Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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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 transpoiters 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, 5042-, 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 Barrier 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
(SGLTl), 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
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techniques and for specific targeting of radioisotopes to the thyroid gland
(Levy, O. et al. (1997) Proc.
Natl. Acad. Sci. USA 94:5568-5573). SMVT is expressed in the intestinal
mucosa, kidney, and
placenta, and is implicated in the transport of the water-soluble vitamins,
e.g., biotin and pantothenate
(Prasad, P.D. et al. (1998) J. Biol. Chem. 273:7501-7506).
One of the largest families of transporters is the major facilitator
superfamily (MFS), also
called the uniporter-symporter-annporter family. MFS transporters are single
polypepnde 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 selecnvines. In particular, cardiac muscle and tumor
cells have transporters that
differ in their Km values for certain substrates, including stereoselecnvity
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 axe specific and selective transporters for
organic canons and organic anions
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in organs including the kidney, intestine and liver. Organic anion
transporters are selective for
hydrophobic, charged molecules with electron-attracting side groups. Organic
canon transporters, such
as the ammonium transporter, mediate the secretion of a variety of drugs and
endogenous metabolites,
and contribute to the maintenance of intercellular pH (Poole, R.C. and A.P.
Halestrap (1993) Am. J.
Physiol. 264:C761-C782; Price, N.T. et al. (1998) Biochem. J. 329:321-328; and
Martinelle, K. and I.
Haggstrom (1993) J. Biotechnol. 30:339-350).
ATP-binding cassette (ABC) transporters are members of a superfamily of
membrane proteins
that transport substances ranging from small molecules such as ions, sugars,
amino acids, peptides, and
phospholipids, to lipopeptides, large proteins, and complex hydrophobic drugs.
ABC transporters
consist of four modules: two nucleotide-binding domains (NBD), which hydrolyze
ATP to supply the
energy required for transport, and two membrane-spanning domains (MSD), each
containing six
putative transmembrane segments. These four modules may be encoded by a single
gene, as is the case
for the cystic fibrosis transmembrane regulator (CFTR), or by separate genes.
When encoded by
separate genes, each gene product contains a single NBD and MSD. These "half
molecules" form
homo- and heterodimers, such as Tap1 and Tap2, the endoplasmic reticulum-based
major
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 fibxosis
(CFTR, an ion channel), adrenoleukodystrophy (adrenoleukodystrophy protein,
ALDP), Zellweger
syndrome (peroxisomal membrane protein-70, PMP70), and hyperinsulinemic
hypoglycemia
(sulfonylurea receptor, SUR). Overexpression of the multidrug resistance (MDR)
protein, another
ABC transporter, in human cancer cells makes the cells resistant to a variety
of cytotoxic drugs used in
chemotherapy (Taglicht, D. and S. Michaelis (1998) Meth. Enzymol. 292:130-
162).
A number of metal ions such as iron, zinc, copper, cobalt, manganese,
molybdenum, selenium,
nickel, and chromium are important as cofactors for a number of enzymes. For
example, copper is
involved in hemoglobin synthesis, connective tissue metabolism, and bone
development, by acting as a
cofactor in oxidoreductases such as superoxide dismutase, ferroxidase
(ceruloplasmin), and lysyl
oxidase. Copper and other metal ions must be provided in the diet, and are
absorbed by transporters in
the gastrointestinal tract. Plasma proteins transport the metal ions to the
liver and other target organs,
where specific transporters move the ions into cells and cellular organelles
as needed. Imbalances in
metal ion metabolism have been associated with a number of disease states
(banks, D.M. (1986) J.
Med. Genet. 23:99-106).
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.
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Fatty acid transport protein (FATP), an integral membrane protein with four
transmembrane segments,
is expressed in tissues exhibiting high levels of plasma membrane fatty acid
flux, such as muscle, heart,
and adipose. Expression of FATP is upregulated in 3T3-L1 cells during adipose
conversion, and
expression in COS7 fibroblasts elevates uptake of long-chain fatty acids (Hui,
T.Y. et al. (1998) J.
Biol. Chem. 273:27420-27429).
Mitochondria) carrier proteins are transmembrane-spanning proteins which
transport ions and
charged metabolites between the cytosol and the mitochondria) matrix. Examples
include the ADP,
ATP carrier protein; the 2-oxoglutarate/malate carrier; the phosphate carriex
protein; the pyruvate
carrier; the dicarboxylate carrier which transports malate, succinate,
fumarate, and phosphate; the
tricarboxylate carrier wluch transports citrate and malate; and the Grave's
disease carrier protein, a
pxotein recognized by IgG in patients with active Grave's disease, an
autoimmune disorder resulting in
hyperthyroidism. Pxoteins 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 Mitochondria)
energy transfer
proteins signature; Online Mendelian Inheritance in Man (OMIM) *275000 Graves
Disease).
This class of transporters also includes the mitochondria) uncoupling
proteins, which create
proton leaks across the inner mitochondria) membrane, thus uncoupling
oxidative phosphorylation from
ATP synthesis. The result is energy dissipation in the form of heat.
Mitochondria) uncoupling proteins
have been implicated as modulators of thermoregulation and metabolic rate, and
have been proposed as
potential targets for drugs against metabolic diseases such as obesity
(Ricquier, D. et al. (1999) J. Int.
Med. 245:637-642).
Ion Channels
The electrical potential of a cell is generated and maintained by controlling
the movement of
ions across the plasma membrane. The movement of ions requires ion channels,
which form ion-
selective pores within the membrane. There are two basic types of ion
channels, ion transporters and
gated ion channels. Ion transporters utilize the energy obtained from ATP
hydrolysis to actively
transport an ion against the ion's concentration gradient. Gated ion channels
allow passive flow of an
ion down the ion's electrochemical gradient under restricted conditions.
Together, these types of ion
channels generate, maintain, and utilize an electrochemical gradient that is
used in 1) electrical impulse
conduction down the axon of a nerve cell, 2) transport of molecules into cells
against concentration
gradients, 3) initiation of muscle contraction, and 4) endocrine cell
secretion.
Ion Trans op rters
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.
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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 Nay 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 are responsible for
generating the low pH within
the lumen of these organelles that is required for function. The coupling
factor (F) class consists of H+
pumps in the mitochondria. F-class ion transporters utilize a proton gradient
to generate ATP from
ADP and inorganic phosphate (P;).
The P-ATPases are hexamers of a 100 kD subunit with ten transmembrane domains
and
several large cytoplasmic regions that may play a role in ion binding
(Scarborough, G.A. (1999) Curr.
Opin. Cell Biol. 11:517-522). The V-ATPases are composed of two functional
domains: the V 1
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 V o domain
contains three types of homologous c subunits with four or five transmembrane
domains and the
essential carboxyl group in TM4 or TM3. Both types of complex also contain a
single a subunit that
may be involved in regulating the pH dependence of activity (Forgac, M. (1999)
J. Biol. Chem.
274:12951-12954).
The resting potential of the cell is utilized in many processes involving
carrier proteins and
gated ion channels. Carrier proteins utilize the resting potential to
transport molecules into and out of
the cell. Amino acid and glucose transport into many cells is linked to sodium
ion co-transport
(symport) so that the movement of Na+ down an electrochemical gradient drives
transport of the other
molecule up a concentration gradient. Similarly, caxdiac muscle links transfer
of Ca2+ out of the cell
with transport of Na+ into the cell (antiport).
Gated Ion Channels
Gated ion channels control ion flow by regulating the opening and closing of
pores. The
ability to control ion flux through various gating mechanisms allows ion
channels to mediate such
diverse signaling and homeostatic functions as neuronal and endocrine
signaling, muscle contraction,
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.,
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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 canon channels form a
superfamily of ion
channel proteins. The characteristic domain of these channel proteins
comprises six transmembrane
domains (S 1-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 Nay and K+ channels are necessary for the function of
electrically excitable cells,
such as nerve and muscle cells. Action potentials, which lead to
neurotransmitter release and muscle
contraction, arise from large, transient changes in the permeability of the
membrane to Na ~ and K+
ions. Depolarization of the membrane beyond the threshold level opens voltage-
gated Na+ channels.
Sodium ions flow into the cell, further depolarizing the membrane and opening
more voltage-gated Na+
channels, which propagates the depolarization down the length of the cell.
Depolarization also opens
voltage-gated potassium channels. Consequently, potassium ions flow outward,
which leads to
repolarization of the membrane. Voltage-gated channels utilize charged
residues in the fourth
transmembrane segment (S4) to sense voltage change. The open state lasts only
about 1 millisecond, at
which time the channel spontaneously converts into an inactive state that
cannot be opened irrespective
of the membrane potential. Inactivation is mediated by the channel's 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, j31 and ~i2.
The J32 subunit is a integral
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membrane glycoprotein that contains an extracellular Ig domain, and its
association with a and (31
subunits correlates with increased functional expression of the channel, a
change in its gating
properties, as well as an increase in whole cell capacitance due to an
increase in membrane surface area
(Isom, L.L. et al. (1995) Cell 83:433-442).
Non voltage-gated Na+ channels include the members of the amiloride-sensitive
Nay
channel/degenerin (NaC/DEG) family. Channel subunits of this family axe
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 NaCIDEG 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 Nay-permeable channels. These channels require
acid pH fluctuations
for activation. ASIC subunits show homology to the degenerins, a family of
mechanically-gated
channels originally isolated from C. elegans. Mutations in the degenerins
cause neurodegeneration.
ASIC subunits may also have a role in neuronal function, or in pain
perception, since tissue acidosis
causes pain (Waldmann, R. and M. Lazdunski (1998) Curr. Opin. Neurobiol. 8:418-
424; Eglen, R.M.
et al. (1999) Trends Pharmacol. Sci. 20:337-342).
K+ channels are located in all cell types, and may be regulated by voltage,
ATP concentration,
or second messengers such as Ca2+ and cAMP. In non-excitable tissue, K+
channels are involved in
protein synthesis, control of endocrine secretions, and the maintenance of
osmotic equilibrium across
membranes. In neurons and other excitable cells, in addition to regulating
action potentials and
repolarizing membranes, K+ channels are responsible for setting resting
membrane potential. The
cytosol contains non-diffusible anions and, to balance this net negative
charge, the cell contains a Na+-
K+ pump and ion channels that provide the redistribution of Na+, K+, and Cl-.
The pump actively
transports Na+ out of the cell and K+ into the cell in a 3:2 ratio. Ion
channels in the plasma membrane
allow K+ and Cl - to flow by passive diffusion. Because of the high negative
charge within the cytosol,
Cl- flows out of the cell. The flow of K+ is balanced by an electromotive
force pulling K+ into the cell,
and a K+ concentration gradient pushing K+ out of the cell. Thus, the resting
membrane potential is
primarily regulated by K+flow (Salkoff, L. and T. Jegla (1995) Neuron 15:489-
492).
Potassium channel subunits of the Shaker-like superfamily all have the
characteristic six
transmembranell 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-
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gated K+ channels as well as the delayed rectifier type channels such as the
human ether-a-go-go
related gene (HERG) associated with long QT, a cardiac dysrythmia syndrome
(Curran, M.E. (1998)
Curr. Opin. Biotechnol. 9:565-572; Kaczorowski, G.J. and M.L. Garcia (1999)
Curr. Opin. Chem.
Biol. 3:448-458).
A second superfamily of K+ channels is composed of the inward rectifying
channels (Kir).
Kir channels have the property of preferentially conducting K'~ currents in
the inward direction. These
proteins consist of a single potassium selective pore domain and two
transmembrane domains, which
correspond to the fifth and sixth transmembrane domains of voltage-gated K+
channels. Kir subunits
also assbciate as tetramers. The Kir family includes ROMK1, 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) Cmr. Opin.
Neurobiol. 5:268-277; Curran, supra).
The recently recognized TWIK K+ channel family includes the mammalian TWIK-1,
TREK-1
and TASK proteins. Members of this family possess an overall structure with
four transmembrane
domains and two P domains. These proteins are probably involved in controlling
the resting potential
in a large set of cell types (Duprat, F. et al. (2997) 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 aZb, and four (3 genes, A
fourth subunit, 'y, has been
identified in skeletal muscle (Walker, D. et al. (1998) J. Biol. Chem.
273:2361-2367; McCleskey, E.W.
(1994) Curr. Opin. Neurobiol. 4:304-312).
The 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 and/or related proteins may
form mammalian CCC
entry channels (Zhu, X. et al. (1996) Cel185:661-671; Boulay, G. et al. (2997)
J. Biol. Chem.
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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 for developing
metastatic disease (Duncan, L.M. et al (2001) J. Clin. Oncol. 19:568-576). '
Chloride channels are necessary in endocrine secretion and in regulation of
cytosolic and
organelle pH. In secretory epithelial cells, Cl- enters the cell across a
basolateral membrane through an
Na ~, K+/Cl' cotransporter, accumulating in the cell above its electrochemical
equilibrium concentration.
Secretion of C1- 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
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
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transmembrane domains and probably function as pentamers (Jentsch, suura).
Amino acids in the
second transmembrane domain appear to be important in determining channel
permeation and
selectivity (Sather, W.A. et al. (1994) Curr. Opin. Neurobiol. 4:313-323).
Ligand-gated channels can be regulated by intracellular second messengers. For
example,
calcium-activated K+ channels are gated by internal calcium ions. In nerve
cells, an influx of calcium
during depolarization opens K+ channels to modulate the magnitude of the
action potential (Ishi et al.,
supra). The large conductance (BK) channel has been purified from brain and
its subunit composition
determined. The a, subunit of the BK channel has seven rather than six
transmembrane domains in
contrast to voltage-gated K+ channels. The extra transmembrane domain is
located at the subunit N-
terminus. A 28-amino-acid stretch in the C-terminal region of the subunit (the
"calcium bowl" region)
contains many negatively charged residues and is thought to be the region
responsible for calcium
binding. The ~i subunit consists of two transmembrane domains connected by a
glycosylated
extracellular loop, with intracellular N- and C-termini (Kaczorowski, 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 are tetraxners containing at least two types of subunits, an a
subunit which can form
functional homomeric channels, and a (3 subunit, which modulates the channel
properties. All CNG
subunits have six transmembrane domains and a pore forming region between the
fifth and sixth
transmembrane domains, similar to voltage-gated K+ channels. A large C-
terminal domain contains a
cyclic nucleotide binding domain, while the N-terminal domain confers
variation among channel
subtypes (Zufall, F. et al. (1997) Curr. Opin. Neurobiol. 7:404-412).
The activity of other types of ion channel proteins may also be modulated by a
variety of
intracellular signalling proteins. Many channels have sites for
phosphorylation by one or more protein
kinases including protein kinase A, protein kinase C, tyrosine kinase, and
casein kinase II, all of
which regulate ion channel activity in cells. Kir channels are activated by
the binding of the G~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 MAGUKs
(membrane-associated
guanylate kinases) which regulate the clustering of ion channels at neuronal
synapses (Craven, S.E. and
D.S. Bredt (1998) Cell 93:495-498).
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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 resultaing in an inability to transport
small molecules across
membranes include, e.g., cystinuria, iminoglycinuria, Hartup disease, and
Fanconi disease (van't Hoff,
W.G. (1996) Exp. Nephrol. 4:253-262; Talente, G.M. et al. (1994) Ann. Intern.
Med. 120:218-226;
and Chillon, M. et al. (1995) New Engl. J. Med. 332:1475-1480).
Human diseases caused by mutations in ion channel genes include disorders of
skeletal muscle,
cardiac muscle, and the central nervous system. Mutations in the pore-forming
subunits of sodium and
chloride channels cause myotonia, a muscle disorder in which relaxation after
voluntary contraction is
delayed. Sodium channel myotonias have been treated with channel blockers.
Mutations in muscle
sodium and calcium channels cause forms of periodic paralysis, while mutations
in the sarcoplasmic
calcium release channel, T-tubule calcium channel, and muscle sodium channel
cause malignant
hyperthermia. Cardiac arrythmia disorders such as the long QT syndromes and
idiopathic ventricular
fibrillation are caused by mutations in potassium and sodium channels (Cooper,
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, suura).
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 Nay 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
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can inhibit secretion of Iymphokines, cell proliferation, and killing of
target cells. A peptide antagonist
of the T-cell potassium channel Kvl.3 was found to suppress delayed-type
hypersensitivity and
allogenic responses in pigs, validating the idea of channel blockers as safe
and efficacious
immunosuppressants (Cahalan, M.D. and K.G. Chandy (1997) Curr. Opin.
Biotechnol. 8:749-756).
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,
referxed to
collectively as "TRICH" and individually as "TRICH-1," "TRICH-2," "TRICH-3,"
"TRICH-4,"
"TRICH-5," "TRICH-6," "TRICH-7," "TRICH-8," "TRICH-9," "TRICH-10," "TRICH-11,"
"TRICH-12," "TRTCH-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," "TRICH-27," "TRICH-28," "TRICH-29," "TRICH-30," "TRICH-31," and
"TRICH-
32." 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
N0:1-32, b) a polypeptide comprising a naturally occurring amino acid sequence
at least 90% identical
to an amino acid sequence selected from the group consisting of SEQ ID N0:1-
32, c) a biologically
active fragment of a polypeptide having an amino acid sequence selected from
the group consisting of
SEQ ID NO:1-32, and d) an immunogenic fragment of a polypeptide having an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-32. In one alternative, the
invention provides an
isolated polypeptide comprising the amino acid sequence of SEQ ID N0:1-32.
The invention further provides an isolated polynucleotide encoding a
polypeptide selected from
the group consisting of a) a polypeptide comprising an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-32, b) a polypeptide comprising a naturally
occurring amino acid sequence
at least 90% identical to an amino acid sequence selected from the group
consisting of SEQ ID NO:1-
32, c) a biologically active fragment of a polypeptide having an amino acid
sequence selected from the
group consisting of SEQ ID NO:1-32, and d) an immunogenic fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID N0:1-32. In
one alternative, the
polynucleotide encodes a polypeptide selected from the group consisting of SEQ
ID NO:1-32. In
another alternative, the polynucleotide is selected from the group consisting
of SEQ ID N0:33-64. ,
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Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide selected
from the group consisting
of a) a polypeptide comprising an amino acid sequence selected from the group
consisting of SEQ ID
NO:l-32, b) a polypeptide comprising a naturally occurring amino acid sequence
at least 90% identical
to an amino acid sequence selected from the group consisting of SEQ ID NO:1-
32, c) a biologically
active fragment of a polypeptide having an amino acid sequence selected from
the group consisting of
SEQ ID N0:1-32, and d) an immunogenic fragment of a polypeptide having an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-32. 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 N0:1-32, b) a polypeptide comprising a naturally occurring amino acid
sequence at least 90%
identical to an amino acid sequence selected from the group consisting of SEQ
ID NO:1-32, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-32, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ID NO:1-32. 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 N0:1-32, b) a polypeptide
comprising a naturally
occurring amino acid sequence at least 90% identical to an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-32, c) a biologically active fragment of a
polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID NO:1-32, and d) an
immunogenic fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID N0:1-32.
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
N0:33-64, b) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:33-64, 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
N0:33-64, b) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:33-64, c) a
polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
complementary to the
polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises
a) hybridizing the
sample with a probe comprising at least 20 contiguous nucleotides comprising a
sequence
complementary to said target polynucleotide in the sample, and which probe
specifically hybridizes to
said target polynucleotide, under conditions whereby a hybridization complex
is formed between said
probe and said target polynucleotide or fragments thereof, and b) detecting
the presence or absence of
said hybridization complex, and optionally, if present, the amount thereof. In
one alternative, the probe
comprises at least 60 contiguous nucleotides.
The invention further provides a method for detecting a target polynucleotide
in a sample, said
target polynucleotide having a sequence of a polynucleotide selected from the
group consisting of a) a
polynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:33-64, b) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:33-64, 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 N0:1-32, b) a polypeptide comprising a
naturally occurring amino acid
sequence at least 90% identical to an amino acid sequence selected from the
group consisting of SEQ
ID NO:1-32, c) a biologically active fragment of a polypeptide having an amino
acid sequence selected
from the group consisting of SEQ ID N0:1-32, and d) an immunogenic fragment of
a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-32, and a
pharmaceutically acceptable excipient. In one embodiment, the composition
comprises an amino acid
sequence selected from the group consisting of SEQ ID N0:1-32. 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 N0:1-32, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected from
the group consisting of SEQ ID N0:1-32, c) a biologically active fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID N0:1-32, and
d) an immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ ID
NO:l-32. 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 N0:1-32, b) a
polypeptide
comprising a naturally occurring amino acid sequence at least 90% identical to
an amino acid sequence
selected from the group consisting of SEQ ID NO:1-32, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1-32, and
d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-32. 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 N0:1-32, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected from
the group consisting of SEQ ID NO:1-32, c) a biologically active fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID N0:1-32, and
d) an immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ ID
N0:1-32. The method comprises a) combining the polypeptide with at least one
test compound under
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suitable conditions, and b) detecting binding of the polypeptide to the test
compound, thereby
identifying a compound that specifically binds to the polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1-32, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected from
the group consisting of SEQ ID NO:1-32, c) a biologically active fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID N0:1-32, and
d) an immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ ID
N0:1-32. 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 ID N0:33-64, 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 N0:33-64, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:33-64,
iii) a
polynucleotide having a sequence complementary to i), iv) a polynucleotide
complementary to the
polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization
occurs under conditions
whereby a specific hybridization complex is formed between said probe and a
target polynucleotide in
the biological sample, said target polynucleotide selected from the group
consisting of i) a
polynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:33-64, ii) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:33-64, iii) a
polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide
complementary to the
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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 GenBank homolog 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
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
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reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same meanings
as commonly understood by one of ordinary skill in the art to which this
invention belongs. Although
any machines, materials, and methods similar or equivalent to those described
herein can be used to
practice or test the present invention, the preferred machines, materials and
methods are now described.
All publications mentioned herein are cited for the purpose of describing and
disclosing the cell lines,
protocols, reagents and vectors which are xeported 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, murine, equine, and
human, and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
TRICH. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of TRICH either by
directly interacting with
TRICH or by acting on components of the biological pathway in which TRICH
participates.
An "allelic variant" is an alternative form of the gene encoding TRICH.
Allelic variants may
result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in
polypeptides whose structure or function may or may not be altered. A gene may
have none, one, or
many allelic variants of its naturally occurring form. Common mutational
changes which give rise to
allelic variants are generally ascribed to natural deletions, additions, or
substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the
others, one or more times in
a given sequence.
"Altered" nucleic acid sequences encoding TRICH include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as TRICH or a
polypeptide with at least one functional characteristic of TRICH. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe of
the polynucleotide encoding TRICH, and improper or unexpected hybridization to
allelic variants, with
a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding TRICH.
The encoded protein may also be "altered," and may contain deletions,
insertions, or substitutions of
amino acid residues which produce a silent change and result in a functionally
equivalent TRICH.
Deliberate amino acid substitutions may be made on the basis of similarity in
polarity, charge,
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solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of
the residues, as long as the
biological or immunological activity of TRICH is retained. For example,
negatively charged amino
acids may include aspartic acid and glutamic acid, and positively charged
amino acids may include
lysine and arginine. Amino acids with uncharged polar side chains having
similar hydrophilicity
S 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
1S 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
2S 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 (KhH). The coupled peptide is then used to immunize the
animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies which
bind specifically to antigenic determinants (particular regions or three-
dimensional structures on the
protein). An antigenic determinant may compete with the intact antigen (i.e.,
the immunogen used to
elicit the immune response) for binding to an antibody.
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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-S'.
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., NaCl), detergents
(e.g., sodium dodecyl sulfate;
SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm
DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to repeated
DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit
(Applied Biosystems,
Foster City CA) in the S' and/or the 3' direction, and resequenced, or which
has been assembled from
one or more overlapping cDNA, EST, or genoznic DNA fragments using a computer
program for
fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison
WI) or Phrap
(University of Washington, Seattle WA). Some sequences have been both extended
and assembled to
produce the consensus sequence.
