga cccgaggctc tgagccccgc agtgtgaacg atcccgggaa catgtccttt 120
gtgaaggaga cggtggacaa gctgttgaaa ggctacgaca ttcgcctaag acccgacttc 180
gggggtcccc cggtctgcgt ggggatgaac atcgacatcg ccagcatcga catggtttcc 240
gaagtcaaca tgagattctg gctgcaggaa aggggaacga agacagtggt ctgtgcgttc 300
caggggtgtc tctgcggttt ttccaaggct gcctcctgga ctgggagacc cgggcccggc 360
accgccagtc tctgtccgag gtgctgacag gcttccgctg cttcagagag cgggaggccg 420
cgcctaggcg ggctctcaga ggagccgctc taccgggaga gtcggaggct ggtgacccag 480
tgtcacttag gtcttctgtg aatgcagact ggattcaata ttctgacctg tgggaagcgg 540
aggtcagtac cccgaggtgc gaagcgggct tttgccagga gtgctttagg acgccaggga 600
atcaggagaa ggatggccct ttcatttgtt aattgagctg aaacgccgtg ggatttgaaa 660
actagtttag ttttctgcgg agggacactc tggaaggagc atttgtaaac aatatttgtt 720
52/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
ttttggaaga aattgtttgg caacttttct ttcggacata caacattgag aatacagtga 780
gacatggttt tagatccact ctgtaggctt cttaactctc gtgtacgtcg gaatcacgtg 840
ggcaaacttg ctttaaatgc gtgcctgcta gttcctcact ccaagggatt ctgatcaaga 900
taggctggag tcttggggtc cctgcattta aaactacttc ttcaaatatt ttgaagcggg 960
tattttgtgg acaatatttt gagaaacttt gcagtaagtc acagatatat gaatatatac 1020
aaataaagta aaactatttt taaaaataga cgttagtgag atacttttaa aatacctacc 1080
actaaggatc ttataggaag aaacttttga ccacgccaag ctgttctcat taatttttca 1140
ctgttaatct aaagctttta aatttacaat cccatgtatt taaaaatgta cttttttcca 1200
aagtatatca cttaggacat ttgtaagtca aatattgtat cagtaaaagt gttagcaagg 1260
aacacagaag gaatgtgact gcataaatta ccttacagta aaaattaaca tgtctatttt 1320
actttttatg taatacatta 1340
<210> 40
<211> 6027
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6355991CB1
<400> 40
atggagcaaa cagtgcttgt accaccagga cctgacagct tcaacttctt caccagagaa 60
tctcttgcgg ctattgaaag acgcattgca gaagaaaagg caaagaatcc caaaccagac 120
aaaaaagatg acgacgaaaa tggcccaaag ccaaatagtg acttggaagc tggaaagaac 180
cttccattta tttatggaga cattcctcca gagatggtgt cagagcccct ggaggacctg 240
gacccctact atatcaataa gcagactttt atagtattga ataaagggaa ggccatcttc 300
cggttcagtg ccacctctgc cctgtacatt ttaactccet tcaatcctct taggaaaata 360
gctattaaga ttttggtaca ttcattattc agcatgctaa ttatgtgcac tattttgaca 420
aactgtgtgt ttatgacaat gagtaaccct cctgattgga caaagaatgt agagtacacc 480
ttcacaggaa tatatacttt tgaatcactt ataaaaatta ttgcaagggg attctgttta 540
gaagatttta ctttccttcg ggatccatgg aactggctcg atttcactgt cattacattt 600
gcgtacgtca cagagtttgt ggacctgggc aatgtctcgg cattgagaac attcagagtt 660
ctccgagcat tgaagacgat ttcagtcatt ccaggcctga aaaccattgt gggagccctg 720
atccagtctg tgaagaagct ctcagatgta atgatcctga ctgtgttctg tctgagcgta 780
tttgctctaa ttgggctgca gctgttcatg ggcaacctga ggaataaatg tatacaatgg 840
cctcccacca atgcttcctt ggaggaacat agtatagaaa agaatataac tgtgaattat 900
aatggtacac ttataaatga aactgtcttt gagtttgact ggaagtcata tattcaagat 960
tcaggatatc attatttcct ggagggtttt ttagatgcac tactatgtgg aaatagctct 1020
gatgcagggc aatgtccaga gggatatatg tgtgtgaaag ctggtagaaa tcccaattat 1080
ggctacacaa gctttgatac cttcagttgg gcttttttgt ccttgtttcg actaatgact 1140
caggacttct gggaaaatct ttatcaactg acattacgtg ctgctgggaa aacgtacatg 1200
atattttttg tattggtcat tttcttgggc tcattctacc taataaattt gatcctggct 1260
gtggtggcca tggcctacga ggaacagaat caggccacct tggaagaagc agaacagaaa 1320
gaggccgaat ttcagcagat gattgaacag cttaaaaagc aacaggaggc agctcagcag 1380
gcagcaacgg caactgcctc agaacattcc agagagccca gtgcagcagg caggctctca 1440
gacagctcat ctgaagcctc taagttgagt tccaagagtg ctaaggaaag aagaaatcgg 1500
aggaagaaaa gaaaacagaa agagcagtct ggtggggaag agaaagatga ggatgaattc 1560
caaaaatctg aatctgagga cagcatcagg aggaaaggtt ttcgcttctc cattgaaggg 1620
aaccgattga catatgaaaa gaggtactcc tccccacacc agtctttgtt gagcatccgt 1680
ggctccctat tttcaccaag gcgaaatagc agaacaagcc ttttcagctt tagagggcga 1740
gcaaaggatg tgggatctga gaacgacttc gcagatgatg agcacagcac ctttgaggat 1800
aacgagagcc gtagagattc cttgtttgtg ccccgacgac acggagagag acgcaacagc 1860
aacctgagtc agaccagtag gtcatcccgg atgctggcag tgtttccagc gaatgggaag 1920
atgcacagca ctgtggattg caatggtgtg gtttccttgg ttggtggacc ttcagttcct 1980
acatcgcctg ttggacagct tctgccagag gtgataatag ataagccagc tactgatgac 2040
aatggaacaa ccactgaaac tgaaatgaga aagagaaggt caagttcttt ccacgtttcc 2100
atggactttc tagaagatcc ttcccaaagg caacgagcaa tgagtatagc cagcattcta 2160
acaaatacag tagaagaact tgaagaatcc aggcagaaat gcccaccctg ttggtataaa 2220
ttttccaaca tattcttaat ctgggactgt tctccatatt ggttaaaagt gaaacatgtt 2280
gtcaacctgg ttgtgatgga cccatttgtt gacctggcca tcaccatctg tattgtctta 2340
aatactcttt tcatggccat ggagcactat ccaatgacgg accatttcaa taatgtgctt 2400
acagtaggaa acttggtatt cactgggatc tttacagcag aaatgtttct gaaaattatt 2460
53167
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
gccatggatc cttactatta tttccaagaa ggctggaata tctttgacgg ttttattgtg 2520
acgcttagcc tggtagaact tggactcgcc aatgtggaag gattatctgt tctccgttca 2580
tttcgattgc tgcgagtttt caagttggca aaatcttggc caacgttaaa tatgctaata 2640
aagatcatcg gcaattccgg gggggctctg ggaaatttaa ccctcgtctt ggccatcatc 2700
gtcttcattt ttgccgtggt cggcatgcag ctctttggta aaagctacaa agattgtgtc 2760
tgcaagatcg ccagtgattg tcaactccca cgctggcaca tgaatgactt cttccactcc 2820
ttcctgattg tgttccgcgt gctgtgtggg gagtggatag agaccatgtg ggactgtatg 2880
gaggttgctg gtcaagccat gtgccttact gtcttcatga tggtcatggt gattggaaac 2940
ctagtggtac tgaatctctt tctggccttg cttctgagct catttagtgc agacaacctt 3000
gcagccactg atgatgataa tgaaatgaat aatctccaaa ttgctgtgga taggatgcac 3060
aaaggagtag cttatgtgaa aagaaaaata tatgaattta ttcaacagtc cttcattagg 3120
aaacaaaaga ttttagatga aattaaacca cttgatgatc taaacaacaa gaaagacagt 3180
tgtatgtcca atcatacagc agaaattggg aaagatcttg actatcttaa agatgtaaat 3240
ggaactacaa gtggtatagg aactggcagc agtgttgaaa aatacattat tgatgaaagt 3300
gattacatgt cattcataaa caaccccagt cttactgtga ctgtaccaat tgctgtagga 3360
gaatctgact ttgaaaattt aaacacggaa gactttagta gtgaatcgga tctggaagaa 3420
agcaaagaga aactgaatga aagcagtagc tcatcagaag gtagcactgt ggacatcggc 3480
gcacctgtag aagaacagcc cgtagtggaa cctgaagaaa ctcttgaacc agaagcttgt 3540
ttcactgaag gttgtgtaca aagattcaag tgttgtcaaa tcaatgtgga agaaggcaga 3600
ggaaaacaat ggtggaacct gagaaggacg tgtttccgaa tagttgaaca taactggttt 3660
gagaccttca ttgttttcat gattctcctt agtagtggtg ctctggcatt tgaagatata 3720
tatattgatc agcgaaagac gattaagacg atgttggaat atgctgacaa ggttttcact 3780
tacattttca ttctggaaat gcttctaaaa tgggtggcat atggctatca aacatatttc 3840
accaatgcct ggtgttggct ggacttctta attgttgatg tttcattggt cagtttaaca 3900
gcaaatgcct tgggttactc agaacttgga gccatcaaat ctctcaggac actaagagct 3960
ctgagacctc taagagcctt atctcgattt gaagggatga gggtagttgt gaatgccctt 4020
ttaggagcaa ttccatccat catgaatgtg cttctggttt gtcttatatt ctggctaatt 4080
ttcagcatca tgggcgtaaa tttgtttgct ggcaaattct accactgtat taacaccaca 4140
actggtgaca ggtttgacat cgaagacgtg aataatcata ctgattgcct aaaactaata 4200
gaaagaaatg agactgctcg atggaaaaat gtgaaagtaa actttgataa tgtaggattt 4260
gggtatctct ctttgcttca agttgccaca ttcaaaggat ggatggatat aatgtatgca 4320
gcagttgatt ccagaaatgt agaactccag cctaagtatg aagaaagtct gtacatgtat 4380
ctttactttg ttattttcat catctttggg tccttcttca ccttgaacct gtttattggt 4440
gtcatcatag ataatttcaa ccagcagaaa aagaagtttg gaggtcaaga catctttatg 4500
acagaagaac agaagaaata ctataatgca atgaaaaaat taggatcgaa aaaaccgcaa 4560
aagcctatac ctcgaccagg aaacaaattt caaggaatgg tctttgactt cgtaaccaga 4620
caagtttttg acataagcat catgattctc atctgtctta acatggtcac aatgatggtg 4680
gaaacagatg accagagtga atatgtgact accattttgt cacgcatcaa tctggtgttc 4740
attgtgctat ttactggaga gtgtgtactg aaactcatct ctctacgcca ttattatttt 4800
accattggat ggaatatttt tgattttgtg gttgtcattc tctccattgt aggtatgttt 4860
cttgccgagc tgatagaaaa gtatttcgtg tcccctaccc tgttccgagt gatccgtctt 4920
gctaggattg gccgaatcct acgtctgatc aaaggagcaa aggggatccg cacgctgctc 4980
tttgctttga tgatgtCCCt tCCtgCgttg tttaaCatCg gCCtCCtaCt cttcctagtc 5040
atgttcatct acgccatctt tgggatgtcc aactttgcct atgttaagag ggaagttggg 5100
atcgatgaca tgttcaactt tgagaccttt ggcaacagca tgatctgcct attccaaatt 5160
acaacctctg ctggctggga tggattgcta gcacccattc tcaacagtaa gccacccgac 5220
tgtgacccta ataaagttaa ccctggaagc tcagttaagg gagactgtgg gaacccatct 5280
gttggaattt tcttttttgt cagttacatc atcatatcct tcctggttgt ggtgaacatg 5340
tacatcgcgg tcatcctgga gaacttcagt gttgctactg aagaaagtgc agagcctctg 5400
agtgaggatg actttgagat gttctatgag gtttgggaga agtttgatcc cgatgcaact 5460
cagttcatgg aatttgaaaa attatctcag tttgcagctg cgcttgaacc gcctctcaat 5520
ctgccacaac caaacaaact ccagctcatt gccatggatt tgcccatggt gagtggtgac 5580
cggatccact gtcttgatat cttatttgct tttacaaagc gggttctagg agagagtgga 5640
gagatggatg ctctacgaat acagatggaa gagcgattca tggcttccaa tccttccaag 5700
gtctcctatc agccaatcac tactacttta aaacgaaaac aagaggaagt atctgctgtc 5760
attattcagc gtgcttacag acgccacctt ttaaagcgaa ctgtaaaaca agcttccttt 5820
acgtacaata aaaacaaaat caaaggtggg gctaatcttc ttataaaaga agacatgata 5880
attgacagaa taaatgaaaa ctctattaca gaaaaaactg atctgaccat gtccactgca 5940
gcttgtccac cttcctatga ccgggtgaca aagccaattg tggaaaaaca tgagcaagaa 6000
ggcaaagatg aaaaagccaa agggaaa 6027
<210> 41
<211> 2168
54/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 70035348CB1
<400> 41
attagctttg cccgaagttt ttccccacac tcttctttag catgctatta tggggaaagt 60
gaccactcct gggagcgggg gtggtcgggg cggtttggtg gcggggaagc ggctgtaact 120
tctacgtgac catggtacct gttgaaaaca ccgagggccc cagtctgctg aaccagaagg 180
ggacagccgt ggagacggag ggcagcggca gccggcatcc tccctgggcg agaggctgcg 240
gcatgtttac cttcctgtca tctgtcactg ctgctgtcag tggcctcctg gtgggttatg 300
aacttgggat catctctggg gctcttcttc agatcaaaac cttattagcc ctgagctgcc 360
atgagcagga aatggttgtg agCtCCCtCg tCattggagC CCtCCttgCC tcactcaccg 420
gaggggtcct gatagacaga tatggaagaa ggacagcaat catcttgtca tcctgcctgc 480
ttggactcgg aagcttagtc ttgatcctca gtttatccta cacggttctt atagtgggac 540
gcattgccat aggggtctcc atctccctct cttccattgc cacttgtgtt tacatcgcag 600
agattgctcc tcaacacaga agaggccttc ttgtgtcact gaatgagctg atgattgtca 660
tcggcattct ttctgcctat atttcaaatt acgcatttgc caatgttttc catggctgga 720
agtacatgtt tggtcttgtg attcccttgg gagttttgca agcaattgca atgtattttc 780
ttcctccaag ccctcggttt ctggtgatga aaggacaaga gggagctgct agcaaggttc 840
ttggaaggtt aagagcactc tcagatacaa ctgaggaact cactgtgatc aaatcctccc 900
tgaaagatga atatcagtac agtttttggg atctgtttcg ttcaaaagac aacatgcgga 960
cccgaataat gataggacta acactagtat tttttgtaca aatcactggc caaccaaaca 1020
tattgttcta tgcatcaact gttttgaagt cagttggatt tcaaagcaat gaggcagcta 1080
gcctcgcctc cactggggtt ggagtcgtca aggtcattag caccatccct gccactcttc 1140
ttgtagacca tgtcggcagc aaaacattcc tctgcattgg ctcctctgtg atggcagctt 1200
cgttggtgac catgggcatc gtaaatctca acatccacat gaacttcacc catatctgca 1260
gaagccacaa ttctatcaac cagtccttgg atgagtctgt gatttatgga ccaggaaacc 1320
tgtcaaccaa caacaatact ctcagagacc acttcaaagg gatttcttcc catagcagaa 1380.
gctcactcat gcccctgaga aatgatgtgg ataagagagg ggagacgacc tcagcatcct 1440
tgctaaatgc tggattaagc cacactgaat accagatagt cacagaccct ggggacgtcc 1500
cagctttttt gaaatggctg tccttagcca gcttgcttgt ttatgttgct gctttttcaa 1560
ttggtctagg accaagagat gttatcttta tcggacagtc aacaaacttg ccctctgctc 1620
cagagggtga cactatctct atctccaaga ctatttatta tgcagcctac aacaaggcta 1680
ttatacaaac agccttggaa agacagccta gagcaaagac agtcagtgcc ttttcccata 1740
agacatgaag aaatgtgaga gacctacgga gaactggctc ecaacccaaa tatcctaaaa 1800
ctcaaatgtc tttctttcta ttcgaaacaa caaactagaa ttttgaaaaa ctcaaagacc 1860
atagagccta gctttttgct ctgtttggtt ttatggagct gaaccagcct attggagggt 1920
gggtatcaat gttggaagca tgagtcatct gccgtaaaat ttaaacttag atttaaacaa 1980
ataactctgg ctcttaaaaa ttttgttcat tggatatttg cacagctaaa gattatgaca 2040
gctccaagga tgtggagcag caggttctaa tttggaagtt tacctagtgg cttcatttca 2100
agacctactg ggtttaaggc aaagaggctg acattgcaga agcacaggtg tttcaaatca 2160
gattctgg 2168
<210> 42
<211> 2229
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472539CB2
<400> 42
atggaatacc aggcgtccga ggtgatcggg cagcgtcagt cttcagccac taagccagga 60
agatctggga aggagtcagt cacagagccc tgggccagag ttccaggggc tctgggagtg 120
gctgccaggc agatgcaccc caagtcaata atcacattca gagagataaa tggggagtac 180
actggggctg tggattttcc caggctagga gtccgtgctt ctgaggaaac agcgctcaga 240
gagctgaaga tgagcaagga gctggcagca atggggcctg gagcttcagg ggacggggtc 300
aggactgaga cagctccaca catagcactg gactccagag ttggtctgca cgcctacgac 360
atcagcgtgg tggtcatcta ctttgtcttc gtcattgctg tggggatctg gtcgtccatc 420
55/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
cgtgcaagtc gagggaccat tggcggctat ttcctggccg ggagttggag catctctgat 480
gtccagcaat gtgggcagtg gcttgttcat cggcctggct gggacagggg ctgccggagg 540
ccttgccgta ggtggcttcg agtggaactg ctcctggccc ttggctgggt cttcgtccct 600
gtgtacatcg cagcaggtgt ggtcacaatg ccgcagtatc tgaagaagcg atttgggggc 660
cagaggatcc aggtgtacat gtctgtcctg tctctcatcc tctacatctt caccaagatc 720
tcgactgaca tcttctctgg agccctcttc atccagatgg cattgggctg gaacctgtac 780
ctctccacag ggatcctgct ggtggtgact gccgtctaca ccattgcagg tggcctcatg 840
gccgtgatct acacagatgc tctgcagacg gtgatcatgg tagggggagc cctggtcctc 900
atgtttctgg gctttcagga cgtgggctgg tacccaggcc tggagcagcg gtacaggcag 960
gccatcccta atgtcacagt ccccaacacc acctgtcacc tcccacggcc cgatgctttc 1020
cacattcttc gggaccctgt gagcggggac atcccttggc caggtctcat tttcgggctc 1080
acagtgctgg ccacctggtg ttggtgcaca gaccaggtca ttgtgcagcg gtctctctcg 1140
gccaagagtc tgtctcatgc caagggaggc tccgtgctgg ggggctacct gaagatcctc 1200
cccatgttct tcatcgtcat gcctggcatg atcagccggg ccctgttccc agacgaggtg 1260
ggctgcgtgg accctgatgt ctgccaaaga atctgtgggg cccgagtggg atgttccaac 1320