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"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of the
protein is conserved and not significantly changed by such substitutions. The
table below shows amino
acids which may be substituted for an original amino acid in a protein and
which are regarded as
conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of the
side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide. Chemical
modifications of a polynucleotide can include, for example, replacement of
hydrogen by an alkyl, aryl,
hydroxyl, or amino group. A derivative polynucleotide encodes a polypeptide
which retains at least one
biological or immunological function of the natural molecule. A derivative
polypeptide is one modified
by glycosylation, pegylation, or any similar process that retains at least one
biological or immunological
function of the polypeptide from which it Was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
"Differential expression" refers to increased or upregulated; or decreased,
downregulated, or
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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
S 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 nuclebtide/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 S, 10,
15, 16, 20, 25, 30, 40, S0, 60, 7S, 100, 150, ZSO 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 S00 amino acids (or first 2S% or SO%) of a polypeptide
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
1S embodiments.
A fragment of SEQ ID N0:33-64 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:33-64, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:33-64 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID N0:33-64 from related polynucleotide sequences. The precise length of a
fragment of SEQ
ID N0:33-64 and the region of SEQ ID N0:33-64 to which the fragment
corresponds are routinely
deternunable by one of ordinary skill in the art based on the intended purpose
for the fragment.
A fragment of SEQ ID N0:1-32 is encoded by a fragment of SEQ ID N0:33-64. A
fragment
of SEQ ID N0:1-32 comprises a region of unique amino acid sequence that
specifically identifies
SEQ ID N0:1-32. For example, a fragment of SEQ ID N0:1-32 is useful as an
immunogenic peptide
for the development of antibodies that specifically recognize SEQ ID NO:1-32.
The precise length of
a fragment of SEQ ID N0:1-32 and the region of SEQ ID N0:1-32 to which the
fragment
corresponds are routinely determinable by one of ordinary skill in the art
based on the intended
purpose for the fragment.
A "full length" polynucleotide sequence is one containing at least a
translation initiation codon
(e.g., methionine) followed by an open reading frame and a translation
termination codon. A "full
length" polynucleotide sequence encodes a "full length" polypeptide sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between two
or more polynucleotide sequences or two or more polypeptide sequences.
3S The terms "percent identity" and "% identity," as applied to polynucleotide
sequences, refer to
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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 VVI). 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 (NCBI) Basic
Local Alignment Search
Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which
is available from several
sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence analysis
programs including "blastn," that is used to align a known polynucleotide
sequence with other
polynucleotide sequences from a variety of databases. Also available is a tool
called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.htxnl. The
"BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: -2
Open Gap: S afzd Exterasi.ofz Gap: 2 pefialties
Gap x drop-off 50
Expect: 10
Word Size: 11
Filter: ofa
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Percent identity may be measured over the length of an entire defined
sequence, for example, as
defined by a particular SEQ ID number, or may be measured over a shorter
length, for example, over
the length of a fragment taken from a larger, defined sequence, for instance,
a fragment of at least 20, at
least 30, at least 40, at least 50, at least 70, at least 100, or at least 200
contiguous nucleotides. Such
lengths are exemplary only, and it is understood that any fragment length
supported by the sequences
shown herein, in the tables, figures, or Sequence Listing, may be used to
describe a length over which
percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes in
a nucleic acid sequence can be made using this degeneracy to produce multiple
nucleic acid sequences
that all encode substantially the same protein.
The phrases "percent identity" arid "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some alignment
methods take into account conservative amino acid substitutions. Such
conservative substitutions,
explained in more detail above, generally preserve the charge and
hydrophobicity at the site of
substitution, thus preserving the structure (and therefore function) of the
polypeptide.
Percent identity between polypeptide sequences may be determined using the
default parameters
of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e
sequence alignment
program (described and referenced above). For pairwise alignments of
polypeptide sequences using
CLUSTAL V, the default parameters are set as follows: Ktuple=1, gap penalty=3,
window=5, and
"diagonals saved"=5. The PAM250 matrix is selected as the default residue
weight table. As with
polynucleotide alignments, the percent identity is reported by CLUSTAL V as
the "percent similarity"
between aligned polypeptide sequence pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø12
(April-21-2000) with blastp set at default parameters. Such default parameters
may be, for example:
Matrix: BLOSUM62
Open Gap: 1l and Extension Gap: 1 penalties
Gap x drop-off. 50
Expect: 1 D
Word Size: 3
Filter: ofa
Percent identity may be measured over the length of an entire defined
polypeptide sequence, for
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example, as defined by a particular SEQ ID number, or may be measured over a
shorter length, for
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for instance,
a fragment of at least 15, at least 20, at least 30, at least 40, at least 50,
at least 70 or at least 150
contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment length
supported by the sequences shown herein, in the tables, figures or Sequence
Listing, may be used to
describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size and which contain all of the
elements required for
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.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the stringency
of the hybridization process, with more stringent conditions allowing less non-
specific binding, i.e.,
binding between pairs of nucleic acid strands that are not perfectly matched.
Permissive conditions for
annealing of nucleic acid sequences are routinely determinable by one of
ordinary skill in the art and
may be consistent among hybridization experiments, whereas wash conditions may
be varied among
experiments to achieve the desired stringency, and therefore hybridization
specificity. Permissive
annealing conditions occur, for example, at 68°C in the presence of
about 6 x SSC, about 1 % (wlv)
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 perfectly matched probe. An equation for
calculating Tm and conditions
for nucleic acid hybridization are well known and can be found in Sambrook, J.
et al. (1989) Molecular
Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Press,
Plainview NY; specifically
see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present invention
include wash conditions of 68°C in the presence of about 0.2 x SSC and
about 0.1 % SDS, for 1 hour.
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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 ~ ~ml. Organic
solvent, such as
formamide at a concentration of about 35-50% v/v, may also be used under
particular circumstances,
such as for RNA:DNA hybridizations. Useful variations on these wash conditions
will be readily
apparent to those of ordinary skill in the art. Hybridization, particularly
under high stringency
conditions, may be suggestive of evolutionary similarity between the
nucleotides. Such similarity is
strongly indicative of a similar role for the nucleotides and their encoded
polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A hybridization
complex may be formed in solution (e.g., Cot or Rot analysis) or formed
between one nucleic acid
sequence present in solution and another nucleic acid sequence immobilized on
a solid support (e.g.,
paper, membranes, filters, chips, pins or glass slides, or any other
appropriate substrate to which cells
or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide sequence
resulting in the addition of one or more amino acid residues or nucleotides,
respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression of
various factors, e.g., cytokines, chemokines, and other signaling molecules,
which may affect cellular
and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of TRICH
which is
capable of eliciting an immune response when introduced into a living
organism, for example, a
mammal. The term "immunogenic fragment" also includes any polypeptide or
oligopeptide fragment of
TRICH which is useful in any of the antibody production methods disclosed
herein or known in the art.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides, polypeptides,
or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of TRICH. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other biological,
functional, or immunological properties of TRICH.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
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synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition. PNAs
preferentially bind complementary single stranded DNA or RNA and stop
transcript elongation, and
may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an TRICH may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in the
art. These processes may occur synthetically or biochemically. Biochemical
modifications will vary 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
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
Bio- logy> Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M.
et al. (1990) PCR
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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).
S Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. Fox 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
20 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 InstituteJMIT Center for
Genome Research,
Cambridge MA) allows the user to input a "mispriming library," in which
sequences to avoid as primer
15 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
20 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
oIigonucleotides 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
25 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
30 artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, su ra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter sequence.
Such a recombinant nucleic acid may be part of a vector that is used, for
example, to transform a cell.
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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 (UTRs).
Regulatory elements interact with host or viral proteins which control
transcription, translation, or RNA
stability.
"Reporter molecules" are chemical of 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 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,
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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),
s-unra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having at
least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of the
nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version
2Ø9 (May-07-1999)
set at default parameters. Such a pair of nucleic acids may show, for example,
at least 50%, at least
60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at
least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least
99% or greater sequence
identity over a certain defined length. A variant may be described as, for
example, an "allelic" (as
defined above), "splice," "species," or "polymorphic" variant. A splice
variant may have significant
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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 GenBank protein (genpept) database. Columns 1 and 2
show the
polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the
invention. Column 3
shows the GenBank identification number (Genbank ID NO:) of the nearest
GenBank homolog.
Column 4 shows the probability score for the match between each polypeptide
and its GenBank
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homolog. Column 5 shows the annotation of the GenBank 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 residues in each polypeptide. Column 4 shows
potential
phosphorylation sites, and column 5 shows potential glycosylation sites, as
determined by the MOTIFS
program of the GCG sequence analysis software package (Genetics Computer
Group, Madison WI).
Column 6 shows amino acid residues comprising signature sequences, domains,
and motifs. Column 7
shows analytical methods for protein structure/function analysis and in some
cases, searchable
databases to which the analytical methods were applied.
Together, Tables 2 arid 3 summarize the properties of polypeptides of the
invention, and these
properties establish that the claimed polypeptides are transporters and ion
channels. For example, SEQ
ID N0:5 is 83% identical to rat GABA receptor rho-3 subunit precursor (GenBank
ID 81060975) as
determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.)
The BLAST
probability score is 1.7e-206, which indicates the probability of obtaining
the observed polypeptide
sequence alignment by chance. SEQ ID N0:5 also contains a neurotransmitter-
gated ion channel
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
N0:5 is a neurotransmitter-gated ion channel. In an alternate example, SEQ ID
N0:16 is 57%
identical to human Na+/glucose cotransporter (GenBank ID 8338055) as
determined by the Basic Local
Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is
2.4e-181, which
indicates the probability of obtaining the observed polypeptide sequence
alignment by chance. SEQ ID
NO:16 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 N0:16 is a
Na+/glucose cotransporter. In
an alternate example, SEQ ID N0:27 is 53% identical to human ATP-binding
cassette transporter-2
(ABC-1) (GenBank ID 84128033) 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 N0:27 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
32
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BLIMPS, MOTIFS, and PROFILESCAN analyses provide further corroborative
evidence that SEQ ID
N0:27 is an ABC transporter. In an alternate example, SEQ ID N0:12 is 45 %
identical to rat thyroid
sodiumliodide symporter NIS (GenBank ID 81399954) as determined by the Basic
Local Alignment
Search Tool (BLAST). (See Table 2.) The BLAST probability score is 3.0e-143,
which indicates the
probability of obtaining the observed polypeptide sequence alignment by
chance. SEQ ID N0:12 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 and PROFILESCAN analyses
provide further
corroborative evidence that SEQ ID N0:12 is a sodiumaolute symporter. SEQ ID
NO:1-4, SEQ ID
N0:6-11, SEQ ID N0:13-15, SEQ ID N0:17-26, and SEQ ID N0:28-32 were analyzed
and annotated
in a similar manner. The algorithms and parameters for the analysis of SEQ ID
NO:1-32 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.
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 N0:33-64 or that distinguish between SEQ ID
N0:33-64 and
related polynucleotide sequences. Column 5 shows identification numbers
corresponding to cDNA
sequences, coding sequences (exons) predicted from genomic DNA, and/or
sequence assemblages
comprised of both cDNA and genomic 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 libxaries. For example,
6724643H1 is the
identification number of an Incyte cDNA sequence, and LUNLTMT01 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., 71495515V1). Alternatively, the identification
numbers in column 5 may
refer to GenBank cDNAs or ESTs (e.8., 85746200) which contributed to the
assembly of the full length
polynucleotide sequences. In addition, the identification numbers in column 5
may identify sequences
derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e.,
those sequences
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including the designation "ENST"). Alternatively, the identification numbers
in column S may be
derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those
sequences including
the designation "NM" or "NT") or the NCBI RefSeq Protein Sequence Records
(i.e., those sequences
including the designation "NP"). Alternatively, the identification numbers in
column S may refer to
S assemblages of both cDNA and Genscan-predicted exons brought together by an
"exon stitching"
algorithm. For example, FL XXXXXX Nl NZ YYYYY N3 N~ represents a "stitched"
sequence in
which XXXXXX is the identification number of the cluster of sequences to which
the algorithm was
applied, and YI'YYY is the number of the prediction generated by the
algorithm, and N~,2,3,.., if present,
represent specific exons that may have been manually edited during analysis
(See Example V).
Alternatively, the identification numbers in column S may refer to assemblages
of exons brought
together by an "exon-stretching" algorithm. Fox example, FLXXXXXX
gAAAAA_gBBBBB_1 N is the
identification number of a "stretched" sequence, with XXXXXX being the Incyte
project identification
number, gAAAAA being the GenBank identification number of the human genomic
sequence to which
the "exon-stretching" algorithm was applied, gBBBBB being the GenBank
identification number or
1S NCBI RefSeq identification number of the nearest GenBank protein homolog,
and N referring to
specific exons (See Example V). In instances where a RefSeq sequence was used
as a protein homolog
for the "exon-stretching" algorithm, a RefSeq identifier (denoted by "NM,"
"NP," or "NT") may be
used in place of the GenBank identifier (i. e., gBBBBB).
Alternatively, a prefix identifies component sequences that were hand-edited,
predicted from
genomic DNA sequences, or derived from a combination of sequence analysis
methods. The following
Table lists examples of component sequence prefixes and corresponding sequence
analysis methods
associated with the prefixes (see Example IV and Example V).
Prefix Type of analysis and/or examples of programs
GNN, GFG, Exon prediction from genomic sequences using,
2S ENST for example,
GENSCAN (Stanford University, CA, USA) or
FGENES
(Computer Genomics Group, The Sanger Centre,
Cambridge, UK).
GBI Hand-edited analysis of genomic sequences.
FL Stitched or stretched genomic sequences
(see Example V).
In some cases, Incyte cDNA coverage redundant with the sequence coverage shown
in column
S was obtained to confirm the final consensus polynucleotide sequence, but the
relevant Incyte cDNA
identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length
polynucleotide sequences
which were assembled using Incyte cDNA sequences. The representative cDNA
Iibrary is the Incyte
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cDNA library which is most frequently represented by the Incyte cDNA sequences
which were used to
assemble and conf'~rm 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 N0:33-64, which encodes TRICH. The
polynucleotide sequences of
SEQ ID N0:33-64, as presented in the Sequence Listing, embrace the equivalent
RNA sequences,
wherein occurrences of the nitrogenous base thymine are replaced with uracil,
and the sugar backbone
is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding
TRICH. In
particular, such a variant polynucleotide sequence will have at least about
70%, or alternatively at least
about 85 %, or even at least about 95 % polynucleotide sequence identity to
the polynucleotide sequence
encoding TRICH. A particular aspect of the invention encompasses a variant of
a polynucleotide
sequence comprising a sequence selected from the group consisting of SEQ ID
N0:33-64 which has at
least about 70%, or alternatively at least about 85 %, or even at least about
95 % polynucleotide
sequence identity to a nucleic acid sequence selected from the group
consisting of SEQ ID N0:33-64.
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
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
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
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 ID
N0:33-64 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 Biology, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A.
(1995) Molecular
Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853.)
The nucleic acid sequences encoding TRICH may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
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DNA within a cloning vector. (See, e.g., Saxkar, 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. I:1 I 1-119.) In this method, multiple restriction
enzyme digestions and
legations may be used to insert an engineered double-stranded sequence into a
region of unknown
sequence before performing PCR. Other methods which may be used to retrieve
unknown sequences
are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids
Res. 19:3055-3060).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries
(Clontech, Palo
Alto CA) to walk genomic DNA. This procedure avoids the need to screen
libraries and es useful in
fording intron/exon junctions. For all PCR-based methods, primers may be
designed using
commercially available software, such as OLIGO 4.06 primer analysis software
(National Biosciences,
Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides
in length, to have a
GC content of about S0% 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 additeon, random-primed libraries,
which often include
sequences contaening the 5' regions of genes, are preferable for situations in
which an oligo d(T) library
does not yield a full-length cDNA. Genomec 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 wavele baths. Outputllight 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
fragments or functional equivalents thereof, in appropriate host cells. Due to
the inherent degeneracy of
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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 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 Properties, WH
Freeman, New York NY, pp.
55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated
synthesis may be achieved
using the ABI 431A peptide synthesizer (Applied Biosystems). Additionally, the
amino acid sequence
of TRICH, or any part thereof, may be altered during direct synthesis and/or
combined with sequences
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CA 02415808 2003-O1-06
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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 (I990) Methods
Enzymol. 182:392-42I.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by sequencing.
(See, e.g., Creighton, s-u~ra, 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 maybe 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
39
CA 02415808 2003-O1-06
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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.K. et al. (1994) Proc. Natl.
Acad. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu,
N. (1987) EMBO
J. 6:307-311; The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw
Hill, New
York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA
81:3655-3659; and
Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors
derived from retxoviruses,
adenoviruses, or herpes or vaccinia viruses, or from various bacterial
plasmids, may be used for
delivery of nucleotide sequences to the targeted organ, tissue, or cell
population. (See, e.g., Di
Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993)
Proc. Natl. Acad. Sci.
USA 90(13):6340-6344; 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. For example,
vectors containing the
strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of TRICH. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomvces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable integration
of foreign sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, 1995, supra;
Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et
al. (1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of TRICH. Transcription of
sequences encoding
TRICH may be driven by viral promoters, e.g., the 35S and 19S promoters of
CaMV used alone or in
combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO
J. 6:307-311).
CA 02415808 2003-O1-06
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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 ugated into an
adenovirus transcription/translation complex consisting of the late promoter
and tripartite leader
sequence. Insextion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses TRICH in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalian host
cells. SV40 or EBV-
based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino polymers,
or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J. et al.
(1997) Nat. Genet. 15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression of
TRICH in cell lines is preferred. For example, sequences encoding TRICH can be
transformed into cell
lines using expression vectors which may contain viral origins of replication
and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media before
being switched to selective media. The purpose of the selectable marker is to
confer resistance to a
selective agent, and its presence allows growth and recovery of cells which
successfully express the
introduced sequences. Resistant clones of stably transformed cells may be
propagated using tissue
culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These include,
but are not limited to, the herpes simplex virus thymidine kinase and adenine
phosphoribosyltransferase
genes, for use in tk and apr- cells, respectively. (See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232;
Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or
hexbicide resistance can be
used as the basis for selection. For example, dl~ confers resistance to
methotrexate; raeo confers
resistance to the aminoglycosides neomycin and G-418; and als and pat confer
resistance to
chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g.,
Wigler, M. et al. (1980)
41
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Pxoc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. (1981) J.
Mol. Biol. 150:1-14.)
Additional selectable genes have been described, e.g., trpB and hisD, which
alter cellular requirements
for metabolites. (See, e.g., Hartman, S.C. and R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA
85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins
(GFP; Clontech),13
glucuronidase and its substrate 13-glucuronide, or luciferase ,and its
substrate luciferin may be used.
These markers can be used not only to identify transformants, but also to
quantify the amount of
transient or stable protein expression attributable to a specific vector
system. (See, e.g., Rhodes, C.A.
(1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
confirmed. For example, if the
sequence encoding TRICH is inserted witlun 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
oligolabeling, nick translation, end-labeling, or PCR amplification using a
labeled nucleotide.
Alternatively, the sequences encoding TRICH, or any fragments thereof, may be
cloned into a vector for
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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 TRICH may be designed to contain signal sequences
which direct
secretion of TRICH through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of the
polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation, 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-
txanslational activities
(e.g., CHO, HeLa, MDCI~, HEK293, and WI38) are available from the American
Type Culture
Collection (ATCC, Manassas VA) and may be chosen to ensure the correct
modification and processing
of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding TRICH may be ligated to a heterologous sequence resulting
in translation of a
fusion protein in any of the aforementioned host systems. For example, a
chimeric TRICH protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of TRICH
activity. Heterologous protein and
peptide moieties may also facilitate purification of fusion proteins using
commercially available affinity
matrices. Such moieties include, but are not limited to, glutathione S-
transferase (GST), maltose
binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-
His, FLAG, c-rnyc, and
hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their
cognate fusion
proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin,
and metal-chelate resins,
respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity
purification of fusion
proteins using commercially available monoclonal and polyclonal antibodies
that specifically recognize
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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, Droso~hila, or E.
coli. Cells expressing TRICH or cell membrane fractions which contain TRICH
are then contacted
with a test compound and binding, stimulation, or inhibition of activity of
either TRICH or the
compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide,
wherein binding is
detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable
label. For example,
the assay may comprise the steps of combining at least one test compound with
TRICH, either in
solution or affixed to a solid support, and detecting the binding of TRICH to
the compound.
Alternatively, the assay may detect or measure binding of a test compound in
the presence of a
labeled competitor. Additionally, the assay may be carried out using cell-free
preparations, chemical
libraries, or natural product mixtures, and the test compounds) may be free in
solution or affixed to a
solid support.
TRICH of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of TRICH. Such compounds may include agonists,
antagonists, or partial
44.
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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.
I0 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 1291SvJ 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 axe identified and microinjected into mouse Bell
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, fox example, neural cells, hematopoietic lineages, and cardiomyocytes
(Thomson, J.A. et al. (1998)
Science 282:1145-1147).
Polynucleotides encoding TRICH can also be used to create "knockin" humanized
animals
(pigs) or transgenic animals (mice or rats) to model human disease. With
knockin technology, a region
of a polynucleotide encoding TRICH is injected into animal ES cells, and the
injected sequence
integrates into the animal cell genome. Transformed cells are injected into
blastulae, and the blastulae
are implanted as described above. Transgenic progeny or inbred lines are
studied and treated with
CA 02415808 2003-O1-06
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potential pharmaceutical agents to obtain information on treatment of a human
disease. Alternatively, a
mammal inbred to overexpress TRICH, e.g., by secreting TRICH in, its milk, may
also serve as a
convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu.
Rev. 4:55-74).
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 adrenal, testicular, and prostate tumors, Crohn's
disease, teratocarcinoma
and dendritic cells, brain, lung, ileum, small intestine, uterine myometrial,
colon, and pancreatic
tissues. Therefore, TRICH appears to play a role in transport, neurological,
muscle, immunological,
and cell proliferative disorders. In the treatment of disorders associated
with increased TRICH
expression or activity, it is desirable to decrease the expression or activity
of TRICH. In the treatment
of disorders associated with decreased TRICH expression or activity, it is
desirable to increase the
expression or activity of TRICH.
Therefore, in one embodiment, TRICH or a fragment or derivative thereof may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of TRICH. Examples of such disorders include, but are not limited to,
a transport disorder
such as akinesia, amyotrophic lateral sclerosis, ataxia telangiectasia, cystic
fibrosis, Becker's
muscular dystrophy, Bell's palsy, Charcot-Marie Tooth disease, diabetes
mellitus, diabetes insipidus,
diabetic neuropathy, Duchenne muscular dystrophy, hyperkalemic periodic
paralysis, normokalemic
periodic paralysis, Parkinson's disease, malignant hyperthermia, multidrug
resistance, myasthenia
gravis, myotonic dystrophy, catatonia, tardive dyskinesia, dystonias,
peripheral neuropathy, cerebral
neoplasms, prostate cancer, cardiac disorders associated with transport, e.g.,
angina, bradyarrythmia,
tachyarrythmia, hypertension, Long QT syndrome, myocarditis, cardiomyopathy,
nemaline
myopathy, centronuclear myopathy, lipid myopathy, mitochondrial myopathy,
thyrotoxic myopathy,
ethanol myopathy, dermatomyositis, inclusion body myositis, infectious
myositis, polymyositis,
neurological disorders associated with transport, e.g., Alzheimer's disease,
amnesia, bipolar disorder,
dementia, depression, epilepsy, Tourette's disorder, paranoid psychoses, and
schizophrenia, and other
disorders associated with transport, e.g., neurofibromatosis, postherpetic
neuralgia, trigeminal
neuropathy, sarcoidosis, sickle cell anemia, Wilson's disease, cataracts,
infertility, pulmonary artery
stenosis, sensorineural autosomal deafness, hyperglycemia, hypoglycemia,
Grave's disease, goiter,
Cushing's disease, Addison's disease, glucose-galactose malabsorption
syndrome,
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
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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,
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
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
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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,
teratocaxcinoma, 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,
pxostate, 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 txeat or prevent a
disorder associated with decreased expression or activity of TRICH including,
but not limited to, those
provided above.