attgcctacc ctaagttggt catggccctc atgcctgttg gtctgcgggg gctgatgatt 1380
gccgtgatca tggccgctct catgagctca ctcacctcca tcttcaacag cagcagcacc 1440
ctgttcacca ttgatgtgtg gcagcgcttc cgcaggaagt caacagagca ggagctgatg 1500
gtggtgggca gagtgtttgt ggtgttcctg gttgtcatca gcatcctctg gatccccatc 1560
atccaaagct ccaacagtgg gcagctcttc gactacatcc aggctgtcac cagttacctg 1620
gccccaccca tcaccgctct cttcctgctg gccatcttct gcaagagggt cacagagccc 1680
ggagctttct ggggcctcgt gtttggcctg ggagtggggc ttctgcgtat gatcctggag 1740
ttctcatacc cagcgccagc ctgtggggag gtggaccgga ggecagcagt gctgaaggac 1800
ttccactacc tgtactttgc aatcctcctc tgcgggctca ctgccatcgt cattgtcatt 1860
ctcacacgcc tcacatggtg gactcggaac tgccccctct ctgagctgga gaaggaggcc 1920
cacgagagca caccggagat atccgagagg ccagccgggg agtgccctgc aggaggtgga 1980
gcggcagaga actcgagcct gggccaggag cagcctgaag ccccaagcag gtcctgggga 2040
aagttgctct ggagctggtt ctgtgggctc tctggaacac cggagcaggc cctgagccca 2100
gcagagaagg ctgcgctaga acagaagctg acaagcattg aggaggagcc actctggaga 2160
catgtctgca acatcaatgc tgtccttttg ctggccatca acatcttcct ctggggctat 2220
tttgcgtga 2229
<210> 43
<211> 1520
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 817477CB1
<400> 43
gCgCCtCgtC gggCCCttCC tCtCtaCCtg CCtCtCCaaC CCCtCtCggC CCCgagCCaC 60
ccggcagcgg gggtgggtgt gcagaggtgc ggcgtccaga accccggctc ctgcagaggc 120
tctgggtggc agcagccctg ttaccgctta gatggcgcgc aggacagagc cccccgacgg 180
gggctgggga tgggtggtgg tgctctcagc gttcttccag tcggcgcttg tgtttggggt 240
gCtCCgCtCC tttggggtct tcttcgtgga gtttgtggcg gcgtttgagg agcaggcagc 300
gcgcgtctcc tggatcgcct ccataggaat cgcggtgcag cagtttggga gcccggtagg 360
cagtgccctg agcacgaagt tcgggcccag gcccgtggtg atgactggag gcatcttggc 420
tgcgctgggg atgctgctcg cctcttttgc tacttccttg acccacctat acctgagtat 480
tgggttgctg tcaggctctg gctgggcttt gaccttcgct ccgaccctgg cctgcctgtc 540
ctgttatttc tctcgccgac gatccctggc caccgggctg gcactgacag gcgtgggcct 600
ctcctccttc acatttgccc cctttttcca gtggctgctc agccactacg cctggagggg 660
gtccctgctg ctggtgtctg ccctctccct ccacctagtg gcctgtggtg ctctcctccg 720
cccaccctcc ctggctgagg accctgctgt gggtggtccc agggcccaac tcacctctct 780
cctccatcat ggccccttcc tccgttacac tgttgccctc accctgatca acactggcta 840
cttcattccc tacctccacc tggtggccca tctccaggac ctggattggg acccactacc 900
tgctgccttc ctactctcag ttgttgctat ttctgacctc gtggggcgtg tggtctccgg 960
atggctggga gatgcagtcc cagggcctgt gacacgactc ctgatgctct ggaccacctt 1020
gactggggtg tcactagccc tgttccctgt agctcaggct cccacagccc tggtggctct 1080
ggctgtggcc tacggcttca catcaggggc tctggcccca ctggccttct ctgtgctgcc 1140
tgaactaata gggactagaa ggatttactg tggcctggga ctgttgcaga tgatagagag 1200
catcgggggg ctgctggggc ctcctctctc aggctacctc cgggatgtga caggcaacta 1260
56/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
cacggcttct tttgtggtgg ctggggcctt ccttctttca gggagtggca ttctcctcac 1320
cctgccccac ttcttctgct tctcaactac tacctccggg ccccaggacc ttgtaacaga 1380
agcactagat actaaagttc ccctacccaa ggagggactg gaagaggact gaactccaca 1440
gagtcaggcc cagaaagcca aagcttgaca gctccaggtc ttctcttgcc acgtcttggt 1500
ctccacagaa ccacagtgcc 1520
<210> 44
<211> 3950
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1442166CB1
<400> 44
gccagcctgt tctgttgccc tggctcttcc tagtccaggc tgccatggcg gcgctcaggg 60
cttaccggaa gtaaaacttc ggaagtgagg cgttcctctg cccggaagtg agcgcggcgc 120
taggaaagat ggcggcagcg gcggcggtgg gcaacgcggt gCCCtgcggg gcccggcctt 180
gcggggtccg gcctgacggg cagcccaagc ccgggccgca gccgcgcgcg ctccttgccg 240
ccgggccggc gctcatagcg aacggtgacg agctggtggc tgccgtgtgg ccgtaccggc 300
ggttggcgct gttgcggcgc ctcacggtgc tgccattcgc cgggctgctt tacccggcct 360
ggttgggtgc cgcagccgct ggctgctggg gctggggcag cagttgggtg cagatccccg 420
aagctgcgct gctcgtgctt gccaccatct gcctcgcgca cgcgctcact gtcctctcgg 480
ggcattggtc tgtgcacgcg cattgcgcgc tcacctgcac cccggagtac gaccccagca 540
aagcgacctt tgtgaaggtg gtgccaaccc ccaacaatgg ctccacggag ctcgtggccc 600
tgcaccgcaa tgagggcgaa gacgggcttg aggtgctgtc cttcgaattc cagaagatca 660
agtattccta cgatgccctg gagaagaagc agtttctccc cgtggccttt cctgtgggaa 720
acgccttctc atactatcag agcaacagag gcttccagga agactcagag atccgagcag 780
ctgagaagaa atttgggagc aacaaggccg agatggtggt gcctgacttc tcggagcttt 840
tcaaggagag agccacagcc cccttctttg tatttcaggt gttctgtgtg gggctctggt 900
gcctggatga gtactggtac tacagcgtct ttacgctatc catgctggtg gcgttcgagg 960
cctcgctggt gcagcagcag atgcggaaca tgtcggagat ccggaagatg ggcaacaagc 1020
cccacatgat ccaggtctac cgaagccgca agtggaggcc cattgccagt gatgagatcg 1080
taccagggga catcgtctcc atcggccgct ccccacagga gaacctggtg ccatgtgacg 1140
tgcttctgct gcgaggccgc tgcatcgtag acgaggccat gctcacgggg gagtccgtgc 1200
cacagatgaa ggagcccatc gaagacctca gcccagaccg ggtgctggac ctccaggctg 1260
attcccggct gcacgtcatc ttcgggggca ccaaggtggt gcagcacatc cccccacaga 1320
aagccaccac gggcctgaag ccggttgaca gcgggtgcgt ggcctacgtc ctgcggaccg 1380
gattcaacac atcccagggc aagctgctgc gcaccatcct cttcggggtc aagagggtga 1440
ctgcgaacaa cctggagacc ttcatcttca tcctcttcct cctggtgttt gccatcgctg 1500
cagctgccta tgtatggatt gaaggtacca aggaccccag ccggaaccgc tacaagctgt 1560
ttctggagtg CaCCCtgatC CtCaCCtCgg tCgtgCCtCC tgagctgccc atcgagctgt 1620
CCCtggCCgt CaaCaCCtCC CtCatCgCCC tggCCaagCt CtaCatgtaC tgcacagagc 1680
ccttccggat cccctttgct ggcaaggtcg aggtgtgctg ctttgacaag acggggacgt 1740
tgaccagtga cagcctggtg gtgcgcggtg tggccgggct gagagacggg aaggaggtga 1800
ccccagtgtc cagcatccct gtagaaacac accgggccct ggcctcgtgc cactcgctca 1860
tgcagctgga cgacggcacc ctcgtgggtg accctctaga gaaggccatg ctgacggccg 1920
tggactggac gctgaccaaa gatgagaaag tattcccccg aagtattaaa actcaggggc 1980
tgaaaattca ccagcgcttt cattttgcca gtgccctgaa gcgaatgtcc gtgcttgcct 2040
cgtatgagaa gctgggctcc accgacctct gctacatcgc ggccgtgaag ggggcccccg 2100
aaactctgca ctccatgttc tcccagtgcc cgcccgacta ccaccacatc cacaccgaga 2160
tctcccggga aggagcccgc gtcctggcgc tggggtacaa ggagctggga cacctcactc 2220
accagcaggc ccgggaggtc aagcgggagg ccctggagtg cagcctcaag ttcgtcggct 2280
tcattgtggt ctcctgcccg ctcaaggctg actccaaggc cgtgatccgg gagatccaga 2340
atgcgtccca ccgggtggtc atgatcacgg gagacaaccc gctcactgca tgccacgtgg 2400
cccaggagct gcacttcatt gaaaaggccc acacgctgat cctgcagcct ccctccgaga 2460
aaggccggca gtgcgagtgg cgctccattg acggcagcat cgtgctgccc ctggcccggg 2520
gctccccaaa ggcactggcc ctggagtacg cactgtgcct cacaggcgac ggcttggccc 2580
acctgcaggc caccgacccc cagcagctgc tccgcctcat cccccatgtg caggtgttcg 2640
cccgtgtggc tcccaagcag aaggagtttg tcatcaccag cctgaaggag ctgggctacg 2700
tgaccctcat gtgtggggat ggcaccaacg acgtgggcgc cctgaagcat gctgacgtgg 2760
gtgtggcgct cttggccaat gcccctgagc gggttgtcga gcggcgacgg cggccccggg 2820
$7/~7
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
acagcccaac cctgagcaac agtggcatca gagccacctc caggacagcc aagcagcggt 2880