In still another embodiment, an agonist which modulates the activity of TRICH
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or activity
of TRICH including, but not limited to, those listed above.
In a further embodiment, an antagonist of TRICH may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of TRICH.
Examples of such
disorders include, but are not limited to, those transport, neurological,
muscle, immunological, and cell
proliferative disorders described above. In one aspect, an antibody which
specifically binds TRICH
may be used directly as an antagonist or indirectly as a targeting or delivery
mechanism for bringing a
pharmaceutical agent to cells or tissues which express TRICH.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding TRICH may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of TRICH including, but not limited to, those
described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be administered in combination with
other appropriate
therapeutic agents.. Selection of the appropriate agents for use in
combination therapy may be made by
one of ordinary skill in the art, according to conventional pharmaceutical
principles. The combination
of therapeutic agents may act synergistically to effect the treatment or
prevention of the various
disorders described above. Using this approach, one may be able to achieve
therapeutic efficacy with
lower dosages of each agent, thus reducing the potential for adverse side
effects.
4S
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An antagonist of TRICH may be produced using methods which axe 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 Corvnebacteriumparvum 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.,
Morxison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and Takeda,
S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the art, to produce
TRICH-specific single
chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be
49
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generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g., Burton,
D.R. (1991) Proc. Natl. Acid. 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. Acid. Sci.
USA 86:3833-3837; Winter,
G. et aI. (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, su ra).
Various methods such as Scatchaxd 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 Ka ranging from
about 109 to 1012 L/mole are preferred for use in immunoassays in which the
TRICH-antibody complex
must withstand rigorous manipulations. Low-affinity antibody preparations with
I~ ranging from
about 106 to 10' L/mole are preferred for use in immunopurification and
similar procedures which
ultimately require dissociation of TRICH, preferably in active form, from the
antibody (Catty, D.
(1988) Antibodies, Volume I: A Practical Approach, IRL Press, Washington DC;
Liddell, J.E. and A.
Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons,
New York NY).
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The titer and avidity of polyclonal antibody preparations may be further
evaluated to determine
the quality and suitability of such preparations for certain downstream
applications. For example, a
polyclonal antibody preparation containing at least 1~2 mg specific
antibody/ml, preferably 5-10 mg
specific antibody/ml, is generally employed in procedures requiring
precipitation of TRICH-antibody
complexes. Procedures for evaluating antibody specificity, titer, and avidity,
and guidelines for
antibody quality and usage in various applications, are generally available.
(See, e.g., Catty, supra, and
Coligan et al, supra.)
In another embodiment of the invention, the polynucleotides encoding TRICH, or
any fragment
or complement thereof, may be used for therapeutic purposes. In one aspect,
modifications of gene
expression can be achieved by designing complementary sequences or antisense
molecules (DNA, RNA,
PNA, or modified oligonucleotides) to the coding or regulatory regions of the
gene encoding TRICH.
Such technology is well known in the art, and antisense oligonucleotides or
larger fragments can be
designed from various locations along the coding or control regions of
sequences encoding TRICH.
(See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press Inc.,
Totawa NJ.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. (1990) Blood
76:271; Ausubel, su ra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et
al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al. (1997)
Nucleic Acids Res.
25(14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding TRICH may be
used for
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease
characterized by X-linked
inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe
combined
immunodeficiency syndrome associated with an inherited adenosine deanninase
(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) Cel175:207-216; 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
S1
CA 02415808 2003-O1-06
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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 falc~arum and
T~anosoma 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 fransfection, (iv) receptor-
mediated gene transfer, and (v)
the use of DNA txansposons (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
FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible
promoter (Rossi, F.M.V.
and Blau, H.M. s_ upra)), 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 LIPID
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
52
CA 02415808 2003-O1-06
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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. 2:841-845). The introduction of DNA to primary cells requires
modification of these
standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to TRICH expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding TRICH under the control of an independent promoter or
the retrovirus long
terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and
(iii) a Rev-responsive
element (RRE) along with additional retrovirus cis-acting RNA sequences and
coding sequences
required for efficient vector propagation. Retrovirus vectors (e.g., PFB and
PFBNEO) axe
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 axe 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 89:22$3-
2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding TRICH to cells which have one or more genetic
abnormalities with respect to
the expression of TRICH. The construction and packaging of adenovirus-based
vectors are well known
to those with ordinary skill in the art. Replication defective adenovirus
vectors have proven to be
versatile for importing genes encoding immunoregulatory proteins into intact
islets in the pancreas
(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.
53
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Rev. Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature 18:389:239-
242, both
incarporated 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 TCP22.
For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and
Xu, H. et al, (1994) Dev.
Biol. 163:152-261, hereby incorporated by reference. The manipulation of
cloned herpesvirus
sequences, the generation of recombinant virus following the transfection of
multiple plasmids
containing different segments of the large herpesvirus genomes, the growth and
propagation of
herpesvirus, and the infection of cells with herpesvirus are techniques well
known to those of ordinary
skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding TRICH to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based on
the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol. 9:464-
469). During
alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full length
genomic RNA, resulting
in the overproduction of capsid proteins relative to the viral proteins with
enzymatic activity (e.g.,
protease and polymerase). Similarly, inserting the coding sequence for TRICH
into the alphavirus
genome in place of the capsid-coding region results in the production of a
large number of TRICH-
coding RNAs and the synthesis of high levels of TRICH in vector transduced
cells. While 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 (STN) indicates that
the lytic replication of alphaviruses can be altered to suit the needs of the
gene therapy application
54
CA 02415808 2003-O1-06
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(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 Immunoloyc Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-
177.) A
complementary sequence or antisense molecule may also be designed to block
translation of mRNA by
preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding TRICH.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared by
any method known in the art for the synthesis of nucleic acid molecules. These
include techniques for
chemically synthesizing oligonucleotides such as solid phase phosphoramidite
chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA 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.
CA 02415808 2003-O1-06
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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-, thin-, and similarly modified forms
of adenine, cytidine,
guanine, thymine, and uridine which are not as easily recognized by endogenous
endonucleases.
An additional embodiment ofthe 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
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
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CA 02415808 2003-O1-06
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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.
These compositions are generally aerosolized immediately priox 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.
57
CA 02415808 2003-O1-06
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Patent No. 5,997,848). Pulmonary delivery has the advantage of administration
without needle
injection, and obviates the need for potentially toxic penetration enhancers.
Compositions suitable for use in the invention include compositions wherein
the active
ingredients are contained in an effective amount to achieve the intended
purpose. The determination of
an effective dose is well within the capability of those skilled in the art.
Specialized forms of compositions may be prepared for direct intracellular
delivery of
macromolecules comprising TRICH or fragments thereof. Fox example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of the
macromolecule. Alternatively, TRICH or a fragment thereof may be joined to a
short cationic N-
terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated
have been found to
transduce into the cells of all tissues, including the brain, in a mouse model
system (Schwarze, S.R. et
al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs, monkeys,
or pigs. An animal model may also be used to determine the appropriate
concentration range and xoute
of administration. Such information can then be used to determine useful doses
and routes for
administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example TRICH
or fragments thereof, antibodies of TRICH, and agonists, antagonists or
inhibitors of TRICH, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by
standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDso (the dose therapeutically effective in 50% of the
population) or LDSO (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDSO/EDso ratio. Compositions
which exhibit large
therapeutic indices are preferred. The data obtained from cell culture assays
and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDSO
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the 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.
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Long-acting compositions may be administered every 3 to 4 days, every week, or
biweekly depending
on the half life and clearance rate of the particular formulation.
Normal dosage amounts may vary from about 0.1 ~cg to 100,000 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 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
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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 ox amplification will
determine whether the
probe identifies only naturally occurring sequences encoding TRICH, allelic
variants, or related
sequences.
Probes may also be used for the detection of related sequences, and may have
at least 50%
sequence identity to any of the TRICH encoding sequences. The hybridization
probes of the subject
invention may be DNA ox RNA and may be derived from the sequence of SEQ ID
N0:33-64 or from
genomic sequences including promoters, enhancers, and introns of the TRICH
gene.
Means for producing specific hybridization probes for DNAs encoding TRICH
include the
cloning of polynucleotide sequences encoding TRICH or TRICH derivatives into
vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and may
be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a variety
of reporter groups, for example, by radionuclides such as 32P or 35S, or by
enzymatic labels, such as
alkaline phosphatase coupled to the probe via avidin/biotin coupling systems,
and the like.
Polynucleotide sequences encoding TRICH may be used fox the diagnosis of
disorders
associated with expression of TRICH. Examples of such disorders include, but
are not limited to, a
transport disorder such as akinesia, amyotrophic lateral sclerosis, ataxia
telangiectasia, cystic fibrosis,
Becker's muscular dystrophy, Bell's palsy, Charcot-Marie Tooth disease,
diabetes mellitus, diabetes
insipidus, diabetic neuropathy, Duchenne muscular dystrophy, hyperkalemic
periodic paralysis,
normokalemic periodic paralysis, Parkinson's disease, malignant hyperthermia,
multidrug resistance,
myasthenia gravis, myotonic dystrophy, catatonia, tardive dyskinesia,
dystonias, peripheral
neuropathy, cerebral neoplasms, prostate cancer, cardiac disorders associated
with transport, e.g.,
angina, bradyarrythmia, tachyarrythmia, hypertension, Long QT syndrome,
myocarditis,
cardiomyopathy, nemaline myopathy, centronuclear myopathy, lipid myopathy,
mitochondrial
myopathy, thyrotoxic myopathy, ethanol myopathy, dermatomyositis, inclusion
body myositis,
infectious myositis, polymyositis, neurological disorders associated with
transport, e.g., Alzheimer's
disease, amnesia, bipolar disorder, dementia, depression, epilepsy, Tourette's
disorder, paranoid
psychoses, and schizophrenia, and other disorders associated with transport,
e.g., neurofibromatosis,
postherpetic neuralgia, trigeminal neuropathy, sarcoidosis, sickle cell
anemia, Wilson's disease,
cataracts, infertility, 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,
iminoglycinuxia, Hartup disease, and
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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, priors diseases including kuru, Creutzfeldt-
Jakob disease, and
Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional
and metabolic diseases
of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the central
nervous system including Down syndrome, cerebral palsy, neuroskeletal
disorders, autonomic
nervous system disorders, cranial nerve disorders, spinal cord diseases,
muscular dystrophy and other
neuromuscular disorders, peripheral nervous system disorders, dermatomyositis
and polymyositis,
inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis,
periodic paralysis, mental
disorders including mood, anxiety, and schizophrenic disorders, seasonal
affective disorder (SAD),
akathesia, amnesia, catatonia, diabetic neuropathy, 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,
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
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
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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 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.
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Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the development
of the disease, or may provide a means for detecting the disease prior to the
appearance of actual
clinical symptoms. A more definitive diagnosis of this type may allow health
professionals to employ
preventative measures or aggressive treatment earlier thereby preventing the
development or further
progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding TRICH
may involve the use of PCR. These oligomers may be chemically synthesized,
generated enzymatically,
or produced in vitro. Oligomers will preferably contain a fragment of a
polynucleotide encoding
TRICH, or a fragment of a polynucleotide complementary to the polynucleotide
encoding TRICH, and
will be employed under optimized conditions for identification of a specific
gene or condition.
Oligomers may also be employed under less stringent conditions for detection
or quantification of
closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding TRICH may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are
substitutions, insertions and deletions that are a frequent cause of inherited
or acquired genetic disease
in humans. Methods of SNP detection include, but are not limited to, single-
stranded conformation
polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP,
oligonucleotide primers
derived from the polynucleotide sequences encoding TRICH are used to amplify
DNA using the
polymerase chain reaction (PCR). The DNA may be derived, for example, from
diseased or normal
tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause
differences in the secondary
and tertiar y structures of PCR products in single-stranded form, and these
differences are detectable
using gel electrophoresis in non-denaturing gels. In fSCCP, the
oligonucleotide primers are
fluorescently labeled, which allows detection of the amplimers in high-
throughput equipment such as
DNA sequencing machines. Additionally, sequence database analysis methods,
termed in silico SNP
(isSNP), are capable of identifying polymorphisms by comparing the sequence of
individual
overlapping DNA fragments which assemble into a common consensus sequence.
These computer-
based methods filter out sequence variations due to laboratory preparation of
DNA and sequencing
errors using statistical models and automated analyses of DNA sequence
chromatograms. In the
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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 spectrophotornetric 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 rnicroarray
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
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.
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Transcript images may be generated using transcripts isolated from tissues,
cell lines, biopsies,
or other biological samples. The transcript image may thus reflect gene
expression in vivo, as in the
case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the
present invention
may also be used in conjunction with in vitro model systems and preclinical
evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and toxicity
(Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S, and N.L.
Anderson (2000)
Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein).
If a test compound has a
signature similar to that of a compound with known toxicity, it is likely to
share those toxic properties.
These fingerprints or signatures are most useful arid 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 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
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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
2U levels of protein bound to each array element (Lueking, A. et al. (1999)
Anal. Biochem. 270:103-111;
Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be
performed by a variety of
methods known in the art, for example, by reacting the proteins in the sample
with a thiol- or amino-
reactive fluorescent compound and detecting the amount of fluorescence bound
at each array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and should
be analyzed in parallel with toxicant signatures at the transcript level.
There is a poor correlation
between transcript and protein abundances for some proteins in some tissues
(Anderson, N.L. and J.
Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures
may be useful in the
analysis of compounds which do not significantly affect the transcript image,
but which alter the
proteomic profile. In addition, the analysis of transcripts in body fluids is
difficult, due to rapid
degradation of mRNA, so proteomic profiling may be more reliable and
informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated biological
sample are separated so that the amount of each protein can be quantified. The
amount of each protein
is compared to the amount of the corresponding protein in an untreated
biological sample. A difference
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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
S 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 W09S/2S 1116;
Shalom D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-
21SS; and Heller, M.J. et al. (1997) U.S. Patent No. S,60S,662.) Various types
of microarrays are well
known and thoroughly described in DNA Microarrays: A Practical Approach, M.
Schena, ed. (1999)
Oxford University Press, London, hereby expressly incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding TRICH
may be used
to generate hybridization probes useful in mapping the naturally occurring
genomic sequence. Either
coding or noncoding sequences may be used, and in some instances, noncoding
sequences may be
preferable over coding sequences. For example, conservation of a coding
sequence among members
of a multi-gene family may potentially cause undesired cross hybridization
during chromosomal
mapping. The sequences may be mapped to a particular chromosome, to a specific
region of a
chromosome, or to artificial chromosome constructions, e.g., human artificial
chromosomes (HACs),
yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs),
bacterial P1
2S constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop genetic
linkage maps, for example, which correlate the inheritance of a disease state
with the inheritance of a
particular chromosome region or restriction fragment length polymorphism
(RFLP). (See, for
example, Larder, 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, su ra, pp. 96S-968.)
Examples of genetic map
data can be found in various scientific journals or at the Online Mendelian
Inheritance in Man (OMIM)
World Wide Web site. Correlation between the location of the gene encoding
TRICH on a physical
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map and a specific disorder, or a predisposition to a specific disorder, may
help define the region of
DNA associated with that disorder and thus may further positional cloning
efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse, may
reveal associated markers even if the exact chromosomal locus is not known.
This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely localized
by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia
to 11q22-23, any sequences
mapping to that area may represent associated or regulatory genes for further
investigation. (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 TRICH and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with TRICH,
or fragments thereof,
and washed. Bound TRICH is then detected by methods well known in the art.
Purified TRICH can
also be coated directly onto plates for use in the aforementioned drug
screening techniques.
Alternatively, non-neutralizing antibodies can be used to capture the peptide
and immobilize it on a
solid support.
In another embodiment, one may use competitive drug screening assays in which
neutralizing
antibodies capable of binding TRICH specifically compete with a test compound
for binding TRICH.
In this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with TRICH.
In additional embodiments, the nucleotide sequences which encode TRICH may be
used in any
molecular biology techniques that have yet to be developed, provided the new
techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
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Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following embodiments are,
therefore, to be construed as merely illustrative, and not limitative of the
remainder of the disclosure
in any way whatsoever.
The disclosures of all patents, applications and publications, mentioned above
and below
including U.S. Ser. No. 60/216,547, U.S. Ser. No. 60/21$,232, U.S. Ser. No.
60/220,112, and U.S.
Ser. No. 601221,839 are expressly incorporated by reference herein., are
expressly incorporated by
reference herein.
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 Iysates were centrifuged over CsCI
cushions or extracted
with chloroform. RNA was precipitated from the lysates with either isopropanol
or sodium acetate and
ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A)+ RNA was isolated
using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, s.~ra, 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 CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
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PBLUESCRIPT plasmid (Stratagene), PSPORTl plasmid (Life Technologies),
PCDNA2.1 plasmid
(Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), or pINCY (Incyte
Genomics, Palo AIto
CA), or derivatives thereof. Recombinant plasmids were transformed into
competent E. coli cells
including XL1-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
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
carried 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,
su ra, unit 7.7). Some of the cDNA sequences were selected for extension using
the techniques
disclosed in Example VIII.
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The polynucleotide sequences derived from Incyte cDNAs were validated by
removing vector,
linker, and poly(A) sequences and by masking ambiguous bases, using
algoritluns and programs based
on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis. The
Incyte cDNA
sequences or translations thereof were then queried against a selection of
public databases such as the
GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and
BLOCKS, PRINTS,
DOMO, PRODOM, 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) Curr. Opin. Struct. Biol. 6:361-365.) The
queries were
performed using programs based on BLAST, FASTA, BLIMPS, and HMMER. The Incyte
cDNA
sequences were assembled to produce full length polynucleotide sequences.
Alternatively, GenBank
cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-
predicted coding
sequences (see Examples IV and V) were used to extend Incyte cDNA assemblages
to full length.
Assembly was performed using programs based on Phred, Phrap, and Conned, 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 CLUSTAL algorithm as
incorporated into the
MEGALIGN multisequence alignment program (DNASTAR), which also calculates the
percent
identity between aligned sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis
and assembly of
Incyte cDNA and full length sequences and provides applicable descriptions,
references, and threshold
parameters. The first column of Table 7 shows the tools, programs, and
algorithms used, the second
column provides brief descriptions thereof, the third column presents
appropriate references, all of
which are incorporated by reference herein in their entirety, and the fourth
column presents, where
applicable, the scores, probability values, and other parameters used to
evaluate the strength of a match
between two sequences (the higher the score or the lower the probability
value, the greater the identity
between two sequences).
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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
N0:33-64. 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 predicted by
Genscan, such as extra or omitted exons. BLAST analysis was also used to find
any Incyte cDNA or
public cDNA coverage of the Genscan-predicted sequences, thus providing
evidence for transcription.
When Incyte cDNA coverage was available, this information was used to correct
or confirm the
Genscan predicted sequence. Full length polynucleotide sequences were obtained
by assembling
Genscan-predicted coding sequences with Incyte cDNA sequences andlor public
cDNA sequences using
the assembly process described in Example III. Alternatively, full length
polynucleotide sequences were
derived entirely from edited or unedited Genscan-predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
"Stitched" Seguences
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.
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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" Sequences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example III were
queried against public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases using
the BLAST program. The nearest GenBank protein homolog was then compared by
BLAST analysis
to either Incyte cDNA sequences or GenScan exon predicted sequences described
in Example IV. A
chimeric protein was generated by using the resultant high-scoring segment
pairs (HSPs) to map the
translated sequences onto the GenBank protein homolog. Insertions or deletions
may occur in the
chimeric protein with respect to the original GenBank protein homolog. The
GenBank protein homolog,
the chimeric protein, or both were used as probes to search for homologous
genomic sequences from the
public human genome databases. Partial DNA sequences were therefore
"stretched" or extended by the
addition of homologous genomic sequences. The resultant stretched sequences
were examined to
determine whether it contained a complete gene.
VI. Chromosomal Mapping of TRICH Encoding Polynucleotides
The sequences which were used to assemble SEQ ID N0:33-64 were compared with
sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ ID N0:33-64 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
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had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ ID NO:, to that
map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map
position of 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.gov/genemapn, can be employed to determine if
previously identified
disease genes map within or in proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a gene
and involves the hybridization of a labeled nucleotide sequence to a membrane
on which RNAs from a
particular cell type or tissue have been bound. (See, e.g., Sambrook, supra,
ch. 7; Ausubel (1995)
supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the computer
search can be modified to determine whether any particular match is
categorized as exact or similar.
The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity
5 x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two
sequences and the length
of the sequence match. The product score is a normalized value between 0 and
100, and is calculated
as follows: the BLAST score is multiplied by the percent nucleotide identity
and the product is divided
by (5 times the length of the shorter of the two sequences). The BLAST score
is calculated by
assigning a score of +5 for every base that matches in a high-scoring segment
pair (HSP), and -4 for
every mismatch. Two sequences may share more than one HSP (separated by 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
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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 SO% 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 III).
Each cDNA sequence is
derived from a cDNA library constructed from a human tissue. Each human tissue
is classified into one
of the following organltissue categories: cardiovascular system; connective
tissue; digestive system;
embryonic structures; endocrine system; exocrine glands; genitalia, female;
genitalia, male; germ cells;
heroic and immune system; liver; musculoskeletal system; nervous system;
pancreas; respiratory
system; sense organs; skin; stomatognathic system; unclassified/mixed; or
urinary tract. The number of
libraries in each category is counted and divided by the total number of
libraries across all categories.
Similarly, each human tissue is classified into one of the following
disease/condition categories: cancer,
cell line, developmental, inflammation, neurological, trauma, cardiovascular,
pooled, and other, and the
number of libraries in each category is counted and divided by the total
number of libraries across all
categories. The resulting percentages reflect the tissue- and disease-specific
expression of cDNA
encoding TRICH. cDNA sequences and cDNA library/tissue information are found
in the LIFESEQ
GOLD database (Incyte Genomics, Palo Alto CA).
VIII. Extension of TRICH Encoding Polynucleotides
Full length polynucleotide sequences were also produced by extension of an
appropriate
fragment of the full length molecule using oligonucleotide primers designed
from this fragment. One
primer was synthesized to initiate 5' extension of the known fragment, and the
other primer was
synthesized to initiate 3' extension of the known fragment. The initial
primers were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target sequence
at temperatures of about 68 ° C to about 72 ° C. Any stretch of
nucleotides which would result in hairpin
structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one extension
was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well lmown 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
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(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, 1 S 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 ~1 of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ~1 to 10 ~l aliquot of the reaction mixture was
analyzed by electrophoresis
on a 1 % agarose gel to determine which reactions were successful in extending
the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to relegation into pUC 18 vectox (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%) agarose
gels, fragments were excised, and agar digested with Agar ACE (Promega).
Extended clones were
relegated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector
(Amersham
Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in
restriction site overhangs,
and transfected into competent E. cole cells. Transformed cells were selected
on antibiotic-containing
media, and individual colonies were picked and cultured overnight at 37
°C in 384-well plates in LB/2x
carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham
Phaxmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following
parameters: Step 1:
94°C, 3 min; Step 2: 94°C, 1S 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
wexe reaxnplified
using the same conditions as described above. Samples were diluted with 20%
dimethysulfoxide (1:2,
v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the
DYENAMIC
DIRECT kit (Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator
cycle
sequencing ready reaction kit (Applied Biosystems).
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In like manner, full length polynucleotide sequences are verified using the
above procedure or
are used to obtain S' 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 N0:33-64 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 ~CCi of [~
32P] adenosine
triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase
(DuPont NEN, Boston
MA). The labeled oligonucleotides are substantially purified using a SEPHADEX
G-25 superfine size
exclusion dextran bead column (Amersham Pharmacia Biotech). An aliquot
containing 10' counts per
minute of the labeled probe is used in a typical membrane-based 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
photolithography, piezoelectric printing (ink-jet printing, See, e.g.,
Baldeschweiler, supra.), mechanical
microspotting technologies, and derivatives thereof. The substrate in each of
the aforementioned
technologies should be uniform and solid with a non-porous surface (Schena
(1999), supra). Suggested
substrates include silicon, silica, glass slides, glass chips, and silicon
wafers. Alternatively, a procedure
analogous to a dot or slot blot may also be used to arrange and link elements
to the surface of a
substrate using thermal, UV, chemical, or mechanical bonding procedures. A
typical array may be
produced using available methods and machines well known to those of ordinary
skill in the art and may
contain any appropriate number of elements. (See, e.g., Schena, M. et al.
(1995) Science 270:467-470;
Shalom D, et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson
(1998) Nat. Biotechnol.
16:27-31.)
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Full length cDNAs, Expressed Sequence Tags (SSTs), 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, Iaser
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/~Q oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 units/pl RNase inhibitor, 500 ~M dATP, 500 ~M dGTP, 500 ~M
dTTP, 40 ~M
dCTP, 40 ~.~M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)+ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M 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
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 girl 5X SSC/0.2% SDS.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each
array element is
amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses
primers complementary to the vector sequences flanking the cDNA insert. Array
elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5
fig. Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia
Biotech).
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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 Carporation (VWR), West Chester PA), washed extensively in
distilled water, and
coated with 0.05 % aminopxopyl 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 Eal of the array
element DNA, at an average
concentration of 100 ng/Eil, 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) fox 30 minutes at 60°C
followed by washes in
0.2% SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 ~.il of sample mixture consisting of 0.2 ~g
each of Cy3 and
Cy5 labeled cDNA synthesis pxoducts in 5X SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65 ° C for 5 minutes and is aliquoted onto the
microarray surface and covered with
an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber
having a cavity just slightly
Iarger than a microscope slide. The chamber is kept at 100% humidity
internally by the addition of
140 ~l of 5X SSC in a corner of the chamber. The chamber containing the arrays
is incubated for
about 6.5 hours at 60° C. The arrays are washed for 10 min at
45° C in a first wash buffer (1X SSC,
0.1 % SDS), three times for 10 minutes each at 45 ° C in a second wash
buffer (0.1X SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores. Appropriate
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filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are S6S nm for Cy3 and 6S0 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
fiuorophore, are hybridized to a single array for the purpose of identifying
genes that are differentially
expressed, the calibration is done by labeling samples of the calibrating cDNA
with the two
fiuorophores and adding identical amounts of each to the hybridization
mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-83SH
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
1S computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping
emission spectra) between the fluorophores using each fluorophore's emission
spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each spot
is centered in each element of the grid. The fluorescence signal within each
element is then integrated
to obtain a numerical value corresponding to the average intensity of the
signal. The software used
for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
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 1S 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 S' 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
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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~raphica 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 Spodo~tera frugiperda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus. (See Engelhard, E.K. et
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-
txansferase (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 jaQonicum, enables the purification of fusion proteins
on immobilized
glutathione under conditions that maintain protein activity and antigenicity
(Amersham Pharmacia
Biotech). Following purification, the GST moiety can be proteolytically
cleaved from TRICH at
specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity purification
using commercially available monoclonal and polyclonal anti-FLAG antibodies
(Eastman Kodak). 6-
His, a stretch of six consecutive histidine residues, enables purification on
metal-chelate resins
(QIAGEN). Methods for protein expression and purification are discussed in
Ausubel (1995, su ra,
ch. 10 and 16). Purified TRICH obtained by these methods can be used directly
in the assays shown in
Examples XVI, XVII, and XVIII 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 CA),
both of which
81
CA 02415808 2003-O1-06
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contain the cytomegalovirus promoter. S-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 ~cg of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate the
apoptotic state of the cells and other cellular properties. FCM detects and
quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events include
changes in nuclear DNA content as measured by staining of DNA with propidium
iodide; changes in
cell size and granularity as measured by forward light scatter and 90 degree
side light scatter; down-
regulation of DNA synthesis as measured by decrease in bromodeoxyuridine
uptake; alterations in
expression of cell surface and intracellular proteins as measured by
reactivity with specific antibodies;
and alterations in plasma membrane composition as measured by the binding of
fluorescein-conjugated
Annexin V protein to the cell surface. Methods in flow cytometry are discussed
in Ormerod, M.G.
(1994) Flow Cytometry, Oxford, New York NY.
The influence of TRICH on gene expression can be assessed using highly
purified populations
of cells transfected with sequences encoding TRICH and either CD64 or CD64-
GFP. CD64 and
CD64-GFP are expressed on the surface of transfected cells and bind to
conserved regions of human
immunoglobulin G (IgG). Transfected cells are efficiently separated from
nontransfected cells using
magnetic beads coated with either human IgG or antibody against CD64 (DYNAL,
Lake Success NY).
mRNA can be purified from the cells using methods well known by those of skill
in the art. Expression
of mRNA encoding TRICH and other genes of interest can be analyzed by northern
analysis or
microarray techniques.
XIV. Production of TRICK 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 are well
described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
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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, ssupra.) 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~3~y proteins (Reimann, supra) or
proteins involved in TRICH
localization or clustering such as MAGUKs (Craven, su ra). TRICH, or
biologically active fragments
thereof, are labeled with l2sl Bolton-Hunter reagent. (See, e.g., Bolton A.E.
and W.M. Hunter (1973)
Biochem. J. 133:529-539.) Candidate molecules previously arrayed in the wells
of a multi-well plate
are incubated with the labeled TRICH, washed, and any wells with labeled TRICH
complex are
assayed. Data obtained using different concentrations of TRICH are used to
calculate values for the
number, affinity, and association of TRICH with the candidate molecules.
Alternatively, proteins that interact with TRICH axe isolated using the yeast
2-hybrid system
(Fields, S. and O. Song (1989) Nature 340:245-246). TRICH, or fragments
thereof, are expressed as
fusion proteins with the DNA binding domain of Gal4 or lexA, and potential
interacting proteins are
expressed as fusion proteins with an activation domain. Interactions between
the TRICH fusion protein
and the TRICH interacting proteins (fusion proteins with an activation domain)
reconstitute a
transactivation function that is observed by expression of a reporter gene.
Yeast 2-hybrid systems are
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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
S between the proteins encoded by two large libraries of genes (Nandabalan, K,
et al. (2000) U.S. Patent
No. 6,0S7,101).
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 eukaryotic expression vector encoding TRICH. Eukaryotic
expression vectors are
commercially available, and the techniques to introduce them into cells are
well known to those .
skilled in the art. A second plasmid which expresses any one of a number of
marker genes, such as 13-
galactosidase, is co-transformed into the cells to allow rapid identification
of those cells which have
taken up and expressed the foreign DNA. The cells are incubated for 48-72
hours after transformation
under conditions appropriate for the cell line to allow expression and
accumulation of TRICH and 13-
galactosidase.
Transformed cells expressing 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, andlor cells transformed with either
vector sequences alone or
13-galactosidase sequences alone, are used as controls and tested in parallel.
Cells expressing TRICH
will have higher anion or canon conductance relative to control cells. The
contribution of TRICH to
conductance can be confirmed by incubating the cells using antibodies specific
for TRICH. The
antibodies will bind to the extracellular side of TRICH, thereby blocking the
pore in the ion channel,
and the associated conductance.
Alternatively, ion channel activity of TRICH is measured as current flow
across a TRICH-
containing 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 O.S-S ng of
mRNA is injected into
mature stage IV oocytes. Injected oocytes are incubated at 18°C for 1-S
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
3S of the TRICH mediator, such as CAMP, cGMP, or Ca+2 (in the form of CaCI~,
where appropriate.
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Electrode resistance is set at 2-5 MSS 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 activities of TRICH-1, TRICH-2, and TRICH-10, are measured
as K
conductance, the activities of TRICH-6 and TRICH-9 are measured as K*
conductance in the presence
of membrane stretch or free fatty acids, the activities of TRICH-18, TRICH-25
and TRICH-31 are
measured as voltage-gated K+ conductance, TRICH-5 activity is measured as CT
conductance in the
presence of GABA, TRICH-11 activity is measured as canon conductance in the
presence of heat, and
the activity of TRICH-9, TRICH-28 is measured as Ca2+ 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 NaCl,
2.5 mM KCl, 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
KCl, 1mM CaCl2, 1mM MgCl2, 10 mM Hepes/Tris pH 7.5). Uptake of various
substrates (e.g.,
amino acids, sugars, drugs, ions, and neurotransmitters) is initiated by
adding labeled substrate (e.g.
radiolabeled with 3H, fluorescently labeled with rhodamine, etc.) to the
oocytes. After incubating for 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 pigment
precursors and related
molecules for TRICH-3, aminophospholipids for TRICH-4., fructose and glucose
for TRICH-7 and
TRICH-15, amino acids for TRICH-8, Nay and iodide for TRICH-12, Na+and H~for
TRICH-13 and
TRICH-21, Na+ and glucose for TRICH-16 and TRICH-19, and glucose for TRICH-23,
TRICH-26,
TRICH-29, TRICH-30, and TRICH-32.
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
recovelred 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
CA 02415808 2003-O1-06
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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 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, ox the Cl-
indicator MQAE (all
available from Molecular Probes) in combination with the FLIPR fluorimetric
plate reading system
(Molecular Devices). In a more generic version of this assay, changes in
membrane potential caused by
ionic flux across the plasma membrane are measured using oxonyl dyes such as
DiBAC4 (Molecular
Probes). DiBAC4 equilibrates between the extracellular solution and cellular
sites according to the
cellular membrane potential. The dye's fluorescence intensity is 20-fold
greater when bound to
hydrophobic intracellular sites, allowing detection of DiBAC4 entry into the
cell (Gonzalez, J.E. and
P.A. Negulescu (1998) Curr. Opin. Biotechnol. 9:624-631). Candidate agonists
or antagonists may be
selected from known ion channel agonists or antagonists, peptide libraries, or
combinatorial chemical
libraries.
Various modifications and variations of the described 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 fox 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.
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CA 02415808 2003-O1-06
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RAUMANN, Brigitte E.
THORNTON, Michael l
DING, Li
YUE, Henry
TANG, Y.Tom
HARLAND, Lee
BURFORD, Neil
GREENE, Barrie D.
SANJANWALA, Madhu S.
BAUGHN, Mariah R.
YAO, Monique G.
YANG, Junming
PATTERSON, Chandra
GANDHI, Ameena R.
HAFALIA, April J.A.
TRIBOULEY, Catherine M.
WALIA, Narinder K.
AU-YOUNG, Janice
WALSH, Roderick T.
RAMKUMAR, Jayala}ani
LU, Yan
LU, Dyung Aina M.
AZIMZAI, Yalda
LAL, Preeti
ELLIOTT, Vicki S.
NGUYEN, Danniel B.
XU, Yuming
SEILHAMER, Jeffrey J.
BOROWSKY, Mark L.
KHAN, Farrah A.
KEARNEY, Liam
THANGAVELU, Kavitha
DAs, Debopriya
POLICKY, Jennifer L.
<120> TRANSPORTERS AND ION CHANNELS
<130> PI-0149 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/216,547; 60/218,232; 60/220,112; 60/221,839
<151> 2000-07-07; 2000-07-14; 2000-07-21; 2000-07-28
<160> 64
<170> PERL Program
<210> 1
<211> 332
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3474673CD1
<400> 1
Met Tyr Arg Pro Arg Ala Arg Ala Ala Pro Glu Gly Arg Val Arg
1 5 10 15
Gly Cys Ala Val Pro Ser Thr Val Leu Leu Leu Leu A1a Tyr Leu
20 25 30
Ala Tyr Leu Ala Leu Gly Thr Gly Val Phe Trp Thr Leu Glu Gly
35 40 45
Arg Ala Ala Gln Asp Ser Ser Arg Ser Phe Gln Arg Asp Lys Trp
1/g~
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
50 55 60
Glu Leu Leu Gln Asn Phe Thr Cys Leu Asp Arg Pro Ala Leu Asp
65 70 75
Ser Leu Ile Arg Asp Val Val Gln A1a Tyr Lys Asn Gly Ala Ser
80 85 90
Leu Leu Ser Asn Thr Thr Ser Met Gly Arg Trp Glu Leu Val Gly
95 100 105
Ser Phe Phe Phe Ser Val Ser Thr Ile Thr Thr Ile Gly Tyr Gly
110 115 120
Asn Leu Ser Pro Asn Thr Met Ala Ala Arg Leu Phe Cys Ile Phe
125 130 135
Phe Ala Leu Val Gly I1e Pro Leu Asn Leu Val Val Leu Asn Arg
140 145 150
Leu Gly His Leu Met Gln Gln Gly Val Asn His Trp Ala Ser Arg
155 160 165
Leu Gly Gly Thr Trp Gln Asp Pro Asp Lys Ala Arg Trp Leu Ala
170 175 180
Gly Ser Gly Ala Leu Leu Ser Gly Leu Leu Leu Phe Leu Leu Leu
185 190 195