cggggctccc tccctccgag gagcagccaa cctcccagag ggaccgcctg agccaggtgc 2940
tgcgagacct cgaggacgag agtacgccca ttgtgaaact gggggatgcc agcatcgcag 3000
cacccttcac ctccaagctc tcatccatcc agtgcatctg ccacgtgatc aagcagggcc 3060
gctgcacgct ggtgaccacg ctacagatgt tcaagatcct ggcgctcaat gccctcatcc 3120
tggcctacag ccagagcgtc ctctacctgg agggagtcaa gttcagtgac ttccaggcca 3180
ccctacaggg gctgctgctg gccggctgct tcctcttcat ctcccgttcc aageccctca 3240
agaccctctc ccgagaacgg cccctgccca acatcttcaa cctgtacacc atcctcaccg 3300
tcatgctcca gttctttgtg cacttcctga gccttgtcta cctgtaccgt gaggcccagg 3360
cccggagccc cgagaagcag gagcagttcg tggacttgta caaggagttt gagccaagcc 3420
tggtcaacag caccgtctac atcatggcca tggccatgca gatggccacc ttcgccatca 3480
attacaaagg cccgcccttc atggagagcc tgcccgagaa caagcccctg gtgtggagtc 3540
tggcagtttc actcctggcc atcattggcc tgctcctcgg ctcctcgccc gacttcaaca 3600
gccagtttgg cctcgtggac atccctgtgg aggtcctgct cctggacttc tgcctggcgc 3660
tcctggccga ccgcgtcctg cagttcttcc tggggacccc gaagctgaaa gtgccttcct 3720
gagatggcag tgctggtacc cactgcccac cctggctgcc gctgggcggg aaccccaaca 3780
gggccccggg agggaaccct gcccccaacc ccccacagca aggctgtaca gtctcgccct 3840
tggaagactg agctgggacc cccacagcca tccgctggct tggccagcag aaccagcccc 3900
aagccagcac ctttggtaaa taaagcagca tctgagattt taaaaaaaaa 3950
<210> 45
<211> 5540
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_~eature
<223> Incyte ID No: 2311751CB1
<400> 45
tctacttcct ctacggcttc gtctggatcc aggacatgat ggagcgcgcc atcatcgaca 60
cttttgtggg gcacgacgtg gtggagccag gcagctacgt gcagatgttc ccctacccct 120
gctacacacg cgatgacttc ctgtttgtca ttgagcacat gatgccgctg tgcatggtga 180
tctcctgggt ctactccgtg gccatgacca tccagcacat cgtggcggag aaggagcacc 240
ggctcaagga ggtgatgaag accatgggcc tgaacaacgc ggtgcactgg gtggcctggt 300
tcatcaccgg ctttgtgcag ctgtccatct ccgtgacagc actcaccgcc atcctgaagt 360
acggccaggt gcttatgcac agccacgtgg tcatcatctg gctcttcctg gcagtctacg 420
cggtggccac catcatgttc tgcttcctgg tgtctgtgct gtactccaag gccaagctgg 480
cctcggcctg cggtggcatc atctacttcc tgagctacgt gccctacatg tacgtggcga 540
tccgagagga ggtggcgcat gataagatca cggccttcga gaagtgcatc gcgtccctca 600
tgtccacgac ggcctttggt ctgggctcta agtacttcgc gctgtatgag gtggccggcg 660
tgggcatcca gtggcacacc ttcagccagt ccccggtgga gggggacgac ttcaacttgc 720
tcctggctgt caccatgctg atggtggacg ccgtggtcta tggcatcctc acgtggtaca 780
ttgaggctgt gcacccaggc atgtacgggc tgCCCCggCC CtggtaCttC CCa.CtgCaga 840
agtcctactg gctgggcagt gggcggacag aagcctggga gtggagctgg ccgtgggcac 900
gcaccccccg cctcagtgtc atggaggagg accaggcctg tgccatggag agccggcgct 960
ttgaggagac ccgtggcatg gaggaggagc ccacccacct gcctctggtt gtctgcgtgg 1020
acaaactcac caaggtctac aaggacgaca agaagctggc cctgaacaag ctgagcctga 1080
acctctacga gaaccaggtg gtctccttct tgggccacaa cggggcgggc aagaccacca 1140
ccatgtccat cctgaccggc ctgttccctc caacgtcggg ttccgccacc atetacgggc 1200
acgacatccg cacggagatg gatgagatcc gcaagaacct gggcatgtgc ccgcageaca 1260
atgtgctctt tgaccggctc acggtggagg aacacctctg gttctactca cggctcaaga 1320
gcatggctca ggaggagatc cgcagagaga tggacaagat gatcgaggac ctggagctct 1380
ccaacaaacg gcactcactg gtgcagacat tgtcgggtgg catgaagcgc aagctgtccg 1440
tggccatcgc cttcgtgggc ggctctcgcg ccatcatcct ggacgagccc acggcgggcg 1500
tggaccccta cgcgcgccgc gccatctggg acctcatcct gaagtacaag ccaggccgca 1560
ccatccttct gtccacccac cacatggatg aggctgacct gcttggggac cgcattgcca 2620
tcatctccca tgggaagctc aagtgctgcg gctccccgct cttcctcaag ggcacctatg 1680
gcgacgggta ccgcctcacg ctggtcaagc ggcccgccga gccggggggc ccccaagagc 1740
cagggctggc atccagcccc ccaggtcggg ccccgctgag cagctgctcc gagctccagg 1800
tgtcccagtt catccgcaag catgtggcct cctgcctgct ggtctcagac acaagcacgg 1860
agctctccta catcctgccc agcgaggccg ccaagaaggg ggctttcgag cgcctcttcc 1920
agcacctgga gcgcagcctg gatgcactgc acctcagcag cttcgggctg atggacacga 1980
58/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
ccctggagga agtgttcctc aaggtgtcgg aggaggatca gtcgctggag aacagtgagg 2040
ccgatgtgaa ggagtccagg aaggatgtgc tccctggggc ggagggcccg gcgtctgggg 2100
agggtcacgc tggcaatctg gcccggtgct cggagctgac ccagtcgcag gcatcgctgc 2160
agtcggcgtc atctgtgggc tctgcccgtg gcgacgaggg agctggctac accgacgtct 2220
atggcgacta ccgccccctc tttgataacc cacaggaccc agacaatgtc agcctgcaag 2280
aggtggaggc agaggccctg tcgagggtcg gccagggcag ccgcaagctg gacggcgggt 2340
ggctgaaggt gcgccagttc cacgggctgc tggtcaaacg cttccactgc gcccgccgca 2400
actccaaggc actCttCtcc cagatcttgc tgCCagCCtt CttCgtCtgC gtggccatga 2460
ccgtggccct gtccgtcccg gagattggtg atctgccccc gctggtcctg tcaccttccc 2520
agtaccacaa ctacacccag ccccgtggca atttcatccc ctacgccaac gaggagcgcc 2580
gcgagtaccg gctgcggcta tcgcccgacg ccagccccca gcagctcgtg agcacgttcc 2640
ggctgccgtc gggggtgggt gccacctgcg tgctcaagtc tcccgccaac ggctcgctgg 2700
ggcccacgtt gaacctgagc agcggggagt cgcgcctgct ggcggctcgg ttcttcgaca 2760
gcatgtgtct ggagtccttc acacaggggc tgccactgtc caatttcgtg ccacccccac 2.82.0
cctcgcccgc cccatctgac tcgccagcgt ccccggatga ggacctgcag gcctggaacg 2880
tctccctgcc gcccaccgct gggccagaaa tgtggacgtc ggcaccctcc ctgccgcgcc 2940
tggtacggga gcccgtccgc tgcacctgct ctgcgcaggg caccggcttc tcctgcccca 3000
gcagtgtggg cgggcacccg ccccagatgc gggtggtcac aggcgacatc ctgaccgaca 3060
tcaccggcca caatgtctct gagtacctgc tCttCaCCtC CgaCCgCttC CgaCtgCa.CC 3120
ggtatggggc catcaccttt ggaaacgtcc tgaagtccat cccagcctca tttggcacca 3180
gggccccacc catggtgcgg aagatcgcgg tgcgcagggc tgcccaggtt ttctacaaca 3240
acaagggcta tcacagcatg cccacctacc tcaacagcct caacaacgcc atcctgcgtg 3300
ccaacctgcc caagagcaag ggcaacccgg cggcttacgg catcaccgtc accaaccacc 3360
ccatgaataa gaccagcgcc agcctctccc tggattacct gctgcagggc acggatgtcg 3420
tcatcgccat cttcatcatc gtggccatgt ccttcgtgcc ggccagcttc gttgtcttcc 3480
tcgtggccga gaagtccacc aaggccaagc atctgcagtt tgtcagcggc tgcaacccca 3540
tcatctactg gctggcgaac tacgtgtggg acatgctcaa ctacctggtc cccgctacct 3600
gctgtgtcat catcctgttt gtgttcgacc tgccggccta cacgtcgccc accaacttcc 3660
ctgccgtcct ctccctcttc ctgctctatg ggtggtccat cacgcccatc atgtacccgg 3720
cctccttctg gttcgaggtc cccagc~ccg cctacgtgtt cctcattgtc atcaatctct 3780
tcatcggcat caccgccacc gtggccacct tcctgctaca gctcttcgag cacgacaagg 3840
acctgaaggt tgtcaacagt tacctgaaaa gctgcttcct cattttcccc aactacaacc 3900
tgggccacgg gctcatggag atggcctaca acgagtacat caacgagtac tacgccaaga 3960
ttggccagtt tgacaagatg aagtccccgt tcgagtggga cattgtcacc cgcggactgg 4020
tggccatggc ggttgagggc gtcgtgggct tcctcctgac catcatgtgc cagtacaact 4080
tcctgcggcg gccacagcgc atgcctgtgt ctaccaagcc tgtggaggat gatgtggacg 4140
tggccagtga gcggcagcga gtgctccggg gagacgccga caatgacatg gtcaagattg 4200
agaacctgac caaggtctac aagtcccgga agattggccg tatcctggcc gttgaccgcc 4260
tgtgcctggg tgtgcgtcct ggcgagtgct tcgggctcct gggcgtcaac ggtgcgggca 4320