Pro Pro Leu Leu Phe Ser His Met G1u Gly Trp Ser Tyr Thr Glu
200 205 210
Gly Phe Tyr Phe A1a Phe Ile Thr Leu her Thr Val G1y Phe Gly
215 220 225
Asp Tyr Val Ile Gly Met Asn Pro Ser Gln Arg Tyr Pro Leu Trp
230 235 240
Tyr Lys Asn Met Val Ser Leu Trp Ile Leu Phe Gly Met A1a Trp
245 250 255
Leu Ala Leu Ile Ile Lys Leu Ile Leu Ser Gln Leu Glu Thr Pro
260 265 270
G1y Arg Val Cys Ser Cys Cys His His Ser Ser Lys Glu Asp Phe
275 280 285
Lys Ser Gln Ser Trp Arg Gln Gly Pro Asp Arg Glu Pro Glu Ser
290 295 300
His Ser Pro Gln Gln Gly Cys Tyr Pro Glu Gly Pro Met G1y Ile
305 310 315
Ile Gln His Leu Glu Pro Ser Ala His Ala Ala G1y Cys Gly Lys
320 325 330
Asp Ser
<210> 2
<211> .226
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4588877CD1
<400> 2
Met Val Glu Met Gly Trp Asp Trp Ala Asp Arg Lys Asp Met Arg
1 5 10 15
His Arg Leu Gln Ala Gly Asn Leu Glu Asn Thr Asp Gln Val Lys
20 25 30
Ser Pro Leu Leu Thr Gly Asp Ser Ser Gly Leu Pro Pro Ala Pro
35 40 45
Ser Ala Pro Thr His Gly Val Lys Ala Ser Gly Gly Leu Gly Thr
50 55 60
Ile Leu His Pro Gln Asp Pro Asp Lys Ala Arg Trp Leu Ala Gly
65 70 75
Ser Gly Ala Leu Leu Ser Gly Leu Leu Leu Phe Leu Leu Leu Pro
80 85 90
Pro Leu Leu Phe Ser His Met Glu Gly Trp Ser Tyr Thr Glu Gly
95 100 105
Phe Tyr Phe Ala Phe Ile Thr Leu Ser Thr Val Gly Phe Gly Asp
110 115 120
Tyr Val Ile G1y Met Asn Pro Ser Gln Arg Tyr Pro Leu Trp Tyr
2/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
125 130 135
Lys Asn Met Val Ser Leu Trp I1e Leu Phe Gly Met Ala Trp Leu
140 145 150
Ala Leu Ile Ile Lys Leu Ile Leu Ser Gln Leu G1u Thr Pro Gly
155 160 165
Arg Val Cys Ser Cys Cys His His Ser Ser Lys Glu Asp Phe Lys
170 175 180
Ser Gln Ser Trp Arg Gln Gly Pro Asp Arg Glu Pro Glu Ser His
185 190 195
Ser Pro Gln Gln Gly Cys Tyr Pro Glu Gly Pro Met Gly Ile Ile
200 205 210
Gln His Leu Glu Pro Ser Ala His Ala Ala GIy Cys Gly Lys Asp
215 220 225
Ser
<210> 3
<211> 646
<212 > PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Tncyte ID No: 7472214CD1
<400> 3
Met Ala Glu Lys Ala Leu Glu Ala Val G1y Cys Gly Leu Gly Pro
1 5 10 15
Gly Ala Val Ala Met Ala Val Thr Leu Glu Asp Gly Ala Glu Pro
20 25 30
Pro Val Leu Thr Thr His Leu Lys Lys Val Glu Asn His Ile Thr
35 40 45
Glu Ala Gln Arg Phe Ser His Leu Pro Lys Arg Ser A1a Val Asp
50 55 60
Ile Glu Phe Val Glu Leu Ser Tyr Ser Val Arg Glu Gly Pro Cys
65 70 75
Trp Arg Lys Arg Gly Tyr Lys Thr Leu Leu Lys Cys Leu Ser Gly
80 85 90
Lys Phe Cys Arg Arg Glu Leu Ile Gly I1e Met Gly Pro Ser Gly
95 100 105
A1a Gly Lys Ser Thr Phe Met Asn Ile Leu Ala Gly Tyr Arg Glu
110 115 120
Ser Gly Met Lys Gly Gln Ile Leu Val Asn Gly Arg Pro Arg Glu
125 13 0 13 5
Leu Arg Thr Phe Arg Lys Met Ser Cys Tyr Ile Met Gln Asp Asp
140 145 150
Met Leu Leu Pro His Leu Thr Val Leu Glu Ala Met Met Val Ser
155 160 165
Ala Asn Leu Asn Leu Thr Glu Asn Pro Asp Val Lys Asn Asp Leu
170 175 180
VaI Thr GIu Ile Leu Thr Ala Leu GIy Leu Met Ser Cys Ser His
185 190 195
Thr Arg Thr Ala Leu Leu Ser Gly Gly Gln Arg Lys Arg Leu Ala
200 205 210
Ile Ala Leu Glu Leu Val Asn Asn Pro Pro Val Met Phe Phe Asp
215 220 225
Glu Pro Thr Ser Gly Leu Asp Ser Ala Ser Cys Phe Gln Val Val
230 235 240
Ser Leu Met Lys Ser Leu Ala Gln Gly Gly Arg Thr Ile Ile Cys
245 250 255
Thr IIe His Gln Pro Ser Ala Lys Leu Phe Glu Met Phe Asp Lys
260 265 270
Leu Tyr Ile Leu Ser Gln Gly Gln Cys Ile Phe Lys Gly Val Val
275 280 285
Thr Asn Leu Ile Pro Tyr Leu Lys Gly Leu Gly Leu His Cys Pro
290 295 300
Thr Tyr His Asn Pro Ala Asp Phe Val Ile Glu Val Ala Ser Gly
3/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
305 310 315
Glu Tyr Gly Asp Leu Asn Pro Met Leu Phe Arg Ala Val Gln Asn
320 325 330
Gly Leu Cys Ala Met Ala Glu Lys Lys Ser Ser Pro Glu Lys Asn
335 340 345
Glu Val Pro Ala Pro Cys Pro Pro Cys Pro Pro Glu Val Asp Pro
350 355 360
Tle Glu Ser His Thr Phe Ala Thr Ser Thr Leu Thr Gln Phe Cys
365 370 375
Tle Leu Phe Lys Arg Thr Phe Leu Ser Tle Leu Arg Asp Thr Val
380 385 390
Leu Thr His Leu Arg Phe Met Ser His Val Val Ile Gly Val Leu
395 400 405
Ile Gly Leu Leu Tyr Leu His Ile Gly Asp Asp Ala Ser Lys Val
410 415 420
Phe Asn Asn Thr Gly Cys Leu Phe Phe Ser Met Leu Phe Leu Met
425 430 435
Phe Ala Ala Leu Met Pro Thr Val Leu Thr Val Pro Leu Glu Met
440 445 450
Ala Val Phe Met Arg Glu His Leu Asn Tyr Trp Tyr Ser Leu Lys
455 460 465
Ala Tyr Tyr Leu A1a Lys Thr Met Ala Asp Val Pro Phe Gln Val
470 475 480
Val Cys Pro Val Val Tyr Cys Ser Tle Val Tyr Trp Met Thr Gly
485 490 495
Gln Pro Ala Glu Thr Ser Arg Phe Leu Leu Phe Ser Ala Leu Ala
500 505 510
Thr Ala Thr Ala Leu Val Ala Gln Ser Leu Gly Leu Leu I1e Gly
515 520 525
Ala Ala Ser Asn Ser Leu Gln Val Ala Thr Phe Val Gly Pro Val
530 535 540
Thr Ala Ile Pro Val Leu Leu Phe Ser Gly Phe Phe Val Ser Phe
545 550 555
Lys Thr Ile Pro Thr Tyr Leu Gln Trp Ser Ser Tyr Leu Ser Tyr
560 565 570
Val Arg Tyr Gly Phe Glu Gly Val Ile Leu Thr Ile Tyr Gly Met
575 580 585
Glu Arg Gly Asp Leu Thr Cys Leu Glu Glu Arg Cys Pro Phe Arg
590 595 600
Glu Pro Gln Ser Ile Leu Arg Ala Leu Asp Val G1u Asp A1a Lys
605 610 615
Leu Tyr Met Asp Phe Leu Val Leu Gly Ile Phe Phe Leu Ala Leu
620 625 630
Arg Leu Leu Ala Tyr Leu Val Leu Arg Tyr Arg Val Lys Ser Glu
635 640 645
Arg
<210> 4
<211> 1190
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473053CD1
<400> 4
Met Ala Val Cys Ala Lys Lys Arg Pro Pro Glu Glu Glu Arg Arg
1 5 10 15
Ala Arg Ala Asn Asp Arg Glu Tyr Asn Glu Lys Phe Gln Tyr Ala
20 25 30
Ser, Asn Cys Ile Lys Thr Ser Lys Tyr Asn Ile Leu Thr Phe Leu
35 40 45
Pro Val Asn Leu Phe Glu Gln Phe Gln Glu Val Ala Asn Thr Tyr
50 55 60
Phe Leu Phe Leu Leu Ile Leu Gln Leu Ile Pro G1n Ile Ser Ser
4/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
65 70 75
Leu Ser Trp Phe Thr Thr Ile Val Pro Leu Val Leu Val Leu Thr
80 85 90
Ile Thr Ala Val Lys Asp Ala Thr Asp Asp Tyr Phe Arg His Lys
95 100 105
Ser Asp Asn Gln Val Asn Asn Arg Gln Ser Gln Val Leu Ile Asn
110 115 120
Gly Ile Leu Gln Gln Glu Gln Trp Met Asn Val Cys Val Gly Asp
125 130 135
Ile Ile Lys Leu Glu Asn Asn GIn Phe Val Ala Ala Asp Leu Leu
140 145 150
Leu Leu Ser Ser Ser Glu Pro His Gly Leu Cys Tyr Ile Glu Thr
155 l60 165
Ala Glu Leu Asp Gly Glu Thr Asn Met Lys Val Arg Gln Ala Ile
170 175 180
Pro Val Thr Ser Glu Leu Gly Asp Ile Ser Lys Leu Ala Lys Phe
185 190 195
Asp Gly Glu Val Ile Cys Glu Pro Pro Asn Asn Lys Leu Asp Lys
200 205 210
Phe Ser G1y Thr Leu Tyr Trp Lys Glu Asn Lys Phe Pro Leu Ser
215 220 225
Asn Gln Asn Met Leu Leu Arg Gly Cys Val Leu Arg Asn Thr Glu
230 235 240
Trp Cys Phe Gly Leu Val Ile Phe Ala Gly Pro Asp Thr Lys Leu
245 250 255
Met Gln Asn Ser Gly Arg Thr Lys Phe Lys Arg Thr Ser Ile Asp
260 265 270
Arg Leu Met Asn Thr Leu Val Leu Trp Ile Phe G1y Phe Leu Val
275 280 285
Cys Met G1y Val Ile Leu Ala Ile Gly Asn Ala Ile Trp Glu His
290 295 300
Glu Val Gly Met Arg Phe Gln Val Tyr Leu Pro Trp Asp G1u Ala
305 310 315
Val Asp Ser Ala Phe Phe Ser Gly Phe Leu Ser Phe Trp Ser Tyr
320 325 330
Ile Ile I1e Leu Asn Thr Val Val Pro Ile Ser Leu Tyr Val Ser
335 340 345
Val Glu Val Ile Arg Leu Gly His Ser Tyr Phe Tle Asn Trp Asp
350 355 360
Lys Lys Met Phe Cys Met Lys Lys Arg Thr Pro Ala Glu Ala Arg
365 370 375
Thr Thr Thr Leu Asn Glu G1u Leu Gly Gln Val Glu Tyr Ile Phe
380 385 390
Ser Asp Lys Thr Gly Thr Leu Thr Gln Asn Ile Met Val Phe Asn
395 400 405
Lys Cys Ser Ile Asn Gly His Ser Tyr Gly Asp Val Phe Asp Val
410 415 420
Leu Gly His Lys Ala Glu Leu Gly Glu Arg Pro Glu Pro Val Asp
425 430 435
Phe Ser Phe Asn Pro Leu Ala Asp Lys Lys Phe Leu Phe Trp Asp
440 445 450
Pro Ser Leu Leu Glu Ala Val Lys Ile Gly Asp Pro His Thr His
455 460 465
Glu Phe Phe Arg Leu Leu Ser Leu Cys His Thr Val Met Ser Glu
470 475 480
Glu Lys Asn Glu Gly Glu Leu Tyr Tyr Lys Ala Gln Ser Pro Asp
485 490 495
Glu Gly Ala Leu Val Thr A1a Ala Arg Asn Phe Gly Phe Va1 Phe
S00 505 510
Arg Ser Arg Thr Pro Lys Thr Ile Thr Val His Glu Met Gly Thr
515 520 525
Ala Ile Thr Tyr Gln Leu Leu A1a Ile Leu Asp Phe Asn Asn Ile
530 535 540
Arg Lys Arg Met Ser Val I1e Val Arg Asn Pro Glu Gly Lys Ile
545 550 555
Arg Leu Tyr Cys Lys Gly Ala Asp Thr Tle Leu Leu Asp Arg Leu
560 565 570
7
CA 02415808 2003-O1-06
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His His Ser Thr Gln Glu Leu Leu Asn Thr Thr Met Asp His Leu
575 580 585
Asn Glu Tyr Ala Gly Glu Gly Leu Arg Thr Leu Val Leu Ala Tyr
590 595 600
Lys Asp Leu Asp Glu Glu Tyr Tyr Glu Glu Trp Ala Glu Arg Arg
605 610 615
Leu Gln Ala Ser Leu Ala Gln Asp Ser Arg Glu Asp Arg Leu Ala
620 625 630
Ser Ile Tyr Glu G1u Val Glu Asn Asn Met Met Leu Leu Gly Ala
635 640 645
Thr Ala Ile Glu Asp Lys Leu Gln Gln Gly Val Pro Glu Thr Ile
650 65'5 660
Ala Leu Leu Thr Leu Ala Asn Ile Lys Ile Trp Val Leu Thr Gly
665 670 675
Asp Lys Gln Glu Thr AIa Val Asn Ile Gly Tyr Ser Cys Lys Met
680 685 690
Leu Thr Asp Asp Met Thr Glu Val Phe Ile Val Thr Gly His Thr
695 700 705
Val Leu Glu Val Arg Glu Glu Leu Arg Lys Ala Arg Glu Lys Met
710 715 720
Met Asp Ser Ser Arg Ser Val Gly Asn Gly Phe Thr Tyr Gln Asp
725 730 735
Lys Leu Ser Ser Ser Lys Leu Thr Ser Val Leu Glu Ala Val Ala
740 745 750
Gly GIu Tyr Ala Leu Val Ile Asn Gly His Ser Leu Ala His Ala
755 760 765
Leu Glu Ala Asp Met Glu Leu Glu Phe Leu Glu Thr Ala Cys Ala
770 775 780
Cys Lys Ala Val Ile Cys,Cys Arg Val Thr Pro Leu Gln Lys Ala
785 790 795
Gln Val Val Glu Leu Val Lys Lys Tyr Lys Lys Ala Val Thr Leu
800 805 810
Ala Ile G1y Asp Gly Ala Asn Asp Val Ser Met Ile Lys Thr Ala
815 820 825
His Ile Gly Val Gly Ile Ser Gly Gln Glu Gly Ile Gln Ala Val
830 835 840
Leu Ala Ser Asp Tyr Ser Phe Ser Gln Phe Lys Phe Leu Gln Arg
845 850 855
Leu Leu Leu Val His Gly Arg Trp Ser Tyr Leu Arg Met Cys Lys
860 865 870
Phe Leu Cys Tyr Phe Phe Tyr Lys Asn Phe Ala Phe Thr Met Val
875 880 885
His Phe Trp Phe Gly Phe Phe Cys Gly Phe Ser Ala Gln Thr Val
890 895 900
Tyr Asp Gln Tyr Phe Ile Thr Leu Tyr Asn Tle Val Tyr Thr Ser
905 910 915
Leu Pro Val Leu Ala Met Gly Val Phe Asp Gln Asp Val Pro Glu
92 0 925 93 0
Gln Arg Ser Met Glu Tyr Pro Lys Leu Tyr Glu Pro Gly Gln Leu
935 940 945
Asn Leu Leu Phe Asn Lys Arg Glu Phe Phe Ile Cys Ile Ala Gln
950 955 960
Gly Ile Tyr Thr Ser Val Leu Met Phe Phe Ile Pro Tyr Gly Val
965 970 975
Phe Ala Asp Ala Thr Arg Asp Asp Gly Thr Gln Leu Ala Asp Tyr
980 985 990
Gln Ser Phe Ala Val Thr Val Ala Thr Ser Leu Val Ile Val Val
995 1000 1005
Ser Val Gln Ile Gly Leu Asp Thr Gly Tyr Trp Thr Ala Ile Asn
1010 1015 1020
His Phe Phe Ile Trp Gly Ser Leu Ala Val Tyr Phe Ala Ile Leu
1025 1030 1035
Phe AIa Met His Ser Asn Gly Leu Phe Asp Met Phe Pro Asn Gln
1040 1045 1050
Phe Arg Phe Val Gly Asn Ala Gln Asn Thr Leu Ala Gln Pro Thr
1055 1060 1065
Val Trp Leu Thr Ile Va1 Leu Thr Thr Val Val Cys Ile Met Pro
6/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
1070 1075 1080
Val Va1 Ala Phe Arg Phe Leu Arg Leu Asn Leu Lys Pro Asp Leu
1085 1090 1095
Ser Asp Thr Val Arg Tyr Thr Gln Leu Val Arg Lys Lys Gln Lys
1100 1105 1110
Ala Gln His Arg Cys Met Arg Arg Val Gly Arg Thr Gly Ser Arg
1115 1120 1125
Arg Ser Gly Tyr Ala Phe Ser His Gln Glu Gly Phe Gly Glu Leu
1130 1135 1240
Ile Met Ser Gly Lys Asn Met Arg Leu Ser Ser Leu Ala Leu Ser
1145 1150 1155
Ser Phe Thr Thr Arg Ser Ser Ser Ser Trp Ile Glu Ser Leu Arg
1160 1165 1170
Arg Lys Lys Ser Asp Ser AIa Ser Ser Pro Ser Gly Gly Ala Asp
1175 1180 1185
Lys Pro Leu Lys Gly
1190
<210> 5
<211> 467
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473347CD1
<400> 5
Met Val Leu Ala Phe G1n Leu Val Ser Phe Thr Tyr Ile Trp Ile
l 5 10 15
Ile Leu Lys Pro Asn Val Cys Ala Ala Ser Asn Ile Lys Met Thr
20 25 30
His Gln Arg Cys Ser Ser Ser Met Lys Gln Thr Cys Lys Gln Glu
35 40 45
Thr Arg Met Lys Lys Asp Asp Ser Thr Lys Ala Arg Pro Gln Lys
50 55 60
Tyr Glu Gln Leu Leu His Ile Glu Asp Asn Asp Phe A1a Met Arg
65 70 75
Pro Gly Phe Gly Gly Ser Pro Val Pro Val Gly Ile Asp Val His
80 85 90
Val Glu Ser Ile Asp Ser Ile Ser G1u Thr Asn Met Asp Phe Thr
95 100 105
Met Thr Phe Tyr Leu Arg His Tyr Trp Lys Asp Glu Arg Leu Ser
110 115 120
Phe Pro Ser Thr Ala Asn Lys Ser Met Thr Phe Asp His Arg Leu
125 13 0 13 5
Thr Arg Lys Ile Trp Val Pro Asp Ile Phe Phe Val His Ser Lys
140 145 150
Arg Ser Phe Ile His Asp Thr Thr Met Glu Asn Ile Met Leu Arg
155 160 165
Val His Pro Asp Gly Asn Val Leu Leu Ser Leu Arg Ile Thr Val
170 175 180
Ser Ala Met Cys Phe Met Asp Phe Ser Arg Phe Pro Leu Asp Thr
185 190 195
Gln Asn Cys Ser Leu Glu Leu Glu Ser Tyr Ala Tyr Asn Glu Asp
200 205 210
Asp Leu Met Leu Tyr Trp Lys His Gly Asn Lys Ser Leu Asn Thr
215 220 225
G1u Glu His Met Ser Leu Ser Gln Phe Phe Ile Glu Asp Phe Ser
230 ~ 235 240
Ala Ser Ser Gly Leu Ala Phe Tyr Ser Ser Thr Gly Trp Tyr Asn
245 250 255
Arg Leu Phe Ile Ile Ser Val Leu Arg Arg His Val Phe Phe Phe
260 265 270
Val Leu Pro Thr Tyr Tyr Pro Ala Ile Leu Met Val Met Leu Ser
275 280 285
Trp Va1 Ser Phe Trp Ile Asp Arg Arg Ala Val Pro Ala Arg Val
7/$7
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
290 295 300
Ser Leu Gly Ile Thr Thr Val Leu Thr Met Ser Thr Ile Ile Thr
305 310 315
Ala Val Ser Ala Ser Met Pro Gln Val Ser Tyr Leu Lys Ala Val
320 325 330
Asp Val Tyr Leu Trp Val Ser Ser Leu Phe Val Phe Leu Ser Val
335 340 345
Ile Glu Tyr Ala Ala Val Asn Tyr Leu Thr Thr Val Glu Glu Arg
350 355 360
Lys Gln Phe Lys Lys Thr Gly Lys Ile Ser Arg Met Tyr Asn Ile
365 370 375
Asp Ala Va1 Gln A1a Met Ala Phe Asp Gly Cys Tyr His Asp Ser
380 385 390
Glu Ile Asp Met Asp Gln Thr Ser Leu Ser Leu Asn Ser GIu Asp
395 400 405
Phe Met Arg Arg Lys Ser I1e Cys Ser Pro Ser Thr Asp Ser Ser
410 415 420
Arg Ile Lys Arg Arg Lys Ser Leu Gly G1y His Val Gly Arg Ile
425 430 435
Tle Leu Glu Asn Asn His Val Ile Asp Thr Tyr Ser Arg Ile Leu
440 445 450
Phe Pro Tle Val Tyr Ile Leu Phe Asn Leu Phe Tyr Trp Gly Val
455 460 465
Tyr Val
<210> 6
<211> 1196
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7474240CD1
<400> 6
Met Pro Val Arg Arg Gly His VaI Ala Pro Gln Asn Thr Phe Leu
1 5 10 15
Gly Thr Ile Ile Arg Lys Phe G1u Gly Gln Asn Lys Lys Phe Ile
20 25 30
Ile Ala Asn Ala Arg Va1 Gln Asn Cys Ala Ile Ile Tyr Cys Asn
35 40 45
Asp Gly Phe Cys Glu Met Thr Gly Phe Ser Arg Pro Asp Val Met
50 55 60
Gln Lys Pro Cys Thr Cys Asp Phe Leu His Gly Pro Glu Thr Lys
65 70 75
Arg His Asp Ile Ala Gln Ile Ala Gln Ala Leu Leu Gly Ser G1u
80 85 90
Glu Arg Lys Va1 Glu Val Thr Tyr Tyr His Lys Asn Gly Ser Thr
95 100 105
Phe Ile Cys Asn Thr His Ile Tle Pro Val Lys Asn Gln Glu Gly
110 115 120
Val Ala Met Met Phe Ile Ile Asn Phe Glu Tyr Va1 Thr Asp Asn
125 130 135
Glu Asn Ala Ala Thr Pro G1u Arg Val Asn Pro Ile Leu Pro Ile
140 145 150
Lys Thr Val Asn Arg Lys Phe Phe Gly Phe Lys Phe Pro Gly Leu
155 160 165
Arg Val Leu Thr Tyr Arg Lys Gln Ser Leu Pro Gln Glu Asp Pro
170 175 180
Asp Val Val Val Ile Asp Ser Ser Lys His Ser Asp Asp Ser Val
185 190 195
Ala Met Lys His Phe Lys Ser Pro Thr Lys Glu Ser Cys Ser Pro
200 205 210
Ser Glu Ala Asp Asp Thr Lys Ala Leu Ile Gln Pro Ser Lys Cys
215 220 225
Ser Pro Leu Val Asn Ile Ser Gly Pro Leu Asp His Ser Ser Pro
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
230 235 240
Lys Arg Gln Trp Asp Arg Leu Tyr Pro Asp Met Leu Gln Ser Ser
245 250 255
Ser Gln Leu Ser His Ser Arg Ser Arg Glu Ser Leu Cys Ser Ile
260 265 270
Arg Arg Ala Ser Ser Val His Asp Ile Glu Gly Phe Gly Val His
275 280 285
Pro Lys Asn Ile Phe Arg Asp Arg His Ala Ser Glu Asp Asn G1y
290 295 300
Arg Asn Val Lys Gly Pro Phe Asn His Ile Lys Ser Ser Leu Leu
305 310 315
Gly Ser Thr Ser Asp Ser Asn Leu Asn Lys Tyr Ser Thr Ile Asn
320 325 330
Lys Ile Pro Gln Leu Thr Leu Asn Phe Ser Glu Val Lys Thr Glu
335 340 345
Lys Lys Asn Ser Ser Pro Pro Ser Ser Asp Lys Thr Ile Ile Ala
350 355 360
Pro Lys Val Lys Asp Arg Thr His Asn Val Thr Glu Lys Val Thr
365 370 375
Gln Val Leu Ser Leu Gly A1a Asp Val Leu Pro Glu Tyr Lys Leu
380 385 390
Gln Thr Pro Arg Ile Asn Lys Phe Thr Ile Leu His Tyr Ser Pro
395 400 405
Phe Lys Ala Val Trp Asp Trp Leu Ile Leu Leu Leu Val Ile Tyr
410 415 420
Thr Ala Ile Phe Thr Pro Tyr Ser Ala Ala Phe Leu Leu Asn Asp
425 430 435
Arg Glu Glu Gln Lys Arg Arg Glu Cys Gly Tyr Ser Cys Ser Pro
440 445 450
Leu Asn Val Val Asp Leu Ile Val Asp Ile Met Phe Ile Ile Asp
455 460 465
Ile Leu Ile Asn Phe Arg Thr Thr Tyr Val Asn Gln Asn Glu Glu
470 475 480
Val Val Ser Asp Pro Ala Lys Ile Ala Ile His Tyr Phe Lys Gly
485 490 495
Trp Phe Leu Ile Asp Met Val Ala Ala Ile Pro Phe Asp Leu Leu
500 505 510
Ile Phe Gly Ser G1y Ser Asp Glu Thr Thr Thr Leu Ile Gly Leu
515 520 525
Leu Lys Thr Ala Arg Leu Leu Arg Leu Val Arg Val Ala Arg Lys
530 535 540
Leu Asp Arg Tyr Ser Glu Tyr Gly Ala Ala Val Leu Met Leu Leu
545 550 555
Met Cys Ile Phe Ala Leu Ile Ala His Trp Leu Ala Cys Ile Trp
560 565 570
Tyr Ala Ile Gly Asn Val Glu Arg Pro Tyr Leu Thr Asp Lys I1e
575 580 585
Gly Trp Leu Asp Ser Leu Gly Gln Gln Ile Gly Lys Arg Tyr Asn
590 595 600
Asp Ser Asp ser Ser Ser Gly Pro Ser Ile Lys Asp Lys Tyr Val
605 610 615
Thr Ala Leu Tyr Phe Thr Phe Ser Ser Leu Thr Ser Val Gly Phe
620 625 630
Gly Asn Val Ser Pro Asn Thr Asn Ser Glu Lys Ile Phe Ser Ile
635 640 645
Cys Val Met Leu Ile Gly Ser Leu Met Tyr Ala Ser Ile Phe Gly
650 655 660
Asn Val Ser Ala Ile Ile Gln Arg Leu Tyr Ser Gly Thr Ala Arg
665 670 675
Tyr His Met Gln Met Leu Arg Val Lys Glu Phe Ile Arg Phe His
680 685 690
Gln Ile Pro Asn Pro Leu Arg Gln Arg Leu Glu Glu Tyr Phe Gln
695 700 705
His Ala Trp Thr Tyr Thr Asn Gly Ile Asp Met Asn Met Val Leu
710 715 720
Lys Gly Phe Pro Glu Cys Leu Gln Ala Asp Ile Cys Leu His Leu
725 730 735
9187
CA 02415808 2003-O1-06
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Asn G1n Thr Leu Leu Gln Asn Cys Lys Ala Phe Arg Gly Ala Ser
740 745 750
Lys Gly Cys Leu Arg Ala Leu Ala Met Lys Phe Lys Thr Thr His
755 760 765
Ala Pro Pro Gly Asp Thr Leu Val His Cys Gly Asp Val Leu Thr
770 775 780
Ala Leu Tyr Phe Leu Ser Arg Gly Ser Ile G1u Ile Leu Lys Asp
785 790 795
Asp Ile Val Val Ala Ile Leu Gly Lys Asn Asp Ile Phe Gly Glu
800 805 810
Met Val His Leu Tyr A1a Lys Pro Gly Lys Ser Asn Ala Asp Val
815 820 825
Arg Ala Leu Thr Tyr Cys Asp Leu His Lys Ile Gln Arg Glu Asp
830 835 840
Leu Leu Glu Val Leu Asp Met Tyr Pro Glu Phe Ser Asp His Phe
845 850 855
Leu Thr Asn Leu Glu Leu Thr Phe Asn Leu Arg His G1u Ser Ala
860 865 870
Lys Ala Asp Leu Leu Arg Ser Gln Ser Met Asn Asp Ser Glu Gly
875 880 885
Asp Asn Cys Lys Leu Arg Arg Arg Lys Leu Ser Phe Glu Ser Glu
890 895 900
Gly Glu Lys Glu Asn Ser Thr Asn Asp Pro Glu Asp Ser Ala Asp
905 910 915
Thr Ile Arg His Tyr Gln Ser Ser Lys Arg His Phe Glu Glu Lys
920 925 930
Lys Ser Arg Ser Ser Ser Phe Ile Ser Ser I1e Asp Asp Glu Gln
935 940 945
Lys Pro Leu Phe Ser Gly Ile Val Asp Ser Ser Pro Gly Ile Gly
950 955 960
Lys A1a Ser Gly Leu Asp Phe Glu Glu Thr Va1 Pro Thr Ser Gly
9&5 970 975
Arg Met His Ile Asp Lys Arg Ser His Ser Cys Lys Asp Ile Thr
980 985 990
Asp Met Arg Ser Trp G1u Arg Glu Asn Ala His Pro Gln Pro Glu
995 1000 1005
Asp Ser Ser Pro Ser Ala Leu Gln Arg Ala Ala Trp Gly Tle Ser
1010 1015 1020
G1u Thr Glu Ser Asp Leu Thr Tyr Gly Glu Val Glu Gln Arg Leu
2025 1030 1035
Asp Leu Leu G1n Glu Gln Leu Asn Arg Leu Glu Ser Gln Met Thr
1040 1045 1050
Thr Asp Ile Gln Thr Ile Leu Gln Leu Leu Gln Lys Gln Thr Thr
1055 1060 1065
Val Val Pro Pro Ala Tyr Ser Met Val Thr Ala Gly Ser Glu Tyr
1070 1075 2080
Gln Arg Pro Ile Ile Gln Leu Met Arg Thr Ser Gln Pro Glu Ala
1085 1090 1095
Ser Ile Lys Thr Asp Arg Ser Phe Ser Pro Ser Ser Gln Cys Pro
1100 1105 1110
Glu Phe Leu Asp Leu Glu Lys Ser Lys Leu Lys Ser Lys Glu Ser
1115 1120 1125
Leu Ser Ser Gly Val His Leu Asn Thr Ala Ser Glu Asp Asn Leu
1130 1135 1140
Thr Ser Leu Leu Lys Gln Asp Ser Asp Leu Ser Leu Glu Leu His
1145 1150 1155
Leu Arg Gln Arg Lys Thr Tyr Va1 His Pro Ile Arg His Pro Ser
1160 1165 1170
Leu Pro Asp Ser Ser Leu Ser Thr Val Gly Ile Val Gly Leu His
1275 1180 1185
Arg His Val Ser Asp Pro Gly Leu Pro Gly Lys
1190 1195
<210> 7
<211> 512
<212> PRT
<213> Homo sapiens
10/7
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
<220>
<221> misc_feature
<223> Incyte TD No: 7475338CD1
<400> 7
Met G1u Asn Lys Glu Ala G1y Thr Pro Pro Pro I1e Pro Ser Arg
1 5 20 25
Glu Gly Arg Leu Gln Pro Thr Leu Leu Leu Ala Thr Leu Ser Ala
20 25 30
Ala Phe Gly Ser Ala Phe Gln Tyr G1y Tyr Asn Leu Ser Val Val
35 40 45
Asn Thr Pro His Lys Val Phe Lys Ser Phe Tyr Asn Glu Thr Tyr
50 55 60
Phe Glu Arg His Ala Thr Phe Met Asp Gly Lys Leu Met Leu Leu
65 70 75
Leu Trp Ser Cys Thr Val Ser Met Phe Pro Leu Gly Gly Leu Leu
80 85 90
Gly Ser Leu Leu Val Gly Leu Leu Val Asp Ser Cys Gly Arg Lys
95 100 105
Gly Thr Leu Leu Tle Asn Asn Ile Phe Ala Ile Ile Pro Ala Ile
110 115 120
Leu Met Gly Val Ser Lys Val Ala Lys Ala Phe Glu Leu Ile Val
125 130 135
Phe Ser Arg Val Va1 Leu Gly Val Cys Ala Gly Tle Ser Tyr Ser
140 145 150
Ala Leu Pro Met Tyr Leu Gly Glu Leu Ala Pro Lys Asn Leu Arg
155 160 165
Gly Met Val Gly Thr Met Thr Glu Val Phe Val Ile Val Gly Val
170 175 180
Phe Leu Ala Gln Ile Phe Ser Leu Gln Ala Ile Leu Gly Asn Pro
185 190 195
Ala Gly Trp Pro Val Leu Leu Ala Leu Thr G1y Val Pro Ala Leu
200 205 210
Leu Gln Leu Leu Thr Leu Pro Phe Phe Pro Glu Ser Pro Arg Tyr
215 220 225
Ser Leu Ile Gln Lys Gly Asp Glu Ala Thr Ala Arg'GIn Ala Leu
230 235 240
Arg Arg Leu Arg Gly His Thr Asp Met Glu Ala Glu Leu Glu Asp
245 250 255
Met Arg Ala Glu Ala Arg Ala Glu Arg Ala Glu Gly His Leu Ser
260 265 270
Val Leu His Leu Cys Ala Leu Arg Ser Leu Arg Trp Gln Leu Leu
275 280 285
Ser Ile Ile Val Leu Met Ala Gly Gln Gln Leu Ser Gly I1e Asn
290 295 300
Ala Ile Asn Tyr Tyr Ala Asp Thr Ile Tyr Thr Ser Ala Gly Val
305 310 315
Glu Ala A1a His Ser Gln Tyr Val Thr Val Gly Ser Gly Val Val
320 325 330
Asn Ile Val Met Thr Ile Thr Ser Ala Val Leu Val Glu Arg Leu
335 340 345
Gly Arg Arg His Leu Leu Leu Ala Gly Tyr Gly Ile Cys Gly Ser
350 355 360
Ala Cys Leu Val Leu Thr Val Val Leu Leu Phe G1n Asn Arg Val
365 370 375
Pro Glu Leu Ser Tyr Leu Gly Ile Ile Cys Val Phe Ala Tyr Ile
380 385 390
Ala Gly His Ser Ile Gly Pro Ser Pro Val Pro Ser Val Val Arg
395 400 405
Thr Glu Ile Phe Leu Gln Ser Ser Arg Arg Ala Ala Phe Met Val
410 415 420
Asp Gly Ala Val His Trp Leu Thr Asn Phe Ile Ile Gly Phe Leu
425 430 435
Phe Pro Ser Ile Gln Glu Ala Ile Gly Ala Tyr Ser Phe Ile Ile
440 445 450
Phe Ala Gly Ile Cys Leu Leu Thr Ala Ile Tyr Ile Tyr Val Val
455 460 465
11/87
CA 02415808 2003-O1-06
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Ile Pro Glu Thr Lys Gly Lys Thr Phe Va1 Glu Ile Asn Arg Ile
470 475 480
Phe Ala Lys Arg Asn Arg Val Lys Leu Pro Glu Glu Lys Glu Glu
485 490 495
Thr Ile Asp Ala Gly Pro Pro Thr Ala Ser Pro Ala Lys Glu Thr
500 505 510
Ser Phe
<210> 8
<211> 568
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7476747CD1
<400> 8
Met Thr Ala Ser Thr Pro Glu Ala Thr Pro Asn Met Glu Leu Lys
1 5 l0 15
Ala Pro Ala Ala Gly Gly Leu Asn Ala Gly Pro Val Pro Pro Ala
20 25 30
Ala Met Ser Thr Gln Arg Leu Arg Asn Glu Asp Tyr His Asp Tyr
35 40 45
Ser Ser Thr Asp Val Ser Pro Glu Glu Ser Pro Ser Glu Gly Leu
50 55 60
Asn Asn Leu Ser Ser Pro Gly Ser Tyr Gln Arg Phe Gly Gln Ser
65 70 75
Asn Ser Thr Thr Trp Phe Gln Thr Leu Ile His Leu Leu Lys Gly
80 85 90
Asn Ile Gly Thr Gly Leu Leu Gly Leu Pro Leu Ala Val Lys Asn
95 100 105
Ala Gly Ile Val Met Gly Pro Ile Ser Leu Leu Ile I1e Gly I1e
110 1l5 120
VaI Ala Val His Cys Met Gly Ile Leu Val Lys Cys Ala His His
125 130 135
Phe Cys Arg Arg Leu Asn Lys Ser Phe Val Asp Tyr Gly Asp Thr
140 145 150
Val Met Tyr G1y Leu Glu Ser Ser Pro Cys Ser Trp Leu Arg Asn
155 160 165
His Ala His Trp Gly Arg Arg Val Val Asp Phe Phe Leu Ile Val
170 175 180
Thr Gln Leu Gly Phe Cys Cys Va1 Tyr Phe Val Phe Leu Ala Asp
185 190 195
Asn Phe Lys Gln Val Ile Glu Ala Ala Asn Gly Thr Thr Asn Asn
200 205 210
Cys His Asn Asn GIu Thr Val Ile Leu Thr Pro Thr Met Asp Ser
215 220 225
Arg Leu Tyr Met Leu Ser Phe Leu Pro Phe Leu Val Leu Leu Val
230 235 240
Phe Ile Arg Asn Leu Arg Ala Leu Ser Ile Phe Ser Leu Leu Ala
245 250 255
Asn Ile Thr Met Leu Val Ser Leu Val Met Ile Tyr Gln Phe Ile
260 265 270
Val Gln Arg Ile Pro Asp Pro Ser His Leu Pro Leu Val Ala Pro
275 280 285
Trp Lys Thr Tyr Pro Leu Phe Phe Gly Thr Ala Ile Phe Ser Phe
290 295 300
Glu G1y Ile Gly Met Val Leu Pro Leu Glu Asn Lys Met Lys Asp
305 310 315
Pro Arg Lys Phe Pro Leu Ile Leu Tyr Leu Gly Met Val Ile Val
320 325 330
Thr Ile Leu Tyr Ile Ser Leu Gly Cys Leu Gly Tyr Leu Gln Phe
335 340 345
Gly Ala Asn Ile Gln Gly Ser Ile Thr Leu Asn Leu Pro Asn Cys
350 355 360
12l $7
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
Trp Leu Tyr Gln Ser Val Lys Leu Leu Tyr Ser Ile Gly Ile Phe
365 370 375
Phe Thr Tyr Ala Leu Gln Phe Tyr Val Pro Ala Glu Tle Tle Ile
380 385 390
Pro Phe Phe Val Ser Arg Ala Pro Glu Pro Cys Glu Leu Val Val
395 400 405
Asp Leu Phe Val Arg Pro Val Leu Val Cys Leu Thr Ser Leu Ser
410 415 420
Gly Ser Val Asp Asn Gly Trp Tyr Gly Thr Glu Ala Asp Gly Thr
425 430 435
Ser Cys Gly Ser Ala Pro Leu Val Phe Val Ser Ser Ser Phe Leu
440 445 450
Ala His Pro Trp Leu Ser Phe Arg Cys Glu Ser Gln Trp Val Ser
455 460 465
Cys His Arg Asp Thr Val Val Val Trp Gly Phe Ala Arg Gly Ile
470 475 480
Leu Ala Ile Leu Ile Pro Arg Leu Asp Leu Val Ile Ser Leu Val
485 490 495
Gly Ser Val Ser Ser Ser Ala Leu Ala Leu Tle I1e Pro Pro Leu
500 505 510
Leu Glu Val Thr Thr Phe Tyr Ser Glu Gly Met Ser Pro Leu Thr
515 520 525
Ile Phe Lys Asp Ala Leu I1e Ser Ile Leu Gly Phe Val Gly Phe
530 535 540
Val Val Gly Thr Tyr Glu Ala Leu Tyr Glu Leu Ile Gln Pro Ser
545 550 555
Asn Ala Pro Ile Phe Ile Asn Ser Thr Cys Ala Phe Ile
560 565
<210> 9
<211> 958
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7477898CD1
<400> 9
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
I1e Ala Asn Ala Gln Met Glu Asn Cys Ala Ile Ile Tyr Cys Asn
35 40 45
Asp Gly Phe Cys Glu Leu Phe Gly Tyr Ser Arg Val Glu Val Met
50 55 60
Gln Gln Pro Cys Thr Cys Asp Phe Leu Thr Gly Pro Asn Thr Pro
65 70 75
Ser Ser Ala Va1 Ser Arg Leu Ala Gln Ala Leu Leu Gly Ala Glu
80 85 90
Glu Cys Lys Va1 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
110 225 120
Ala Val Ile Met Phe Ile Leu Asn Phe Glu Asp Leu Ala Gln Leu
12 5 13 0 13 5
Leu Ala Lys Cys Ser Ser Arg Ser Leu Ser Gln Arg Leu Leu Ser
140 145 150
Gln Ser Phe Leu Gly Ser Glu Gly Ser His Gly Arg Pro Gly Gly
155 160 165
Pro Gly Pro Gly Thr Gly Arg Gly Lys Tyr Arg Thr Ile Ser Gln
170 175 180
Ile Pro Gln Phe Thr Leu Asn Phe Val Glu Phe Asn Leu Glu Lys
285 190 195
His Arg Ser Ser Ser Thr Thr Glu Ile Glu Tle Ile Ala Pro His
200 205 210
13/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
Lys Val Val Glu Arg Thr G1n 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 A1a 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 I1e
305 310 315
Val Ile Asn Phe Arg Thr Thr Tyr Val Asn Thr Asn Asp Glu Va1
320 325 330
Val Ser His Pro Arg Arg Ile Ala Val His Tyr Phe Lys Gly Trp
335 340 345
Phe Leu Ile Asp Met Val Ala Ala Ile Pro Phe Asp Leu Leu Ile
350 355 360
Phe Arg Thr Gly Ser Asp Glu Thr Thr Thr Leu Ile Gly Leu Leu
365 370 375
Lys Thr Ala Arg Leu Leu Arg Leu Val Arg Val Ala Arg Lys Leu
380 385 390
Asp Arg Tyr Ser Glu Tyr Gly Ala Ala Val Leu Phe Leu Leu Met
395 400 405
Cys Thr Phe Pro Leu Ile Ala His Trp Leu Ala Cys Ile Trp Tyr
410 415 420
A1a Ile Gly Asn Val Glu Arg Pro Tyr Leu Glu His Lys Ile Gly
425 430 435
Trp Leu Asp Ser Leu Gly Val Gln Leu Gly Lys Arg Tyr Asn Gly
440 445 450
Ser Asp Pro Ala Ser Gly Pro Ser Val Gln Asp Lys Tyr Val Thr
455 460 465
Ala Leu Tyr Phe Thr Phe Ser Ser Leu Thr Ser Val Gly Phe Gly
470 475 480
Asn Va1 Ser Pro Asn Thr Asn Ser Glu Lys Val Phe Ser Ile Cys
485 490 495
Va1 Met Leu Ile Gly Ser Leu Met Tyr Ala Ser Ile Phe Gly Asn
500 505 510
Val Ser Ala Ile Ile Gln Arg Leu Tyr Ser G1y Thr Ala Arg Tyr
515 520 525
His Thr G1n Met Leu Arg Val Lys Glu Phe Ile Arg Phe His Gln
530 535 540
Ile Pro Asn Pro Leu Arg Gln Arg Leu G1u Glu Tyr Phe Gln His
545 550 555
Ala Trp Ser Tyr Thr Asn Gly Ile Asp Met Asn Ala Val Leu Lys
560 565 570
Gly Phe Pro Glu Cys Leu Gln Ala Asp Ile Cys Leu His Leu His
575 580 585
Arg Ala Leu Leu Gln His Cys Pro Ala Phe Ser Gly Ala Gly Lys
590 595 600
Gly Cys Leu Arg Ala Leu Ala Val Lys Phe Lys Thr Thr His Ala
605 620 615
Pro Pro Gly Asp Thr Leu Val His Leu Gly Asp Val Leu Ser Thr
620 625 630
Leu Tyr Phe Ile Ser Arg Gly Ser Ile Glu Ile Leu Arg Asp Asp
635 640 645
Va1 Val Val Ala Ile Leu Gly Lys Asn Asp Ile Phe Gly Glu Pro
650 655 660
Val Ser Leu His Ala Gln Pro Gly Lys Ser Ser Ala Asp Val Arg
665 670 675
Ala Leu Thr Tyr Cys Asp Leu His Lys Ile Gln Arg AIa Asp Leu
680 685 690
Leu Glu Val Leu Asp Met Tyr Pro Ala Phe Ala Glu Ser Phe Trp
695 700 705
Ser Lys Leu Glu Val Thr Phe Asn Leu Arg Asp Val Thr Gly Gly
14/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
710 715 720
Leu His Ser Ser Pro Arg Gln Ala Pro Gly Ser Gln Asp His Gln
725f 730 735
Gly Phe Phe Leu Ser Asp Asn Gln Ser Asp Ala A1a Pro Pro Leu
740 745 750
Ser Ile Ser Asp Ala Phe Trp Leu Trp Pro Glu Leu Leu Gln Glu
755 760 765
Met Pro Pro Lys His Ser Pro Gln Ser Pro Gln Glu Asp Pro Asp
770 775 780
Cys Trp Pro Leu Lys Leu Gly Ser Arg Leu Glu Gln Leu Gln Ala
785 790 795
Gln Met Asn Arg Leu Glu Ser Arg Val Ser Ser Asp Leu Ser Arg
800 805 810
Ile Leu Gln Leu Leu Gln Lys Pro Met Pro Gln Gly His Ala Ser
815 820 825
Tyr Tle Leu Glu Ala Pro A1a Ser Asn Asp Leu Ala Leu Val Pro
830 835 840
Ile Ala Ser Glu Thr Thr Ser Pro Gly Pro Arg Leu Pro Gln G1y
845 850 855
Phe Leu Pro Pro Ala Gln Thr Pro Ser Tyr Gly Asp Leu Asp Asp
860 865 870
Cys Ser Pro Lys His Arg Asn Ser Ser Pro Arg Met Pro His Leu
875 880 885
Ala Val Ala Met Asp Lys Thr Leu Ala Pro Ser Ser Glu Gln G1u
890 895 900
Gln Pro Glu Gly Leu Trp Pro Pro Leu Ala Ser Pro Leu His Pro
905 910 915
Leu Glu Val Gln Gly Leu Ile Cys Gly Pro Cys Phe Ser Ser Leu
920 925 930
Pro Glu His Leu Gly Ser Val Pro Lys Gln Leu Asp Phe Gln Arg
935 940 945
His Gly Ser Asp Pro Gly Phe Ala Gly Ser Trp Gly His
950 955
<210> 10
<211> 724
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_teature
<223> Incyte ID No: 7472728CD1
<400> 10
Met Gly His Gln Gly Pro Phe Glu Glu Gly Asn Gly Gly Leu Arg
1 5 10 15
Val Ile Ala Thr Trp Arg Arg Lys Glu A1a Trp Arg Arg Asp Cys
20 25 30
Leu Leu Gly Ala Leu Pro Ser Val Ser Cys Gly Gly Trp Gly His
35 40 45
Arg Gly Arg Gln Thr Tyr Gly Arg Ala Cys Gly Val Lys Glu Lys
50 55 60
Pro Phe Ser Leu Leu Gly Pro G1n Ile Thr Val Tyr Ala Val Trp
65 70 75
Pro Gln Ser Glu Gly Pro Gln Glu Gly Arg Leu Arg Val Asn Ser
80 85 90
Ala Cys Leu Pro Pro Glu Arg Gly Leu Thr Asn Ala Cys Thr Asn
95 100 105
His Glu Glu Leu Ser Leu Asp Cys Leu Leu Phe Glu Asn Val Asn
110 115 120
Thr Leu Thr Leu Asp Phe Cys Leu Trp G1u Lys Thr Thr Ile Val
22 5 13 0 13 5
Pro Gly Val Leu Pro Tyr Ala Gly Leu Thr Leu Gln Ser Lys Phe
140 145 150
Leu Leu Gly Arg Ala Leu Leu Ala Gly Val His Val Ile Thr Leu
155 160 165
Thr Pro GIu Arg Val Thr His His Val His Gly Trp Tyr Met Glu
15/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
170 175 180
Asp Gly Phe Lys Gly Asp Arg Thr Glu Gly Cys Arg Ser Asp Ser
185 190 195
Val Ala Val Pro Ala Ala Ala Pro Val Cys Gln Pro Lys Ser Ala
200 205 210
Thr Asn Gly Gln Pro Pro Ala Pro Ala Pro Thr Pro Thr Pro Arg
215 220 225
Leu Ser Ile Ser Ser Arg Ala Thr Val Val Ala Arg Met G1u Gly
230 235 240
Thr Ser Gln Gly Gly Leu Gln Thr Val Met Lys Trp Lys Thr Val
245 250 255
Val Ala Ile Phe Val Va1 Val Val Val Tyr Leu Val Thr G1y Gly
260 265 270
Leu Val Phe Arg Ala Leu Glu Gln Pro Phe Glu Ser Ser Gln Lys
275 280 285
Asn Thr Ile Ala Leu Glu Lys A1a Glu Phe Leu Arg Asp His Val
290 295 300
Cys Val Ser Pro G1n Glu Leu Glu Thr Leu Ile Gln His Ala Leu
305 310 315
Asp Ala Asp Asn Ala Gly Val Ser Pro Ile Gly Asn Ser Ser Asn
320 325 330
Asn Ser Ser His Trp Asp Leu Gly Ser Ala Phe Phe Phe Ala Gly
335 340 345
Thr Val Ile Thr Thr Met Tyr Gly Asn Ile Ala Pro Ser Thr Glu
350 355 360
Gly Gly Lys Ile Phe Cys Ile Leu Tyr Ala Ile Phe Gly Ile Pro
365 370 375
Leu Phe Gly Phe Leu Leu Ala Gly Ile Gly Asp Gln Leu Gly Thr
380 385 390
Ile Phe Gly Lys Ser Ile Ala Arg Val Glu Lys Val Phe Arg Lys
395 400 405
Lys Gln Val Ser Gln Thr Lys Ile Arg Val Ile Ser Thr Ile Leu
410 415 420
Phe Ile Leu Ala Gly Cys Ile Val Phe Val Thr Ile Pro Ala Val
425 430 435
Ile Phe Lys Tyr IIe Glu G1y Trp Thr AIa Leu Glu Ser Ile Tyr
440 445 450
Phe Val Val Val Thr Leu Thr Thr Val Gly Phe Gly Asp Phe Val
455 460 465
AIa Val Val Val Phe Arg Gly Asn Ala Gly Tle Asn Tyr Arg G1u
470 475 480
Trp Tyr Lys Pro Leu Va1 Trp Phe Trp Ile Leu Val Gly Leu Ala
485 490 495
Tyr Phe Ala Ala Val Leu Ser Met Ile Gly Asp Trp Leu Arg Val
500 505 510
Leu Ser Lys Lys Thr Lys Glu Glu Val Gly G1u Ile Lys Ala His
515 520 525
Ala Ala Glu Trp Lys Ala Asn Val Thr Ala Glu Phe Arg Glu Thr
530 535 540
Arg Arg Arg Leu Ser Val Glu Ile His Asp Lys Leu Gln Arg Ala
545 550 555
Ala Thr Ile Arg Ser Met Glu Arg Arg Arg Leu G1y Leu Asp Gln
560 565 570
Arg Ala His Ser Leu Asp Met Leu Ser Pro Glu Lys Arg Ser Val
575 580 585
Phe Ala Ala Leu Asp Thr Gly Arg Phe Lys Ala Ser Ser Gln Glu
590 595 600
Ser Ile Asn Asn Arg Pro Asn Asn Leu Arg Leu Lys Gly Pro Glu