agaccagcac cttcaagatg ctgaccggcg acgagagcac gacggggggc gaggccttcg 4380
tcaatggaca cagcgtgetg aaggagctgc tccaggtgca gcagagcctc ggctactgcc 4440
cgcagtgtga cgcgctgttc gacgagctca cggcccggga gcacctgcag ctgtacacgc 4500
ggctgcgtgg gatctcctgg aaggacgagg cccgggtggt gaagtgggct ctggagaagc 4560
tggagctgac caagtacgca gacaagccgg ctggcaccta cagcggcggc aacaagcgga 4620
agctctccac ggccatcgcc ctcattgggt acccagcctt catcttcctg gacgagccca 4680
ccacaggcat ggaccccaag gcccggcgct tcctctggaa cctcatcctc gacctcatca 4740
agacagggcg ttcagtggtg ctgacatcac acagcatgga ggagtgcgag gcgctgtgca 4800
cgcggctggc catcatggtg aacggtcgcc tgcggtgcct gggcagcatc cagcacctga 4860
agaaccggtt tggagatggc tacatgatca cggtgcggac caagagcagc cagagtgtga 4920
aggacgtggt gcggttcttc aaccgcaact tcccggaagc catgctcaag gagcggcacc 4980
acacaaaggt gcagtaccag ctcaagtcgg agcacatctc gctggcccag gtgttcagca 5040
agatggagca ggtgtctggc gtgctgggca tcgaggacta ctcggtcagc cagaccacac 5100
tggacaatgt gttcgtgaac tttgccaaga agcagagtga caacctggag cagcaggaga 5160
cggagccgcc atccgcactg cagtcccctc tcggctgctt gctcagcctg ctccggcccc 5220
ggtctgcccC cacggagctc cgggcacttg tggcagacga gcccgaggac ctggacacgg 5280
aggacgaggg cctcatcagc ttcgaggagg agcgggccca gctgtccttc aacacggaca 5340
cgctctgctg accacccaga gctgggccag ggaggacacg CtCCdCtgaC CdCCCagdgC 5400
tgggccaggg actcaacaat ggggacagaa gtcccccagt gcctgccagg gcctggagtg 5460
gaggttcagg accaaggggc ttctggtcct ccagcccctg tactcggcca tgtcctgcgg 5520
tcactgcggt tgccggccct 5540
<210> 46
<211> 2074
59/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472537CB1
<220>
<221> unsure
<222> 1869, 1898, 1988, 2000
<223> a, t, c, g, or other
<400> 46
ggaatcacag tgcctaggca tataataaat attcgttgaa ttaataaaat catctgatta 60
tggtatggta gtagttcaga aaattctgtc atgaccctgt actctttctt tggaagggct 120
ctaaatggga acaacaatat agtatgtagt ctctctgcat agctaatgtg cagcaaagca 180
gggcaatgta ggtatacaac caatctattt ttcaactcag aaacatcaca tcatttccat 240
tcctttataa ccatccttct tccatcccaa agtatagttt gtcaacctgg aactcaa.aca 300
ttgtatggtc tggaatgacc gtacagtgtg aaggaggaaa agaaaattgg ggtgtcttat 360
ttcccctcct ctgattcagt tacttagatc acctgaaaca tacatatgat tcagagcata 420
tatttagatg ttttcacttt cttatttgtg tgtgtgtgtg ttcagtcaat ttgctaatga 480
agacactgaa agtcagaaat tcctgacaaa tggatttttg gggaaaaaga agctggcaga 540
tcccttcttt ttcaagcatc ccggaaccac ttcctttgga atgtcttcat ttaacctgag 600
taatgccatc atgggcagtg ggatcctggg cttgtcctat gccatggcca acacagggat 660
catacttttt atgttcatgc tgcttgctgt ggcaatatta tcactgtatt cagttcacct 720
tttattaaaa acatctttga ttgtagggtc tttgatttat gaaaaattag gagaaaaggc 780
atttggatgg ccgggaaaaa ttggagcttt tgtttccatt acaatgcaga acattggagc 840
aatgtcaagc tacctcttta tcattaaata tgaactacct gaagtaatca gagcattcat 900
gggacttgaa gaaacttcta gagaatggta cctcaatggc aactacctca tcatatttgt 960
gtctgttgga attattcttc cactttcgct ccttaaaaat ctaggttatc ttggctatac 1020
cagtggattt tctcttacct gcatggtgtt ttttgttagt gtggtgattt acaagaaatt 1080
ccaaataccc tgccctctac ctgagaacca ggccaagggc tctcttcatg acagtggagt 1140
agaatatgaa gctcatagtg atgacaagtg tgaacccaaa tactttgtat tcaactccca 1200
gacggcctat gcaattccta tcctagtatt tgcttttgta tgccaccctg aggtccttcc 1260
catctacagt gaacttaaag atcggtcccg gagaaaaatg caaacggtgt caaatatttc 1320
catcacgggg atgcttgtca tgtaCCtgCt tgCCgCCCtC tttggttacc taaccttcta 1380
tggtagggtt gaagatgaat tacttcatgc ctacagcaaa gtgtatacat tagacatccc 1440
ccttctcatg gttcgcctgg cagtccttgt ggcagtaaca ctaactgtgc ccattgtcct 1500
cttcccagtt cgtacatcag tgatcacact gttatttccc aaacgaccct tcagctggat 1560
acgacatttc ctgattgcag ctgtgcttat tgcacttaat aatgttctgg tcatccttgt 1620
gccaactata aaatacatct tcggattcat aggggcttct tctgccacta tgctgatttt 1680
tattcttcca gcagtttttt atcttaaact tgtcaagaaa gaaactttta ggtcaccccc 1740
tgaattacag gctttaattt tccttgtggt tggaatattc ttcatgattg gaagcatggc 1800
actcattata attgactgga tttatgatcc tccaaattcc aagcatcact aacacaagga 1860
aaaatactnt ctttttctat tggaaatggt tacaagtnat actccaaaag atatttgaat 1920
tatcttgatt ggaatgttat tcataggaaa taacaggaag attccaaaga cgtttaccag 1980
taatatcncc aggcacctgn cagaagaggg aaaatcactg tttttgtcaa ggatggttgt 2040
gtatgtgttt taaaataaaa cctgtggtgc acat 2074
<210> 47
<211> 2259
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472546CB1
<400> 47
atggaatacc aggcgtccga ggtgatcggg cagcgtcagt cttcagccac taagccagga 60
agatctggga aggagtcagt cacagagccc tgggccagag ttccaggggc tctgggagtg 120
gctgccaggc agatgcaccc caagtcaata atcacattca gagagataaa tggggagtac 180
actggggctg tggattttcc caggctagga gtccgtgctt ctgaggaaac agcgctcaga 240
60/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
gagctgaaga tgagcaagga gctggcagca atggggcctg gagcttcagg ggacggggtc 300
aggactgaga cagctccaca catagcactg gactccagag ttggtctgca cgcctacgac 360
atcagcgtgg tggtcatcta ctttgtcttc gtcattgctg tggggatctg gtcgtccatc 420
cgtgcaagtc gagggaccat tggcggctat ttcctggccg ggaggtccat gagctggtgg 480
ccaattggag catctctgat gtccagcaat gtgggcagtg gcttgttcat cggcctggct 540
gggacagggg ctgccggagg ccttgccgta ggtggcttcg agtggaacgc aacctggctg 600
ctcctggccc ttggctgggt cttcgtccct gtgtacatcg cagcaggtgt ggtcacaatg 660
ccgcagtatc tgaagaagcg atttgggggc cagaggatcc aggtgtacat gtctgtcctg 720
tctctcatcc tctacatctt caccaagatc tcgactgaca tcttctctgg agccctcttc 780
atccagatgg cattgggctg gaacctgtac ctctccacag ggatcctgct ggtggtgact 840
gccgtctaca ccattgcagg tggcctcatg gccgtgatct acacagatgc tctgcagacg 900
gtgatcatgg tagggggagc cctggtcctc atgtttctgg gctttcagga cgtgggctgg 960
tacccaggcc tggagcagcg gtacaggcag gccatcccta atgtcacagt ccccaacacc 1020
acctgtcacc tcccacggcc cgatgctttc cacattcttc gggaccctgt gagcggggac 1080
atcccttggc caggtctcat tttcgggctc acagtgctgg ccacctggtg ttggtgcaca 1140
gaccaggtca ttgtgcagcg gtctctctcg gccaagagtc tgtctcatgc caagggaggc 1200
tccgtgctgg ggggctacct gaagatcctc cccatgttct tcatcgtcat gcctggcatg 1260
atcagccggg ccctgttccc agacgaggtg ggctgcgtgg accctgatgt ctgccaaaga 1320
atctgtgggg cccgagtggg atgttccaac attgcctacc ctaagttggt catggccctc 1380
atgcctgttg gtctgcgggg gctgatgatt gccgtgatca tggCCgCtCt catgagctca 1440
ctcacctcca tcttcaacag cagcagcacc ctgttcacca ttgatgtgtg gcagcgcttc 1500
cgcaggaagt caacagagca ggagctgatg gtggtgggca gagtgtttgt ggtgttcctg 1560
gttgtcatca gcatcctctg gatccccatc atccaaagct ccaacagtgg gcagctcttc 1620
gaCtaCatCC aggCtgtCaC cagttacctg gccccaccca tcaccgctct cttcctgctg 1680
gccatcttct gcaagagggt cacagagccc ggagctttct ggggcctcgt gtttggcctg 1740
ggagtggggc ttctgcgtat gatcctggag ttctcatacc cagcgccagc ctgtggggag 1800
gtggaccgga ggccagcagt gctgaaggac ttccactacc tgtactttgc aatcctcctc 1860
tgcgggctca ctgccatcgt cattgtcatt ctcacacgcc tcacatggtg gactcggaac 1920
tgccccctct ctgagctgga gaaggaggcc cacgagagca caccggagat atccgagagg 1980
ccagccgggg agtgccctgc aggaggtgga gcggcagaga actcgagcct gggccaggag 2040
cagcctgaag ccccaagcag gtcctgggga aagttgctct ggagctggtt ctgtgggctc 2100
tctggaacac cggagcaggc cctgagccca gcagagaagg ctgcgctaga acagaagctg 2160
acaagcattg aggaggagcc actctggaga catgtctgca acatcaatgc tgtccttttg 2220
ctggccatca acatcttcct ctggggctat tttgcgtga 2259
<210> 48
<211> 2439
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7474202CB1
<400> 48
ggctctgtga gaggagggcc agttcagccg cagcaggagg actgacaggg gcctgatgga 60
ggagttggtg gggctgcgtg agggcttctc aggggaccct gtgactctgc aggagctgtg 120
gggcccctgt ccccacatcc gccgagccat ccaaggtggc ctggagtggc taaagcagaa 180
ggtgttccgc ctgggagaag actggtactt cctgatgacc ctcggggtgc tcatggccct 240
ggtcagctat gccatgaact ttgccatcgg gtgtgtggtc cgaggcttct cccagagcat 300
cacgccctcc tctggaggtt ctggaatccc ggagctgaag accatgttgg cgggtgtgat 360
cttggaggac tacctggata tcaagaactt tggggccaag gtggtgggcc tctcctgcac 420
cctggccacc ggcagcaccc tgttcctggg caaagtgggc cctttcgtgc acctgtctgt 480
aatgatcgct gcctacctgg gccgtgtgcg caccacgacc atcggggagc ctgagaacaa 540
gagcaagcaa aacgaaatgc tggtggcagc ggcggcagtg ggcgtggcca cagtctttgc 600
agctcccttc agcggcgtcc tgttcagcat cgaggtcatg tcttcccact tctctgtccg 660
ggattactgg aggggcttct ttgcggccac ctgcggggcc ttcatattcc ggctcctggc 720
agtcttcaac agcgagcagg agaccatcac ctccctctac aagaccagtt tccgggtgga 780
cgttcccttc gacctgcctg agatcttctt ttttgtggcg ctgggtggca tctgcggcgt 840
cctgagctgt gcttacctct tctgtcagcg aaccttcctc agcttcatca agaccaatcg 900
gtacagctcc aaactgctgg ctactagcaa gcctgtgtac tccgctctgg ccaccttgct 960
tctcgcctcc atcacctacc cgcctggtgt gggccacttc ctagcttctc ggctgtccat 1020
gaagcagcat ctggactcgc tgttcgacaa ccactcctgg gcgctgatga cccagaactc 1080
6I/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
cagcccaccc tggcccgagg agctcgaccc ccagcacctt tggtgggaat ggtaccaccc 1140
gCggttCaCC atCtttggga CCCttgCCtt cttcctggtt atgaagttct ggatgctgat 1200
tctggccacc accatcccca tgcctgccgg gtacttcatg cccatcttta tccttggagc 1260
tgccatcggg cgcctcttgg gagaggctct tgccgtcgcc ttccctgagg gcattgtgac 1320
tggaggggtt accaatccca tcatgcccgg ggggtatgct ctggcagggg ctgcagcctt 1380
ctcaggggct gtgacccaca ccatctccac ggcgctgctg gcctttgagc tgaccggcca 1440
gatagtgcat gcactgcccg tgctgatggc ggtgctggca gccaacgcca ttgcacagag 1500
ctgccagccc tccttctatg atggcaccat cattgtcaag aagctgccat acctgccacg 1560
gattctgggc cgcaacatcg gctcccacca tgtgagggtg gagcacttca tgaaccacag 1620
catcaccaca ctggccaagg acacgccgct ggaggaggtg gtcaaggttg tgacctccac 1680
agacgtgacc gagtatcccc tggtggagag cacagagtcc cagatcctgg taggcatcgt 1740
gcagagggcc cagctggtgc aggccctcca ggctgagcct ccttccaggg ctccaggaca 1800
ccagtgtctc caggacatct tggccagggg ctgccccacg gaaccagtga ccctgacgct 1860
attctcagag accaccttgc accaggcaca aaacctcttt aagctgttga accttcagtc 1920
cctcttcgtg acatcgcggg gcagagctgt gggctgcgtg tcctgggtgg agatgaagaa 1980
agcaatttcc aacctgacaa atccgccagc tccaaagtga gccggcccag caagatgaaa 2040
cagggcaccc cagctgacct ggtactgagg ttgggctgag accctgcttc tcttccccca 2100
tcaccacctg cccctccctc cagcccagct ccattctttg gcataacagg caactctaac 2160
ctagcccaga agaggatggc tcatcctggg tgggacgatg gctcctgcct tgaaagacaa 2220
aaatcccacc ttgggcagag ctgagtgtga gaagatggaa aaccagtatc tgccaggtgc 2280
tcagtgactg gccatcacat taatgaatga cgagattgga gtacactgtc accaagggca 2340
ggcaaagatg ccctctgggg ttgtctggtt cccagtgaga ggctcctgag aaaaataaag 2400
ctggttccca gagctgctgt ccatccctca aaaaaaaaa 2439
<210> 49
<211> 2762
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Tncyte ID No: 7476280CB1
<400> 49
atggacccca tcacgcctaa ctggactgag atcgtgaaca ggaagctcag cttcccacct 60
ccactcctgg atgccatcca ggagggccga ctgggctttg tgcagcagct gctggagtca 220
gaggttgagg ccgcgagcag tgggccaggc tggcccctgt ggaatgtgga agaggctgag 180
gaccgctgct ggagggaggc actcaacctg gccatccgcc tgggccatga ggccctcacc 240
gatgtgctgt tggccagtgt caagtttgac ttccgccaga tccatgaggc cctgctagtg 300
gcagtggaca caaaccaggc agtggtgcgt cgcctgccgg cccggctgga acgggagaag 360
ggtcgcaaag tagacaccag gtctttctca ctggctttct ttgactcatc aattgatggc 420
tcccgctttg cacctggtgt gactcccctc ccccaggcct gccagaagga cctgtatgag 480
atagcacagc tgctcatgga acagggccac accattgccc ggccccaccc ggtctcctgt 540
gcctgcctcg agtgcagcaa cgcccgccgc tatgacctgc tgaaactctc tctgtcccgc 600
atcaacacct accttggcat cgccagcagg gcccacctct cactggccag tgaggatgcc 660
atgctggctg ccttccagct tagccgtgag ctcaggcgcc ttgcacgcaa ggagcctgaa 720
tttaagcctg agtacattgc tctggagtca ctgagccagg actatggctt tcagctgctg 780
ggcatgtget ggaaccagag tgaggtcact gcagtgctca acgacctggc cgaggacagc 840
gagactgagc ccgaggctga aggcctgggc ctggcctttg aggaaggcat ccccaacctg 900
gtgaggctgc gactggctgt caactacaac cagaagcggt tcgtagcaca cctcatctgc 960
cagcaagtcc tgtcctccat ctggtgtggg aacctggctg gttggcgggg aagcaccacc 1020
agctggaagc tCtttgCtaC CttCCtCatC ttCCtcaCCa tgCCCttCCt CtgCCttggC 1080
tactggctga caccaaagtc ccagctgggc cacctgctaa agatcccagt actgaagttc 1140
ctgctgcact ctgcctccta tctgtggttc ctcatcttcc tgctgggaga gtccctggtc 1200
atggagacac agctgagcac cttccgtggc cgcagccaga gtgtctggga gacttcacta 1260
cacatgattt gtgtcacagg cttcctgtgg tttgagtgca aggaagtgtg gattgagggc 1320
ctgcgcagtt acctcctgga ctggtggaac ttcctggata tggtcgtcct gtccctgtac 1380
ctggcagcct tcgcactgcg cctcctcctg gctgggcttg cccccatgca ctgccgggac 1440
gcctcccaag cggctgcctg ccactatttc accatggctg aaagaagcga gtggcacacc 1500
gaggatcccc agttcttggc tgaggtgctc ttcactgcca ccagcatgct cagcttcacc 1560
cgcctggcct acattctgcc ggcccacgag tcgctgggca ctctgcagat ttccattggc 1620
aagatgattg aagacatgat ccggtttatg ttcatcctca tgatcatcct gaccgccttc 1680
ctctgtggcc tcaacaacat ctatgtgccc taccagaaga cagagtggct gggcaagagt 1740
62/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
ttcaatgaga cgtttcagtt tctgttctgg accatgttcg gtatggaaga gcacagcgtg 1800
gtggacgtgc ctcagtttct ggtgcccgag tttgcaggcc gggccctcta tggcatcttt 1860
accatcatca tggtcattgt gctgctcaac atgctcattg ctatgatcac caactccttc 1920
cagaagattg aggatgatgc tgacgtggag tggacgtttg ctcgctccaa gctgtatctg 1980
ttctacttcc gagagggcct gacactgcct gtgcccttca acatcctgcc ctcctcgaag 2040
gctgtcttct accttctcag gagaatttgc cagttcattt gctgttgctg ttcctgctgc 2100
aaaaccaaga agccagacta tcccccgatc cctacttttg tgaatcccag ggcaggggct 2160
gtgcctgggg agggagagcg tggatcctac cgccttcacg tcatcaaggc cctggtacag 2220
cgctacacag agactgcccg gcgagaattc gaggagaccc ggcggaaaga tctgggcaac 2280
agactcacag agctgaccaa gaccatatct cgactgcaaa gcgaggtagc cggtgtgcgg 2340
agaactctgg cagagggagg gacgccccgg cctcccgacg gtgccagcgt cctcagtcac 2400
tacatcactc aagtgcacaa cagcttccag aacctggggc ctcccatccc tgagacccca 2460
gagctgacag ggcctgggat tgtgaggacc caggaatcat caggaaccgg gcttcaggac 2520
actggagggg tgaggactct ggcttccgga gagtctggcc cctgctcccc agctcatgtg 2580
ctagttcata gggagcagga agcagagggg gctggggacc tgccccaggg ggaggattcg 2640