605 610 615
Gln Leu Asn Lys His Gly Gln Gly Ala Ser Glu Asp Asn Ile Tle
620 625 630
Asn~Lys Phe Gly Ser Thr Ser Arg Leu Thr Lys Arg Lys Asn Lys
635 640 645
Asp Leu Lys Lys Thr Leu Pro Glu Asp Val Gln Lys Ile Tyr Lys
650 655 660
Thr Phe Arg Asn Tyr Ser Leu Asp Glu Glu Lys Lys Glu Glu Glu
665 670 675
16/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
Thr Glu Lys Met Cys Asn Ser Asp Asn Ser Ser Thr Ala Met Leu
680 685 690
Thr Asp Cys Ile Gln Gln His A1a Glu Leu Glu Asn Gly Met Ile
695 700 705
Pro Thr Asp Thr Lys Asp Arg Glu Pro Glu Asn Asn Ser Leu Leu
710 715 720
Glu Asp Arg Asn
<210> 11
<211> 470
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7474322CD1
<220>
<221> unsure
<222> 253
<223> unknown or other
<400> 11
Met Tyr Asn Glu Ile Leu Met Leu Gly Ala Lys Leu His Pro Thr
1 5 10 15
Leu Lys Leu Glu Glu Leu Thr Asn Lys Lys Gly Met Thr Pro Leu
20 25 30
Ala Leu Ala Ala Gly Thr Gly Lys Ile Gly Asn Arg His Asp Met
35 40 45
Leu Leu Val Glu Pro Leu Asn Arg Leu Leu Gln Asp Lys Trp Asp
50 55 60
Arg Phe Val Lys Arg Ile Phe Tyr Phe Asn Phe Leu Val Tyr Cys
65 70 75
Leu Tyr Met Ile Ile Phe Thr Met Ala Ala Tyr Tyr Arg Pro Val
80 85 90
Asp Gly Leu Pro Pro Phe Lys Met G1u Lys Thr Gly Asp Tyr Phe
95 100 105
Arg Val Thr Gly Glu Ile Leu Ser Val Leu Gly Gly Val Tyr Phe
110 115 120
Phe Phe Arg Gly Ile Gln Tyr Phe Leu Gln Arg Arg Pro Ser Met
125 130 135
Lys Thr Leu Phe Val Asp Ser Tyr Ser Glu Met Leu Leu Phe Leu
140 145 150
Gln Ser Leu Phe Met Leu Ala Thr Val Val Leu Tyr Phe Ser His
155 160 165
Leu Lys GIu Tyr Val Ala Ser Met Val Phe Ser Leu Ala Leu Gly
170 175 180
Trp Thr Asn Met Leu Tyr Tyr Thr Arg Gly Phe Gln Gln Met Gly
185 190 195
Ile Tyr Ala Val Met Ile Glu Lys Met Ile Leu Arg Asp Leu Cys
200 205 210
Arg Phe Met Phe Val Tyr Ile Val Phe Leu Phe Gly Phe Ser Thr
215 220 225
Ala Val Val Thr Leu Ile Glu Asp Gly Lys Asn Asp Ser Leu Pro
230 235 240
Ser Glu Ser Thr Ser His Arg Trp Arg Gly Pro AIa Xaa Arg Pro
245 250 255
Asn Ser Ser Tyr Asn Ser Leu Tyr Ser Thr Cys Leu Glu Leu Phe
260 265 270
Lys Phe Thr Ile Gly Met Gly Asp Leu Glu Phe Thr Glu Asn Tyr
275 280 285
Asp Phe Lys A1a Val Phe IIe Ile Leu Leu Leu Ala Tyr'Val Ile
290 295 300
Leu Thr Tyr Ile Val Leu Leu Leu Asn Met Leu Ile Ala Leu Met
305 310 315
Gly Glu Thr Val Glu Asn Val Ser Lys Glu Ser Glu Arg Ile Trp
17/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
320 325 330
Arg Leu Gln Arg Ala Ile Thr Ile Leu Asp Thr Glu Lys Ser Phe
335 340 345
Leu Lys Cys Met Arg Lys Ala Phe Arg Ser Gly Lys Leu Leu Gln
350 355 360
Val Gly Tyr Thr Pro Asp Gly Lys Asp Asp Tyr Arg Trp Cys Phe
365 370 375
Val Asp Glu Val Asn Trp Thr Thr Trp Asn Thr Asn Val G1y Ile
380 385 390
Ile Asn Glu Asp Pro Gly Asn Cys Glu Gly Val Lys Arg Thr Leu
395 400 405
Ser Phe Ser Leu Arg Ser Ser Arg Val Ser Gly Arg His Trp Lys
410 415 420
Asn Phe Ala Leu Val Pro Leu Leu Arg Glu Ala Ser Ala Arg Asp
425 430 435
Arg Gln Ser Ala Gln Pro Glu Glu Val Tyr Leu Arg Gln Phe Ser
440 445 450
Gly Ser Leu Lys Pro Glu Asp Ala Glu Val Phe Lys Ser Pro Ala
455 460 465
Ala Ser Gly Glu Lys
470
<210> 12
<211> 618
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5455621CD1
<400> 12
Met Glu Va1 Lys Asn Phe Ala Val Trp Asp Tyr Val Val Phe Ala
1 5 10 15
Ala Leu Phe°Phe Ile Ser Ser G1y Ile Gly Val Phe Phe Ala Ile
20 25 30
Lys Glu Arg Lys Lys Ala Thr Ser Arg Glu Phe Leu Va1 G1y Gly
35 40 45
Arg Gln Met Ser Phe Gly Pro Val Gly Leu Ser Leu Thr Ala Ser
50 55 60
Phe Met Ser Ala Val Thr Val Leu Gly Thr Pro Ser Glu Val Tyr
65 70 75
Arg Phe Gly Ala Ser Phe Leu Val Phe Phe Ile Ala Tyr Leu Phe
80 85 90
Val Ile Leu Leu Thr Ser Glu Leu Phe Leu Pro Val Phe Tyr Arg
95 100 105
Ser Gly Ile Thr Ser Thr Tyr Glu Tyr Leu Gln Leu Arg Phe Asn
110 115 120
Lys Pro Val Arg Tyr Ala Ala Thr Val I1e Tyr Ile Va1 Gln Thr
125 130 135
Ile Leu Tyr Thr Gly Val Val Val Tyr Ala Pro Ala Leu Ala Leu
140 145 150
Asn Gln Val Thr Gly Phe Asp Leu Trp Gly Ser Val Phe Ala Thr
155 160 165
Gly Ile Val Cys Thr Phe Tyr Cys Thr Leu Gly Gly Leu Lys Ala
170 175 180
Val Val Trp Thr Asp Ala Phe Gln Met Val Val Met Ile Val Gly
185 190 195
Phe Leu Thr Val Leu Ile Gln Gly Ser Thr His Ala Gly Gly Phe
200 205 210
His Asn Val Leu G1u Gln Ser Thr Asn Gly Ser Arg Leu His Ile
215 220 225
Phe Asp Phe Asp Val Asp Pro Leu Arg Arg His Thr Phe Trp Thr
230 235 240
Ile Thr Val Gly Gly Thr Phe Thr Trp Leu Gly Ile Tyr Gly Val
245 250 255
Asn Gln Ser Thr Ile Gln Arg Cys Ile Ser Cys Lys Thr Glu Lys
18/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
260 265 270
His Ala Lys Leu Ala Leu Tyr Phe Asn Leu Leu Gly Leu Trp I1e
275 280 285
Ile Leu Val Cys Ala Va1 Phe Ser Gly Leu Ile Met Tyr Ser His
290 295 300
Phe Lys Asp Cys Asp Pro Trp Thr Ser Gly Ile Ile Ser Ala Pro
305 310 315
Asp Gln Leu Met Pro Tyr Phe Val Met Glu Ile Phe Ala Thr Met
320 325 330
Pro Gly Leu Pro Gly Leu Phe Val Ala Cys Ala Phe Ser Gly Thr
335 340 345
Leu Ser Thr Val Ala Ser Ser Ile Asn Ala Leu Ala Thr Val Thr
350 355 360
Phe Glu Asp Phe Val Lys Ser Cys Phe Pro His Leu Ser Asp Lys
365 370 375
Leu Ser Thr Trp IIe Ser Lys Gly Leu Cys Leu Leu Phe G1y Val
380 385 390
Met Cys Thr Ser Met Ala Val Ala Ala Ser Val Met Gly Gly Val
395 400 405
Val Gln Ala Ser Leu Ser Ile His Gly Met Cys Gly Gly Pro Met
410 415 420
Leu Gly Leu Phe Ser Leu Gly Ile Val Phe Pro Phe Val Asn Trp
425 430 435
Lys Gly Ala Leu Gly Gly Leu Leu Thr G1y Ile Thr Leu Ser Phe
440 445 450
Trp Val Ala Ile G1y Ala Phe Ile Tyr Pro Ala Pro Ala Ser Lys
455 460 465
Thr Trp Pro Leu Pro Leu Ser Thr Asp Gln Cys Ile Lys Ser Asn
470 475 480
Val Thr Ala Thr Gly Pro Pro Val Leu Ser Ser Arg Pro Gly I1e
485 490 495
Ala Asp Thr Trp Tyr Ser Ile Ser Tyr Leu Tyr Tyr Ser Ala Leu
500 505 510
G1y Cys Leu Gly Cys Ile Val A1a Gly Val Ile Ile Ser Leu Ile
515 520 525
Thr Gly Arg Gln Arg Gly Glu Asp Ile Gln Pro Leu Leu Ile Arg
530 535 540
Pro Val Cys Asn Leu Phe Cys Phe Trp Ser Lys Lys Tyr Lys Thr
545 550 555
Leu Cys Trp Cys Gly Va1 Gln His Asp Ser Gly Thr Glu Gln Glu
560 565 570
Asn Leu Glu Asn Gly Ser Ala Arg Lys Gln Gly Ala Glu Ser Val
575 580 585
Leu G1n Asn Gly Leu Arg Arg Glu Ser Leu Val His Val Pro G1y
590 595 600
Tyr Asp Pro Lys Asp Lys Ser Tyr Asn Asn Met Ala Phe Glu Thr
605 610 615
Thr His Phe
<210> 13
<211> 631
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7477248CD1
<400> l3
Met Glu Arg Gln Ser Arg Val Met Ser G1u Lys Asp Glu Tyr Gln
1 5 10 15
Phe Gln His Gln Gly Ala Val Glu Leu Leu Val Phe Asn Phe Leu
20 25 30
Leu Ile Leu Thr Ile Leu Thr Tle Trp Leu Phe Lys Asn His Arg
35 40 45
Phe Arg Phe Leu His Glu Thr Gly Gly Ala Met Val Tyr Gly Leu
1917
CA 02415808 2003-O1-06
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50 55 60
Ile Met Gly Leu Ile Leu Arg Tyr Ala Thr Ala Pro Thr Asp Ile
65 70 75
Glu Ser Gly Thr Val Tyr Asp Cys Val Lys Leu Thr Phe Ser Pro
80 85 90
Ser Thr Leu Leu Val Asn Ile Thr Asp Gln Va1 Tyr Glu Tyr Lys
95 100 105
Tyr Lys Arg Glu Ile Ser Gln His Asn Ile Asn Pro His Gln Gly
210 115 120
Asn Ala Ile Leu Glu Lys Met Thr Phe Asp Pro Glu Ile Phe Phe
125 130 135
Asn Val Leu Leu Pro Pro Ile Tle Phe His Ala Gly Tyr Ser Leu
140 145 . 150
Lys Lys Arg His Phe Phe Gln Asn Leu Gly Ser Ile Leu Thr Tyr
155 160 165
Ala Phe Leu Gly Thr Ala Ile Ser Cys Ile Val Ile Gly Leu Ile
170 175 180
Met Tyr Gly Phe Val Lys Ala Met Ile His Ala Gly Gln Leu Lys
185 190 195
Asn Gly Asp Phe His Phe Thr Asp Cys Leu Phe Phe Gly Ser Leu
200 205 210
Met Ser Ala Thr Asp Pro Va1 Thr Val Leu Ala Ile Phe His Glu
215 220 225
Leu His Val Asp Pro Asp Leu Tyr Thr Leu Leu Phe Gly Glu Ser
230 235 240
Val Leu Asn Asp Ala Val Ala Ile Val Leu Thr Tyr Ser Ile Ser
245 250 255
Ile Tyr Ser Pro Lys Glu Asn Pro Asn Ala Phe Asp Ala Ala Ala
260 265 270
Phe Phe Gln Ser Val Gly Asn Phe Leu Gly Ile Phe Ala Gly Ser
275 280 285
Phe Ala Met Gly Ser Ala Tyr Ala Ile Ile Thr Ala Leu Leu Thr
290 295 300
Lys Phe Thr Lys Leu Cys Glu Phe Pro Met Leu Glu Thr Gly Leu
305 310 315
Phe Phe Leu Leu Ser Trp Ser Ala Phe Leu Ser Ala Glu Ala Ala
320 325 330
Gly Leu Thr Gly Ile Val Ala Val Leu Phe Cys Gly Val Thr Gln
335 340 345
Ala His Tyr Thr Tyr Asn Asn Leu Ser Ser Asp Ser Lys Tle Arg
350 355 360
Thr Lys Gln Leu Phe Glu Phe Met Asn Phe Leu Ala Glu Asn Val
365 370 375
Ile Phe Cys Tyr Met Gly Leu Ala Leu Phe Thr Phe Gln Asn His
380 385 390
Ile Phe Asn Ala Leu Phe Ile Leu Gly Ala Phe Leu Ala Ile Phe
395 400 405
Val Ala Arg Ala Cys Asn Ile Tyr Pro Leu Ser Phe~Leu Leu Asn
410 415 420
Leu Gly Arg Lys Gln Lys Ile Pro Trp Asn Phe Gln His Met Met
425 430 435
Met Phe Ser Gly Leu Arg Gly Ala Ile Ala Phe Ala Leu Ala Ile
440 445 450
Arg Asn Thr Glu Ser Gln Pro Lys Gln Met Met Phe Thr Thr Thr
455 460 465
Leu Leu Leu Val Phe Phe Thr Val Trp Val Phe Gly Gly Gly Thr
470 475 480
Thr Pro Met Leu Thr Trp Leu Gln Ile Arg Val Gly Val Asp Leu
485 490 495
Asp Glu Asn Leu Lys Glu Asp Pro Ser Ser GIn His Gln Glu Ala
500 505 520
Asn Asn Leu Asp Lys Asn Met Thr Lys Ala Glu Ser Ala Arg Leu
515 520 525
Phe Arg Met Trp Tyr Ser Phe Asp His Lys Tyr Leu Lys Pro Ile
530 535 540
Leu Thr His Ser Gly Pro Pro Leu Thr Thr Thr Leu Pro Glu Trp
545 550 555
20/87
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Cys Gly Pro Ile Ser Arg Leu Leu Thr Ser Pro Gln Ala Tyr Gly
560 565 570
Glu Gln Leu Lys Glu Asp Asp Val Glu Cys Ile Val Asn Gln Asp
575 580 585
Glu Leu Ala Ile Asn Tyr Gln Glu Gln Ala Ser Ser Pro Cys Ser
590 595 600
Pro Pro AIa Arg Leu GIy Leu Asp Gln Lys Ala Ser Pro Gln Thr
605 610 615
Pro Gly Lys Glu Asn Ile Tyr Glu Gly Asp Leu Gly Pro Gly Arg
620 625 630
Leu
<210> 14
<211> 1256
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2944004CD1
<400> 14
Met Asp Arg Glu Glu Arg Lys Thr Ile Asn Gln Gly Gln Glu Asp
1 5 10 25
Glu Met G1u Ile Tyr Gly Tyr Asn Leu Ser Arg Trp Lys Leu Ala
20 25 30
Ile Val Ser Leu Gly Val Ile Cys Ser Gly Gly Val Ser Pro Pro
35 40 45
Pro Leu Tyr Trp Met Pro Glu Trp Arg Val Lys Ala Thr Cys Val
50 55 60
Arg Ala Ala IIe Lys Asp Cys Glu VaI Val Leu Leu Arg Thr Thr
65 70 75
Asp Glu Phe Lys Met Trp Phe Cys Ala Lys Ile Arg Val Leu Ser
80 85 90
Leu Glu Thr Tyr Pro Va1 Ser Ser Pro Lys Ser Met Ser Asn Lys
95 100 105
Leu Ser Asn Gly His Ala Val Cys Leu Ile Glu Asn Pro Thr Glu
110 115 120
Glu Asn Arg His Arg Ile Ser Lys Tyr Ser Gln Thr Glu Ser GIn
125 13 0 135
Gln Ile Arg Tyr Phe Thr His His Ser Val Lys Tyr Phe Trp Asn
140 145 150
Asp Thr Ile His Asn Phe Asp Phe Leu Lys Gly Leu Asp Glu Gly
155 160 165
Val Ser Cys Thr Ser Ile Tyr Glu Lys His Ser Ala Gly Leu Thr
170 275 280
Lys Gly Met His Ala Tyr Arg Lys Leu Leu Tyr Gly Val Asn Glu
185 190 195
Ile Ala Val Lys Val Pro Ser Val Phe Lys Leu Leu Ile Lys Glu
200 205 210
Val Leu Asn Pro Phe Tyr Ile Phe G1n Leu Phe Ser Val Ile Leu
215 220 225
Trp Ser Thr Asp Glu Tyr Tyr Tyr Tyr Ala Leu Ala Ile Val Val
230 235 240
Met Ser Ile Val Ser Ile Val Ser Ser Leu Tyr Ser Ile Arg Lys
245 250 255
Gln Tyr Val Met Leu His Asp Met Val Ala Thr His Ser Thr Val
260 265 270
Arg Val Ser Val Cys Arg Val Asn Glu Glu Ile G1u Glu Ile Phe
275 280 285
Ser Thr Asp Leu Va1 Pro Gly Asp Val Met Val Ile Pro Leu Asn
290 295 300
Gly Thr Ile Met Pro Cys Asp Ala Val Leu Ile Asn Gly Thr Cys
305 310 315
Ile Val Asn Glu Ser Met Leu Thr Gly Glu Ser Val Pro Val Thr
320 325 330
21/87
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Lys Thr Asn Leu Pro Asn Pro Ser Val Asp Val Lys Gly Ile Gly
335 340 345
Asp Glu Leu Tyr Asn Pro Glu Thr His Lys Arg His Thr Leu Phe
350 355 360
Cys Gly Thr Thr Val Tle Gln Thr Arg Phe Tyr Thr Gly Glu Leu
365 370 375
Val Lys Ala Ile Val Val Arg Thr Gly Phe Ser Thr Ser Lys Gly
380 385 390
Gln Leu Val Arg Ser Ile Leu Tyr Pro Lys Pro Thr Asp Phe Lys
395 400 405
Leu Tyr Arg Asp Ala Tyr Leu Phe Leu Leu Cys Leu Val Ala Val
410 415 420
Ala Gly Ile Gly Phe Ile Tyr Thr Ile Ile Asn Ser I1e Leu Asn
425 430 435
Glu Val Gln Val Gly Val Ile Ile Ile Glu Ser Leu Asp Ile Ile
440 445 450
Thr Ile Thr Val Pro Pro Ala Leu Pro AIa Ala Met Thr Ala G1y
455 460 465
Ile VaI Tyr Ala Gln Arg Arg Leu Lys Lys I1e Gly Ile Phe Cys
470 475 480
I1e Ser Pro Gln Arg Ile Asn Ile Cys Gly Gln Leu Asn Leu Val
485 490 495
Cys Phe Asp Lys Thr Gly Thr Leu Thr Glu Asp Gly Leu Asp Leu
500 505 510
Trp Gly Ile Gln Arg Val G1u Asn Ala Arg Phe Leu Ser Pro Glu
515 520 525
Glu Asn Val Cys Asn Glu Met Leu Val Lys Ser Gln Phe Val Ala
530 535 540
Cys Met Ala Thr Cys His Ser Leu Thr Lys Ile Glu Gly Val Leu
545 550 555
Ser Gly Asp Pro Leu Asp Leu Lys Met Phe Glu Ala Ile Gly Trp
560 565 570
Ile Leu Glu Glu Ala Thr Glu Glu Glu Thr Ala Leu His Asn Arg
575 580 585
Ile Met Pro Thr Val Val Arg Pro Pro Lys Gln Leu Leu Pro Glu
590 595 600
Ser Thr Pro Ala Gly Asn Gln Glu Met Glu Leu Phe G1u Leu Pro
605 610 615
Ala Thr Tyr Glu Ile GIy Ile Val Arg Gln Phe Pro Phe Ser Ser
620 625 630
Ala Leu GIn Arg Met Ser Val Val Ala Arg Val Leu Gly Asp Arg
635 640 645
Lys Met Asp A1a Tyr Met Lys Gly Ala Pro Glu Ala Ile A1a GIy
650 655 660
Leu Cys Lys Pro G1u Thr Val Pro Val Asp Phe Gln Asn Val Leu
665 670 675
Glu Asp Phe Thr Lys Gln Gly Phe Arg Val Ile Ala Leu Ala His
680 685 690
Arg Lys Leu G1u Ser Lys Leu Thr Trp His Lys Val GIn Asn Ile
695 700 705
Ser Arg Asp A1a Ile Glu Asn Asn Met Asp Phe Met Gly Leu Ile
710 715 720
Ile Met Gln Asn Lys Leu Lys Gln Lys Thr Pro Ala Va1 Leu Glu
725 730 735
Asp Leu His Lys Ala Asn Ile Arg Thr VaI Met Val Thr Gly Asp
740 745 750
Ser Met Leu Thr Ala Val Ser Val Ala Arg Asp Cys GIy Met Ile
755 760 765
Leu Pro Gln Asp Lys Va1 Ile Ile Ala Glu Ala Leu Pro Pro Lys
770 775 780
Asp Gly Lys Val Ala Lys Ile Asn Trp His Tyr Ala Asp Ser Leu
785 790 795
Thr Gln Cys Ser His Pro Ser Ala IIe Asp Pro Glu Ala Ile Pro
800 805 810
Val Lys Leu Val His Asp Ser Leu Glu Asp Leu Gln Met Thr Arg
815 820 825
Tyr His Phe Ala Met Asn Gly Lys Ser Phe Ser VaI Ile Leu GIu
22/87
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830 835 840
His Phe G1n Asp Leu Val Pro Lys Leu Met Leu His Gly Thr Val
845 850 855
Phe Ala Arg Met Ala Pro Asp Gln Lys Thr Gln Leu Ile Glu Ala
860 865 870
Leu Gln Asn Val Asp Tyr Phe Val Gly Met Cys Gly Asp Gly Ala
875 880 885
Asn Asp Cys Gly Ala Leu Lys Arg Ala His Gly Gly I1e Ser Leu
890 895 900
Ser Glu Leu Glu Ala Ser Val Ala Ser Pro Phe Thr Ser Lys Thr
905 910 915
Pro Ser Ile Ser Cys Val Pro Asn Leu Ile Arg G1u Gly Arg Ala
920 925 930
Ala Leu Ile Thr Ser Phe Cys Val Phe Lys Phe Met Ala Leu Tyr
935 940 945
Ser Ile I1e Gln Tyr Phe Ser Val Thr Leu Leu Tyr Ser Ile Leu
950 955 960
Ser Asn Leu Gly Asp Phe Gln Phe Leu Phe Ile Asp Leu Ala Ile
965 ~ 970 975
Ile Leu Val Val Val Phe Thr Met Ser Leu Asn Pro Ala Trp Lys
980 985 990
Glu Leu Val Ala Gln Arg Pro Pro Ser G1y Leu Ile Ser Gly Ala
995 1000 1005
Leu Leu Phe Ser Val Leu Ser Gln Ile Ile Ile Cys Ile Gly Phe
1010 1015 1020
Gln Ser Leu Gly Phe Phe Trp Val Lys Gln Gln Pro Trp Tyr Glu
1025 1030 1035
Val Trp His Pro Lys Ser Asp Ala Cys Asn Thr Thr Gly Ser Gly
1040 1045 1050
Phe Trp Asn Ser Ser His Val Asp Asn Glu Thr Glu Leu Asp Glu
1055 1060 1065
His Asn Ile Gln Asn Tyr Glu Asn Thr Thr Val Phe Phe Ile Ser
1070 1075 1080
Ser Phe G1n Tyr Leu Tle Val Ala Ile Ala Phe Ser Lys Gly Lys
1085 1090 1095
Pro Phe Arg Gln Pro Cys Tyr Lys Asn Tyr Phe Phe Val Phe Ser
1100 1105 1110
Val Ile Phe Leu Tyr Tle Phe Ile Leu Phe Ile Met Leu Tyr Pro
1115 1120 1125
Val Ala Ser Val Asp Gln Val Leu Gln Ile Val Cys Val Pro Tyr
1130 1135 1140
Gln Trp Arg Val Thr Met Leu Ile Ile Val Leu Val Asn Ala Phe
1145 1150 1155
Val Ser Ile Thr Val Glu Asn Phe Phe Leu Asp Met Val Leu Trp
1160 1165 1170
Lys Val Val Phe Asn Arg Asp Lys Gln Gly Glu Tyr Arg Phe Ser
1175 1180 1185
Thr Thr Gln Pro Pro Gln Glu Ser Val Asp Arg Trp Gly Lys Cys
- 1190 1195 1200
Cys Leu Pro Trp Ala Leu Gly Cys Arg Lys Lys Thr Pro Lys Ala
1205 1210 1215
Lys Tyr Met Tyr Leu Ala Gln Glu Leu Leu Val Asp Pro Glu Trp
1220 1225 1230
Pro Pro Lys Pro Gln Thr Thr Thr Glu Ala Lys Ala Leu Val Lys
1235 1240 1245
Glu Asn Gly Ser Cys Gln Ile Ile Thr Ile Thr
1250 1255
<210> 15
<211> 499
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3046849CD1
23/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
<400> 15
Met Leu His Ala Leu Leu Arg Ser Arg Thr Ile Gln Gly Arg Ile
1 5 10 - 15
Leu Leu Leu Thr Ile Cys Ala Ala Gly Ile Gly Gly Thr Phe Gln
20 25 30
Phe Gly Tyr Asn Leu Ser Ile Ile Asn Ala Pro Thr Leu His Ile
35 40 45
Gln Glu Phe Thr Asn Glu Thr Trp Gln Ala Arg Thr Gly Glu Pro
50 55 60
Leu Pro Asp His Leu Val Leu Leu Met Trp Ser Leu Ile Va1 Ser
65 70 75
Leu Tyr Pro Leu Gly G1y Leu Phe Gly Ala Leu Leu Ala Gly Pro