gggactgaga ggaggtcctg atacagtgga agagtccctt cttctgttgc tgagcgtggt 2700
agcctaggag ggtgagggtg gggggcccct tgggaggagc ctgtgctgct tttcttgctt 2760
ca 2762
<210> 50
<211> 1897
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1713377CB1
<400> 50
gcgatctaga actagtgagc tgcaggctgg catggctggg gggatgtcag cggagtgccc 60
tgagcctggg ccaggaggtc tgcagggcca gtccccaggg ccaggcaggc agtgtccccc 120
tcccatcacg cccacctcct ggagcctgcc cccgtggagg gcctacgtgg ctgccgccgt 180
cctctgctac atcaacctcc tgaattacat gaactggttc atcattgcag gagtgctgct 240
ggatatacag gaggttttcc agatcagtga caaccatgct ggtttgcttc agactgtctt 300
cgttagctgc ctgctgctgt ctgcacctgt gtttggctac ctgggcgacc gacatagccg 360
caaggctacc atgagcttcg gtatcttgct gtggtcagga gctggcctct ctagctcctt 420
catctccccc cggtattctt ggctcttctt cctgtcccgg ggcatcgtgg gcactggctc 480
ggccagctac tccaccatcg cgcccaccgt cctgggcgac ctcttcgtga gggaccagcg 540
cacccgcgtg ctggctgtct tctacatctt tatccccgtt ggaagtggtc tgggctacgt 600
gctggggtcg gctgtgacga tgctgactgg gaactggcgc tgggccctcc gagtcatgcc 660
ctgcctggag gccgtggcct tgatcctgct tatcctgctg gttccagacc caccccgggg 720
agctgccgag acacaggggg agggggccgt gggaggcttc agaagcagct ggtgtgagga 780
cgtcagatac ctggggaaaa actggagttt tgtgtggtcg accctcggag tgaccgccat 840
ggcctttgtg actggagccc tggggttctg ggcccccaag tttctgctcg aggcacgcgt 900
ggttcacggg ctgcagcctc cctgcttcca ggagccgtgc agcaaccccg acagcctgat 960
ttttggggca ctgaccatca tgaccggcgt cattggggtc atcttggggg cagaagcttc 1020
gaggaggtac aagaaagtca ttccaggagc tgagcccctc atctgcgcct ccagcctgct 1080
tgccacagcc ccctgcctct acctggctct cgtcctggcc ccgaccaccc tgctggcctc 1140
ctatgtgttc ctgggccttg gggagctgct tctgtcctgc aactgggcag tggttgccga 1200
catcctgctg tctgtggtgg tgcccagatg ccgggggacg gcagaggcac ttcagatcac 1260
ggtgggccac atcctgggag acgetggcag cccctatctc acaggactta tctctagtgt 2320
cctgcgggcc aggcgccctg actcctatct gcagcgcttc cgcagcctgc agcagagctt 1380
cctgtgctgc gcctttgtca tcgccctggg gggcggctgc ttcctgctga ctgcgctgta 1440
cctggagaga gacgagaccc gggcctggca gcctgtcaca gggaccccag acagcaatga 1500
tgtggacagc aacgacctgg agagacaagg cctactttcg ggcgctggcg cctctacaga 1560
ggagccctga ggtccctgcc tacactcgtc ctgcctgcaa gcctcccgtt ggtccccaca 1620
gcagcagtgc ctcggttcct ctttggctgt cctcggggac tccggctgag gcacatctgc 1680
cacttttgaa ttcccggctg gagagctggc aggaccctgt ggctgggctg ggaatggagc 1740
tgtcagcact ctgcgtggga ggcctgggcc tgtgcctgca tcccgctcaa ggctgcccca 1800
gcctggggtc tccagcctgg ctgctgctgg gccctgaata aagagaggcc agtacaaagc 1860
ccatggattt tgggcctgta aaaaaaaaaa aaaaaaa 1897
<210> 51
63/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
<211> 2361
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte TD No: 5842557CB1
<400> 51
gatgatggca gacaggagag ctgactactt tcagaacctg cctgagtctc tgacttccct 60
tcctggtgct tctgaccacg tccaacaacc ccgatgtgat gattcctgcg tattccaaga 120
accgggccta tgccatcttc ttcatagtct tcactgtgat aggaagcctg tttctgatga 180
acctgctgac agccatcatc tacagtcagt tccggggcta cctgatgaaa tctctccaga 240
cctcgctgtt tcggaggcgg ctgggaaccc gggctgcctt tgaagtccta tcctccatgg 300
tgggggaggg aggagccttc cctcaggcag ttggggtgaa gccccagaac ttgctgcagg 360
tgcttcagaa ggtccagctg gacagctccc acaaacaggc catgatggag aaggtgcgtt 420
cctacgacag tgttctgctg tcagctgagg agtttcagaa gctcttcaac gagcttgaca 480
gaagtgtggt taaagagcac ccgccgaggc ccgagtacca gtctccgttt ctgcagagcg 540
cccagttcct cttcggccac tactactttg actacctggg gaacctcatc gccctggcaa 600
acctggtgtc catttgcgtg ttcctggtgc tggatgcaga tgtgctgcct gctgagcgtg 660
atgacttcat cctggggatt ctcaactgcg tcttcattgt gtactacctg ttggagatgc 720
tgctcaaggt ctttgccctg ggcctgcgag ggtacctgtc ctaccccagc aacgtgtttg 780
acgggctcct caccgttgtc ctgctggttt tggagatctc aactctggct gtgtaccgat 840
tgecacaccc aggctggagg ccggagatgg tgggcctgct gtcgctgtgg gacatgaccc 900
gcatgctgaa catgctcatc gtgttccgct tcctgcgtat catccccagc atgaagccga 960
tggccgtggt ggccagtacc gtcctgggcc tggtgcagaa catgcgtgct tttggcggga 1020
tcctggtggt ggtctactac gtatttgcca tcattgggat caacttgttt agaggcgtca 1080
ttgtggctct tcctggaaac agcagcctgg cccctgccaa tggctcggcg ccctgtggga 1140
gcttcgagca gctggagtac tgggccaaca acttcgatga ctttgcggct gccctggtca 1200
ctctgtggaa cttgatggtg gtgaacaact ggcaggtgtt tctggatgca tatcggcgct 1260
actcaggccc gtggtccaag atctattttg tattgtggtg gctggtgtcg tctgtcatct 1320
gggtcaacct gtttctggcc ctgattctgg agaacttcct tcacaagtgg gacccccgca 1380
gccacctgca gccccttgct gggaccccag aggccaccta ccagatgact gtggagctcc 1440
tgttcaggga tattctggag gagcccgagg aggatgagct cacagagagg ctgagccagc 1500
acccgcacct gtggctgtgc aggtgacgtc cgggctgccg tcccagcagg ggcggcagga 1560
gagagaggct ggcctacaca ggtgcccgtc atggaagagg cggccatgct gtggccagcc 1620
aggcaggaag agacctttec tctgacggac cactaagctg gggacaggaa ccaagtcctt 1680
tgcgtgtggc ccaacaaccg tctacagaac agctgctggt gcttcaggga ggcgccgtgc 1740
cctccgcttt cttttatagc tgcttcagtg agaattccct cgtcgactcc acagggacct 1800
ttcagacaaa aatgcaagaa gcagcggcct cccctgtccc ctgcagcttc cgtggtgcct 1860
ttgctgccgg cagcccttgg ggaccacagg cctgaccagg gcctgcacag gttaaccgtc 1920
agacttccgg ggcattcagg tggggatgct ggtggtttga catggagaga accttgactg 1980
tgttttatta tttcatggct tgtatgagtg tgactgggtg tgtttcttta gggttctgat 2040
tgccagttat tttcatcaat aagtcttgca aagaatggga ttgtcattct tcacttcagc 2100
acagttctag tcctgcttct ctggagtagg gttgttgagt aaggttgctt gggttgtgca 2160
tttgcacaag ggcacatggc tgtgaggtgt atcctggcgg ggggctgtct acctgcagtg 2220
aggggcacct tttctgtttt gctcaaaggc atgtataagc caatgggtga ccttatttcc 2280
tgtgtcttca ggtgtgtgca ggggcctggg gtggggagtt gggggagcga gcagtgtgtg 2340
gaaggggatc cactagttct a 2361
<210> 52
<211> 2032
<212> DNA
<223> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7476643CB1
<400> 52
gccttggcag agtctggggt ccctggactg agccatcagc tgggtcactg agacccatgg 60
caaggaaaca aaataggaat tccaaggaac tgggcctagt tcccctcaca gatgacacca 120
gccacgccag gcctccaggg ccagggaggg cactgctgga gtgtgaccac ctgaggagtg 180
64/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
gggtgccagg tggaaggaga agaaaggact ggtcctgctc gctcctcgtg gcctccctcg 240
cgggcgcctt cggctcctcc ttcctctacg gctacaacct gtcggtggtg aatgccccca 300
ccccgtacat caaggccttt tacaatgagt catgggaaag aaggcatgga cgtccaatag 360
acccagacac tctgactttg ctctggtctg tgactgtgtc catattcgcc atcggtggac 420
ttgtggggac gttaattgtg aagatgattg gaaaggttct tgggaggaag cacactttgc 480
tggccaataa tgggtttgca atttctgctg cattgctgat ggcctgctcg ctccaggcag 540
gagcctttga aatgctcatc gtgggacgct tcatcatggg catagatgga ggcgtcgccc 600
tcagtgtgct ccccatgtac ctcagtgaga tctcacccaa ggagatccgt ggctctctgg 660
ggcaggtgac tgccatcttt atctgcattg gcgtgttcac tgggcagctt ctgggcctgc 720
ccgagctgct gggaaaggag agtacctggc catacctgtt tggagtgatt gtggtccctg 780
ccgttgtcca gctgctgagc cttccctttc tcccggacag cccacgctac ctgctcttgg 840
agaagcacaa cgaggcaaga gctgtgaaag ccttccaaac gttcttgggt aaagcagacg 900
tttcccaaga ggtagaggag gtcctggctg agagccgcgt gcagaggagc atccgcctgg 960
tgtccgtgct ggagctgctg agagctccct acgtccgctg gcaggtggtc accgtgattg 1020
tcaccatggc ctgctaccag ctctgtggcc tcaatgcaat ttggttctat accaacagca 1080
tctttggaaa agctgggatc cctctggcaa agatcccata cgtcaccttg agtacagggg 1140
gcatcgagac tttggctgcc gtcttctctg gtttggtcat tgagcacctg ggacggagac 1200
CCCtCCtCat tggtggCttt gggCtCatgg gCCtCttCtt tgggaCCCtC aCCatCaCgC 1260
tgaccctgca ggaccacgcc ccctgggtcc cctacctgag tatcgtgggc attctggcca 1320
tcatcgcctc tttctgcagt gggccaggtg gcatcccgtt catcttgact ggtgagttct 1380
tccagcaatc tcagcggccg gctgccttca tcattgcagg caccgtcaac tggctctcca 1440
actttgctgt tgggctcctc ttcccattca ttcagaaaag tctggacacc tactgtttcc 1500
tagtctttgc tacaatttgt atcacaggtg ctatctacct gtattttgtg ctgcctgaga 1560
ccaaaaacag aacctatgca gaaatcagcc aggcattttc caaaaggaac aaagcatacc 1620
caccagaaga gaaaatcgac tcagctgtca ctgatgctca aaggaactaa gacaaagatc 1680
atggagacca tcgggtgagt ctcaagactt cccccagctc tgcttggctg gtctcctgct 1740
ggtattttct gtctgtagag aggaacaaga acttccattt tatcttgctt acctgcactt 1800
atgaaaagtc aaactgagtc atgctgagag Ccagggaaca taggagtcag ttcttctgca 1860
gcagcactca gccagttgaa ggcaatgtgg agtgatggaa ggagagcagt gatgcagtga 1920
tgctggcacc aactccttta ctatggcatc cattgtacca gctgccatac accaggcaac 1980
ttctacactt tatctctaat catcctagaa taagtattag tttccccatc tt 2032
<210> 53
<212> 2779
<222> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7621651CB2
<400> 53
cgcctgtggc tccgggcagg ggccgcggcc gaaagatgcc ggtccgcagg ggccacgtcg 60
ctccccaaaa cacttacctg gacaccatca tccgcaagtt cgagggccaa agtcggaagt 120
tcctgattgc caatgctcag atggagaact gcgccatcat ttactgcaac gacggettct 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 catcatgttc attctcaact 420
tcgaggacct ggcccagctc ctggccaagt gcagcagccg cagcttgtcc cagcgcctgt 480
tgtcccagag cttcctgggc tccgagggct ctcatggcag gccaggcgga ccagggccag 540
gcacaggcag gggcaagtac aggaccatca gccagatccc acagttcacg ctcaacttcg 600
tggagttcaa cttggagaag caccgctcca gctccaccac ggagattgag atcatcgcgc 660
cccataaggt ggtggagcgg acacagaacg tcactgagaa ggtcacccag gtcctgtccc 720
tgggcgcgga tgtgctgccg gagtacaagc tgcaggcgcc gcgcatccac cgctggacca 780
tcctgcacta cagccccttc aaggccgtgt gggactggct catcctgctg 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
acatggtggc cgccatccct ttcgacctcc tgatcttccg cactggctcc gatgagacca 1140
caaccctgat tgggctattg aagacagcgc ggctgctgcg gctggtgcgc gtagcacgga 2200
agctggaccg ctactctgag tatggggcgg ctgtgctctt cttgctcatg tgcaccttcg 1260
65/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
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 ggacgcagcc gggggtctcc 2040
actcatcccc ccgacaggct cctggcagcc aagaccacca aggtttcttt ctcagtgaca 2100
accagtcaga tgcagcccct cccctgagca tctcagatgc atctggcctc tggcctgagc 2160
tactgcagga aatgccccca aggcacagcc cccaaagccc tcaggaagac ccagattgct 2220
ggcctctgaa gctgggctcc aggctagagc agctccaggc ccagatgaac aggctggagt 2280
cccgcgtgtc ctcagacctc agccgcatct tgcagctcct ccagaagccc atgccccagg 2340
gccacgccag ctacattctg gaagcccctg cctccaatga cctggccttg gttcctatag 2400
cctcggagac gacgagtcca gggcccaggc tgccccaggg ctttctgcct cctgcacaga 2460
ccccaagcta tggagacttg gatgactgta gtccaaagca caggaactcc tcccccagga 2520
tgcctcacct ggctgtggca atggacaaaa ctctggcacc atcctcagaa caggaacagc 2580
ctgaggggct ctggccaccc ctagcctcac ctctacatcc cctggaagta caaggactca 2640
tCtgtggtCC CtgCttCtCC tCCCtCCCtg aacaCCttgg ctctgttccc aagcagctgg 2700
acttccagag acatggctca gatcctggat ttgcagggag ttggggccac tgaactccaa 2760
gataaagaca ccatgaggg 2779
<210> 54
<211> 2430
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2522075CB1
<400> 54
atggccgagg ccgcggagcc ggagggggtt gccccgggtc cccaggggcc gccggaggtc 60
cccgcgcctc tggctgagag acccggagag ccaggagccg cgggcgggga ggcagaaggg 120
ccggagggga gcgagggcgc agaggaggcg ccgaggggcg ccgccgctgt gaaggaggca 180
ggaggcggcg ggccagacag gggcccggag gccgaggcgc ggggcacgag gggggcgcac 240
ggcgagactg aggccgagga gggagccccg gagggtgccg aggtgcccca aggaggggag 300
gagacaagcg gcgcgcagca ggtggagggg gcgagcccgg gacgcggcgc gcagggcgag 360
ccccgcgggg aggctcagag ggagcccgag gactctgcgg cccccgagag gcaggaggag 420
gcggagcaga ggcctgaggt cccggaaggt agcgcgtccg gggaggcggg ggacagcgta 480
gacgcggagg gcccgctggg ggacaacata gaagcggagg gcccggcggg cgacagcgta 540
gaggcggagg gccgggtggg ggacagcgta gacgcggaag gtccggcggg ggacagcgta 600
gacgcggagg gcccgctggg ggacaacata caagccgagg gcccggcggg ggacagcgta 660
gacgcggagg gccgggtggg ggacagcgta gacgcggaag gtccggcggg ggacagcgta 720
gacgcggagg gccgggtggg ggacagcgta gaggcggggg acccggcggg ggacggcgta 780
gaagcggggg tcccggcggg ggacagcgta gaagccgaag gcccggcggg ggacagcatg 840
gacgccgagg gtccggcagg aagggcgcgc cgggtctcgg gtgagccgca gcaatcgggg 900
gacggcagcc tctcgcccca ggccgaggca attgaggtcg cagccgggga gagtgcgggg 960
cgcagccccg gtgagctcgc ctgggacgca gcggaggagg cggaggtccc gggggtaaag 2020
gggtccgaag aagcggcccc cggggacgca agggcagacg ctggcgagga cagggtaggg 1080
gatgggccac agcaggagcc gggggaggac gaagagagac gagagcggag cccggagggg 2140
ccaagggagg aggaagcagc ggggggcgaa gaggaatccc ccgacagcag cccacatggg 1200
gaggcctcca ggggcgccgc ggagcctgag gcccagctca gcaaccacct ggccgaggag 1260
ggccccgccg agggtagcgg cgaggccgcg cgcgtgaacg gccgccggga ggacggagag 1320
gcgtccgagc cccgggccct ggggcaggag cacgacatca ccctcttcgt caaggctggt 1380
tatgatggtg agagtatcgg aaattgcccg ttttctcagc gtctctttat gattctctgg 1440
ctgaaaggcg ttatatttaa tgtgaccaca gtggacctga aaaggaaacc cgcagacctg 1500
cagaacctgg ctcccggaac aaaccctcct ttcatgactt ttgatggtga agtcaagacg 1560
66/67
CA 02410084 2002-11-20
WO 01/92304 PCT/USO1/17065
gatgtgaata agatcgagga gttcttagag gagaaattag ctcccccgag gtatcccaag 1620
ctggggaccc aacatcccga atctaattcc gcaggaaatg acgtgtttgc caaattctca 1680
gcgtttataa aaaacacgaa gaaggatgca aatgagattc atgaaaagaa cctgctgaag 1740
gccctgagga agctggataa ttacttaaat agccctctgc ctgatgaaat agatgcctac 1800
agcaccgagg atgtcactgt ttctggaagg aagtttctgg gtggggacga gctgacgctg 1860
gctgactgca acctcttacc caagctccat attattaaga ttgtggccaa gaagtacaga 1920
gattttgaat ttccttctga aatgactggc atctggagat acttgaataa tgcttatgct 1980
agagatgagt tcacaaatac gtgtccagct gatcaagaga ttgaacacgc atattcagat 2040
gttgcaaaaa gaatgaaatg aagctgggct gttttctgtc ttatttctca gttgagtgag 2100
caaggatacg aaaacagtgt gtttgaaaac aaattaggtt tgggttcaat tccttcaatt 2160
tttaaaaaac tggtctctga gagtttttta aatcattgag agcctgtttt tcttctctaa 2220
aacattagtt taattttctt caaaatgaaa atactgcttt gtaattacaa aatgagacac 2280
acctatcttg atattttaaa gcaatatcag agggtgtaaa gaaggacatt ttaacaatcg 2340
ccttcaattt tactccactt aattaccgaa aacttactgg agaacatgtt ccaaatcttc 2400
agtatcttgt tctctctctc tctctctctc 2430
67/67