80 85 90
Leu Ala Ile Thr Leu Gly Arg Lys Lys Ser Leu Leu Val Asn Asn
95 100 105
Ile Phe Val Val Ser Ala Ala Ile Leu Phe Gly Phe Ser Arg Lys
110 115 120
Ala G1y Ser Phe Glu Met Ile Met Leu Gly Arg Leu Leu Val Gly
125 130 135
Val Asn Ala Gly Val Ser Met Asn Ile Gln Pro Met Tyr Leu Gly
140 145 150
G1u Ser Ala Pro Lys Glu Leu Arg Gly Ala Val Ala Met Ser Ser
155 160 165
Ala I1e Phe Thr Ala Leu Gly Ile Val Met Gly Gln Val Val G1y
170 175 180
Leu Arg Glu Leu Leu Gly Gly Pro Gln Ala Trp Pro Leu Leu Leu
185 190 195
Ala Ser Cys Leu Val Pro Gly Ala Leu Gln Leu Ala Ser Leu Pro
200 205 210
Leu Leu Pro Glu Ser Pro Arg Tyr Leu Leu Ile Asp Cys Gly Asp
215 220 225
Thr Glu Ala Cys Leu Ala Ala Leu Arg Gln Leu Arg Gly Ser Gly
230 235 240
Asp Leu A1a Gly Glu Leu Glu Glu Leu Glu Glu Glu Arg Ala Ala
245 250 255
Cys Gln Gly Cys Arg A1a Arg Arg Pro Trp Glu Leu Phe Gln His
260 265 270
Arg Ala Leu Arg Arg Gln Val Thr Ser Leu Val Val Leu Gly Ser
275 280 285
Ala Met Glu Leu Cys Gly Asn Asp Ser Val Tyr Ala Tyr Ala Ser
290 295 300
Ser Val Phe Arg Lys Ala Gly Va1 Pro Glu Ala Lys Ile Gln Tyr
305 310 315
Ala Ile Ile Gly Thr Gly Ser Cys Glu Leu Leu Thr Ala Val Val
320 325 330
Ser Cys Val Val Ile Glu Arg Val G1y Arg Arg Val Leu Leu Ile
335 340 345
Gly Gly Tyr Ser Leu Met Thr Cys Trp Gly Ser Ile Phe Thr Val
350 355 360
Ala Leu Cys Leu Gln Ser Ser Phe Pro Trp Thr Leu Tyr Leu Ala
365 370 375
Met Ala Cys Ile Phe Ala Phe Ile Leu Ser Phe Gly Ile Gly Pro
380 385 390
Ala Gly Val Thr Gly Ile Leu Ala Thr Glu Leu Phe Asp Gln Met
395 400 405
Ala Arg Pro Ala Ala Cys Met Val Cys Gly Ala Leu Met Trp Ile
410 415 420
Met Leu Ile Leu Val Gly Leu Gly Phe Pro Phe Ile Met Glu Ala
425 430 435
Leu Ser His Phe Leu Tyr Val Pro Phe Leu Gly Val Cys Val Cys
440 445 450
Gly Ala Ile Tyr Thr Gly Leu Phe Leu Pro Glu Thr Lys Gly Lys
455 460 465
Thr Phe Gln Glu Ile Ser Lys Glu Leu His Arg Leu Asn Phe Pro
470 475 480
Arg Arg Ala Gln Gly Pro Thr Trp Arg Ser Leu Glu Val Ile Gln
485 490 495
24/87
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Ser Thr Glu Leu
<210> 16
<211> 596
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Tncyte ID No: 4538363CD1
<400> 16
Met Ala Ala Asn Ser Thr Ser Asp Leu His Thr Pro Gly Thr Gln
1 5 10 15
Leu Ser Val Ala Asp Ile Ile Val I1e Thr Val Tyr Phe Ala Leu
20 25 30
Asn Val Ala Val Gly Tle Trp Ser Ser Cys Arg Ala Ser Arg Asn
35 40 45
Thr Val Asn Gly Tyr Phe Leu Ala Gly Arg Asp Met Thr Trp Trp
50 55 60
Pro Tle Gly Ala Ser Leu Phe Ala Ser Ser Glu Gly Ser Gly Leu
65 70 75
Phe Ile Gly Leu Ala Gly Ser Gly Ala Ala G1y G1y Leu Ala Val
80 85 90
A1a Gly Phe Glu Trp Asn Ala Thr Tyr Val Leu Leu Ala Leu Ala
95 100 105
Trp Val Phe Val Pro Ile Tyr Ile Ser Ser Glu Ile Val Thr Leu
110 115 120
Pro Glu Tyr Ile Gln Lys Arg Tyr Gly Gly Gln Arg Ile Arg Met
125 130 135
Tyr Leu Ser Val Leu Ser Leu Leu Leu Ser Va1 Phe Thr Lys Ile
140 145 150
Ser Leu Asp Leu Tyr Ala Gly Ala Leu Phe Val His Ile Cys Leu
155 160 165
Gly Trp Asn Phe Tyr Leu Ser Thr Ile Leu Thr Leu Gly Ile Thr
170 175 180
Ala Leu Tyr Thr Ile Ala Gly Gly Leu Ala Ala Val Ile Tyr Thr
185 190 195
Asp Ala Leu Gln Thr Leu I1e Met Val Val Gly Ala Val Ile Leu
200 205 210
Thr Ile Lys Ala Phe Asp Gln Ile Gly Gly Tyr Gly Gln Leu Glu
215 220 225
Ala Ala Tyr Ala Gln Ala Ile Pro Sex Arg Thr Ile Ala Asn Thr
230 235 240
Thr Cys His Leu Pro Arg Thr Asp Ala Met His Met Phe Arg Asp
245 250 255
Pro His Thr Gly Asp Leu Pro Trp Thr Gly Met Thr Phe Gly Leu
260 265 270
Thr Ile Met Ala Thr Trp Tyr Trp Cys Thr Asp Gln Va1 Ile Val
275 280 285
Gln Arg Ser Leu Ser Ala Arg Asp Leu Asn His Ala Lys Ala Gly
290 295 300
Ser Ile Leu Ala Ser Tyr Leu Lys Met Leu Pro Met Gly Leu Ile
305 310 315
Ile Met Pro Gly Met Ile Ser Arg Ala Leu Phe Pro Asp Asp Val
320 325 330
Gly Cys Val Val Pro Ser Glu Cys Leu Arg Ala Cys Gly Ala Glu
335 340 345
Val Gly Cys Ser Asn Ile Ala Tyr Pro Lys Leu Val Met Glu Leu
350 355 360
Met Pro Ile Gly Leu Arg Gly Leu Met Ile Ala Val Met Leu Ala
365 370 375
Ala Leu Met Ser Ser Leu Thr Ser Ile Phe Asn Ser Ser Ser Thr
380 385 390
Leu Phe Thr Met Asp Ile Trp Arg Arg Leu Arg Pro Arg Ser Gly
395 400 405
25/87
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Glu Arg Glu Leu Leu Leu Va1 Gly Arg Leu Val Ile Val Ala Leu
410 415 420
Ile Gly Val Ser Val Ala Trp Ile Pro Val Leu Gln Asp Ser Asn
425 430 435
Ser Gly Gln Leu Phe Ile Tyr Met Gln Ser Val Thr Ser Ser Leu
440 445 450
Ala Pro Pro Val Thr Ala Val Phe Val Leu Gly Val Phe Trp Arg
455 460 465
Arg Ala Asn Glu Gln Gly Ala Phe Trp Gly Leu Tle Ala Gly Leu
470 475 480
Val Val Gly Ala Thr Arg Leu Val Leu Glu Phe Leu Asn Pro Ala
485 490 495
Pro Pro Cys Gly Glu Pro Asp Thr Arg Pro Ala Val Leu Gly Ser
500 505 510
Ile His Tyr Leu His Phe Ala Val Ala Leu Phe Ala Leu Ser Gly
515 520 525
Ala Val Val Val Ala Gly Ser Leu Leu Thr Pro Pro Pro Gln Ser
530 535 540
Va1 Gln Ile Glu Asn Leu Thr Trp Trp Thr Leu Ala Gln Asp Val
545 550 555
Pro Leu Gly Thr Lys A1a Gly Asp Gly Gln Thr Pro Gln Lys His
560 565 570
Ala Phe Trp Ala Arg Val Cys Gly Phe Asn Ala Ile Leu Leu Met
575 580 585
Cys Val Asn Ile Phe Phe Tyr Ala Tyr Phe Ala
590 595
<210> 17
<211> 1192
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6427460CD1
<400> 17
Met Asp Cys Ser Leu Val Arg Thr Leu Val His Arg Tyr Cys A1a
1 5 10 15
Gly Glu Glu Asn Trp Va1 Asp Ser Arg Thr Ile Tyr Val Gly His
20 25 30
Arg Glu Pro Pro Pro Gly Ala Glu Ala Tyr I1e Pro Gln Arg Tyr
35 40 45
Pro Asp Asn Arg Ile Val Ser Ser Lys Tyr Thr Phe Trp Asn Phe
50 55 60
Ile Pro Lys Asn Leu Phe Glu Gln Phe Arg Arg Val Ala Asn Phe
65 70 75
Tyr Phe Leu Ile Ile Phe Leu Val Gln Leu Ile Ile Asp Thr Pro
80 85 90
Thr Ser Pro Val Thr Ser Gly Leu Pro Leu Phe Phe Val Ile Thr
95 100 105
Val Thr Ala Ile Lys Gln Gly Tyr Glu Asp Trp Leu Arg His Lys
110 115 120
Ala Asp Asn Ala Met Asn Gln Cys Pro Val His Phe Ile Gln His
125 130 135
Gly Lys Leu Val Arg Lys Gln Ser Arg Lys Leu Arg Val Gly Asp
140 145 150
Ile Val Met Val Lys Glu Asp Glu Thr Phe Pro Cys Asp Leu Ile
155 160 165
Phe Leu Ser Ser Asn Arg G1y Asp Gly Thr Cys His Val Thr Thr
170 175 180
Ala Ser Leu Asp Gly Glu Ser Ser His Lys Thr His Tyr Ala Val
185 190 195
Gln Asp Thr Lys Gly Phe His Thr Glu Glu Asp Ile Gly Gly Leu
200 205 210
His Ala Thr Ile Glu Cys Glu Gln Pro Gln Pro Asp Leu Tyr Lys
215 220 225
26/87
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Phe Val Gly Arg Ile Asn Val Tyr Ser Asp Leu Asn Asp Pro Val
230 235 240
Va1 Arg Pro Leu Gly Ser Glu Asn Leu Leu Leu Arg Gly Ala Thr
245 250 255
Leu Lys Asn Thr Glu Lys Ile Phe Gly Val Ala Ile Tyr Thr Gly
260 265 270
Met Glu Thr Lys Met Ala Leu Asn Tyr Gln Ser Lys Ser Gln Lys
275 280 285
Arg Ser AIa Val Glu Lys Ser Met Asn AIa Phe Leu Ile Val Tyr
290 295 300
Leu Cys Ile Leu Ile Ser Lys Ala Leu Tle Asn Thr Val Leu Lys
305 310 325
Tyr Val Trp Gln Ser Glu Pro Phe Arg Asp Glu Pro Trp Tyr Asn
320 325 330
Gln Lys Thr Glu Ser Glu Arg Gln Arg Asn Leu Phe Leu Lys Ala
335 340 345
Phe Thr Asp Phe Leu Ala Phe Met Val Leu Phe Asn Tyr Ile Tle
350 355 360
Pro Val Ser Met Tyr Val Thr Val Glu Met Gln Lys Phe Leu Gly
365 370 375
Ser Tyr Phe Ile Thr Trp Asp Glu Asp Met Phe Asp Glu Glu Thr
380 385 390
Gly GIu Gly Pro Leu Val Asn Thr Ser Asp Leu Asn Glu Glu Leu
395 400 405
Gly Gln VaI Glu Tyr Ile Phe Thr Asp Lys Thr Gly Thr Leu Thr
410 4l5 420
Glu Asn Asn Met Glu Phe Lys Glu Cys Cys Ile Glu Gly His Val
425 430 435
Tyr Val Pro His Val Ile Cys Asn Gly Gln Val Leu Pro Glu Ser
440 445 450
Ser G1y Tle Asp Met Ile Asp Ser Ser Pro Ser Val Asn Gly Arg
455 460 465
Glu Arg Glu Glu Leu Phe Phe Arg Ala Leu Cys Leu Cys His Thr
470 475 480
Val Gln Val Lys Asp Asp Asp Ser Val Asp Gly Pro Arg Lys Ser
485 490 495
Pro Asp Gly Gly Lys Ser Cys Val Tyr Ile Ser Ser Ser Pro Asp
500 505 510
Glu Val Ala Leu Val Glu Gly Val Gln Arg Leu Gly Phe Thr Tyr
515 520 525
Leu Arg Leu Lys Asp Asn Tyr Met Glu Ile Leu Asn Arg Glu Asn
530 535 540
His Ile Glu Arg Phe Glu Leu Leu Glu Ile Leu Ser Phe Asp Ser
545 550 555
Val Arg Arg Arg Met Ser Val Ile Val Lys Ser Ala Thr Gly Glu
560 565 570
Ile Tyr Leu Phe Cys Lys GIy Ala Asp Ser Ser Ile Phe Pro Arg
575 580 585
Val Ile G1u Gly Lys Val Asp Gln Ile Arg Ala Arg Val Glu Arg
590 595 600
Asn Ala Val Glu Gly Leu Arg Thr Leu Cys Val Ala Tyr Lys Arg
605 610 615
Leu Ile Gln Glu Glu Tyr Glu Gly Ile Cys Lys Leu Leu Gln Ala
620 625 630
Ala Lys Val Ala Leu Gln Asp Arg Glu Lys Lys Leu A1a Glu Ala
635 640 645
Tyr G1u Gln Ile Glu Lys Asp Leu Thr Leu Leu Gly A1a Thr Ala
650 655 660
Val Glu Asp Arg Leu Gln Glu Lys Ala Ala Asp Thr Ile Glu A1a
665 670 675
Leu Gln Lys Ala Gly Ile Lys Val Trp Val Leu Thr Gly Asp Lys
680 685 690
Met Glu Thr AIa Ala Ala Thr Cys Tyr Ala Cys Lys Leu Phe Arg
695 700 705
Arg Asn Thr Gln Leu Leu Glu Leu Thr Thr Lys Arg Ile Glu Glu
710 715 720
G1n Ser Leu His Asp Val Leu Phe Glu Leu Ser Lys Thr Val Leu
27/87
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725 730 735
Arg His Ser Gly Ser Leu Thr Arg Asp Asn Leu Ser Gly Leu Ser
740 745 750
Ala Asp Met Gln Asp Tyr Gly Leu Ile Ile Asp Gly Ala Ala Leu
755 760 765
Ser Leu Ile Met Lys Pro Arg Glu Asp Gly Ser Ser Gly Asn Tyr
770 775 780
Arg Glu Leu Phe Leu Glu Ile Cys Arg Ser Cys Ser Ala Val Leu
785 790 795
Cys Cys Arg Met Ala Pro Leu Gln Lys Ala Gln Ile Val Lys Leu
800 805 810
Ile Lys Phe Ser Lys Glu His Pro Ile Thr Leu Ala Ile Gly Asp
8l5 820 825
Gly Ala Asn Asp Val Ser Met Ile Leu Glu Ala His Val Gly Ile
830 835 840
Gly Val Ile Gly Lys Glu Gly Arg Gln Ala Ala Arg Asn Ser Asp
845 850 855
Tyr Ala Ile Pro Lys Phe Lys His Leu Lys Lys Met Leu Leu Val
860 865° 870
His Gly His Phe Tyr Tyr Ile Arg Ile Ser Glu Leu Val Gln Tyr
875 880 885
Phe Phe Tyr Lys Asn Val Cys Phe Ile Phe Pro Gln Phe Leu Tyr
890 895 900
Gln Phe Phe Cys Gly Phe Ser Gln Gln Thr Leu Tyr Asp Thr Ala
905 910 915
Tyr Leu Thr Leu Tyr Asn Ile Ser Phe Thr Ser Leu Pro Ile Leu
920 925 930
Leu Tyr Ser Leu Met Glu Gln His Val Gly Ile Asp Val Leu Lys
935 940 945
Arg Asp Pro Thr Leu Tyr Arg Asp Val Ala Lys Asn Ala Leu Leu
950 955 960
Arg Trp Arg Val Phe Ile Tyr Trp Thr Leu Leu G1y Leu Phe Asp
965 970 975
Ala Leu Val Phe Phe Phe Gly Ala Tyr Phe Val Phe Glu Asn Thr
980 985 990
Thr Val Thr Ser Asn Gly Gln Ile Phe Gly Asn Trp Thr Phe Gly
995 1000 1005
Thr Leu Val Phe Thr Val Met Val Phe Thr Val Thr Leu Lys Leu
1010 1015 1020
Ala Leu Asp Thr His Tyr Trp Thr Trp Ile Asn His Phe Val Ile
1025 1030 1035
Trp Gly Ser Leu Leu Phe Tyr Val Val Phe Ser Leu Leu Trp Gly
1040 1045 1050
Gly Val Ile Trp Pro Phe Leu Asn Tyr Gln Arg Met Tyr Tyr Val
1055 1060 1065
Phe Ile Gln Met Leu Ser Ser Gly Pro Ala Trp Leu Ala Ile Val
1070 1075 1080
Leu Leu Val Thr Ile Ser Leu Leu Pro Asp Val Leu Lys Lys Val
1085 1090 1095
Leu Cys Arg Gln Leu Trp Pro Thr Ala Thr Glu Arg Val Gln Gln
1100 1105 1110
Asn Gly Cys Ala Gln Pro Arg Asp Arg Asp Ser Glu Phe Thr Pro
1115 1120 1125
Leu Ala S2r Leu Gln Ser Pro Gly Tyr Gln Ser Thr Cys Pro Ser
1130 1135 1140
Ala Ala Trp Tyr Ser Ser His Ser Gln Gln Val Thr Leu Ala Ala
1145 1150 1155
Trp Lys Glu Lys Val Ser Thr Glu Pro Pro Pro Ile Leu Gly Gly
1160 1165 1170
Ser His His His Cys Ser Ser Ile Pro Ser His Ser Cys Pro Arg
1175 1180 1185
Ser Arg Va1 G1y Met Leu Val
1190
<210> 18
<211> 638
<212> PRT
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7474127CD1
<400> 18
Met Gly Lys Ile Glu Asn Asn Glu Arg Val Ile Leu Asn Val Gly
1 5 10 15
Gly Thr Arg His Glu Thr Tyr Arg Ser Thr Leu Lys Thr Leu Pro
20 25 30
Gly Thr Arg Leu Ala Leu Leu Ala Ser Ser Glu Pro Pro Gly Asp
35 40 45
Cys Leu Thr Thr Ala Gly Asp Lys Leu Gln Pro Ser Pro Pro Pro
50 55 60
Leu Ser Pro Pro Pro Arg Ala Pro Pro Leu Ser Pro Gly Pro Gly
65 70 7S
Gly Cys Phe Glu Gly Gly Ala Gly Asn Cys Ser Ser Arg Gly Gly
80 85 90
Arg Ala Ser Asp His Pro Gly Gly Gly Arg Glu Phe Phe Phe Asp
95 100 105
Arg His Pro Gly Val Phe Ala Tyr Val Leu Asn Tyr Tyr Arg Thr
110 215 120
G1y Lys Leu His Cys Pro Ala Asp Val Cys Gly Pro Leu Phe Glu
125 130 135
Glu Glu Leu Ala Phe Trp Gly Ile Asp Glu Thr Asp Val Glu Pro
140 145 150
Cys Cys Trp Met Thr Tyr Arg Gln His Arg Asp Ala Glu Glu Ala
155 160 165
Leu Asp Ile Phe Glu Thr Pro Asp Leu Ile Gly Gly Asp Pro G1y
170 175 180
Asp Asp Glu Asp Leu Ala Ala Lys Arg Leu Gly Ile Glu Asp Ala
185 190 195
Ala Gly Leu Gly Gly Pro Asp Gly Lys Ser Gly Arg Trp Arg Arg
200 205 210
Leu Gln Pro Arg Met Trp Ala Leu Phe G1u Asp Pro Tyr Ser Ser
215 220 225
Arg Ala Ala Arg Phe Ile Ala Phe Ala Ser Leu Phe Phe Ile Leu
230 235 240
Val Ser I1e Thr Thr Phe Cys Leu Glu Thr His Glu Ala Phe Asn
245 250 255
Ile Val Lys Asn Lys Thr Glu Pro Val I1e Asn Gly Thr Ser Val
260 265 270
Val Leu Gln Tyr G1u Ile G1u Thr Asp Pro A1a Leu Thr Tyr Val
275 280 285
Glu Gly Val Cys Val Val Trp Phe Thr Phe Glu Phe Leu Val Arg
290 295 300
Ile Val Phe Ser Pro Asn Lys Leu Glu Phe Ile Lys Asn Leu Leu
305 310 315
Asn Ile Ile Asp Phe Val Ala Ile Leu Pro Phe Tyr Leu Glu Val
320 325 330
Gly Leu Ser Gly Leu Ser Ser Lys Ala A1a Lys Asp Val Leu Gly
335 340 345
Phe Leu Arg Val Val Arg Phe Val Arg Ile Leu Arg Ile Phe Lys
350 355 360
Leu Thr Arg His Phe Val Gly Leu Arg Val Leu Gly His Thr Leu
365 370 375
Arg Ala Ser Thr Asn Glu Phe Leu Leu Leu Ile Ile Phe Leu Ala
380 385 390
Leu Gly Val Leu Ile Phe Ala Thr Met Ile Tyr Tyr Ala Glu Arg
395 400 405
Val Gly Ala Gln Pro Asn Asp Pro Ser Ala Ser Glu His Thr G1n
410 415 420
Phe Lys Asn I1e Pro Ile Gly Phe Trp Trp Ala Val Val Thr Met
425 430 435
Thr Thr Leu Gly Tyr Gly Asp Met Tyr Pro Gln Thr Trp Ser Gly
440 445 450
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Met Leu Val Gly Ala Leu Cys Ala Leu Ala Gly Val Leu Thr Ile
455 460 465
Ala Met Pro Val Pro Val Ile Val Asn Asn Phe Gly Met Tyr Tyr
470 475 480
Ser Leu Ala Met Ala Lys Gln Lys Leu Pro Arg Lys Arg Lys Lys
485 490 495
His Tle Pro Pro Ala Pro Gln Ala Ser Ser Pro Thr Phe Cys Lys
500 505 510
Thr Glu Leu Asn Met A1a Cys Asn Ser Thr Gln Ser Asp Thr Cys
515 520 525
Leu Gly Lys Asp Asn Arg Leu Leu Glu His Asn Arg Ser Val Leu
530 535 540
Ser Gly Asp Asp Ser Thr Gly Ser Glu Pro Pro Leu Ser Pro Pro
545 550 555
Glu Arg Leu Pro Ile Arg Arg Ser Ser Thr Arg Asp Lys Asn Arg
560 565 570
Arg Gly Glu Thr Cys Phe Leu Leu Thr Thr Gly Asp Tyr Thr Cys
575 580 585
Ala Ser Asp Gly Gly Ile Arg Lys Gly Tyr Glu Lys Ser Arg Ser
590 595 600
Leu Asn Asn Ile Ala Gly Leu Ala Gly Asn Ala Leu Arg Leu Ser
605 610 615
Pro Val Thr Ser Pro Tyr Asn Ser Pro Cys Pro Leu Arg Arg Ser
620 625 630
Arg Ser Pro I1e Pro Ser Ile Leu
635
<210> 19
<211> 681
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7476949CD1
<400> 19
Met Ser Lys Asp Leu Ala Ala Met Gly Pro Gly Ala Ser G1y Asp
1 5 10 15
Gly Val Arg Thr Glu Thr Ala Pro His Ile Ala Leu Asp Ser Arg
20 25 30
Val Gly Leu His Ala Tyr Asp Tle Ser Val Val Val Ile Tyr Phe
35 40 45
Val Phe Val Ile Ala Val Gly Ile Trp Ser Ser Tle Arg Ala Ser
50 55 60
Arg Gly Thr Ile Gly Gly Tyr Phe Leu Ala G1y Arg Sex Met Ser
65 70 75
Trp Trp Pro Ile Gly Ala Ser Leu Met Ser Ser Asn Val Gly Ser
80 85 90
Gly Leu Phe Ile Gly Leu Ala Gly Thr Gly Ala Ala Gly Gly Leu
95 100 105
Ala Val G1y Gly Phe Glu Trp Asn Ala Thr Trp Leu Leu Leu A1a
110 115 120
Leu Gly Trp Val Phe Val Pro Val Tyr Ile Ala Ala Gly Val Val
125 130 235
Thr Met Pro Gln Tyr Leu Lys Lys Arg Phe Gly Gly Gln Arg Ile
140 145 150
Gln Val Tyr Met Ser Val Leu Ser Leu Ile Leu Tyr Ile Phe Thr
155 160 165
Lys Ile Ser Thr Asp Ile Phe Ser Gly Ala Leu Phe Ile Gln Met
170 175 180
Ala Leu Gly Trp Asn Leu Tyr Leu Ser Thr Gly I1e Leu Leu Val
185 190 195
Val Thr A1a Val Tyr Thr Ile Ala Gly Gly Leu Met Ala Val Ile
200 205 210
Tyr Thr Asp A1a Leu Gln Thr Val Ile Met Val Gly Gly Ala Leu
215 220 225
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Val Leu Met Phe Leu Gly Phe Gln Asp Val Gly Trp Tyr Pro Gly
230 235 240
Leu G1u Gln Arg Tyr Arg Gln Ala Ile Pro Asn Val Thr Val Pro
245 250 255
Asn Thr Thr Cys His Leu Pro Arg Pro Asp Ala Phe His Ile Leu
260 ~ 265 270
Arg Asp Pro Val Ser Gly Asp Ile Pro Trp Pro Gly Leu Ile Phe
275 280 285
Gly Leu Thr Val Leu Ala Thr Trp Cys Trp Cys Thr Asp Gln Val
290 295 300
Ile Val Gln Arg Ser Leu Ser Ala Lys Ser Leu Ser His Ala Lys
305 310 315
Gly Gly Ser Val Leu Gly Gly Tyr Leu Lys I1e Leu Pro Met Phe
320 325 330
Phe Ile Val Met Pro Gly Met Ile Ser Arg Ala Leu Phe Pro Asp
335 340 345
Glu Val Gly Cys Val Asp Pro Asp Val Cys Gln Arg Ile Cys Gly
350 355 360
Ala Arg Val Gly Cys Ser Asn Ile Ala Tyr Pro Lys Leu Val Met
365 370 375
Ala Leu Met Pro Val Gly Leu Arg Gly Leu Met Ile Ala Val Ile
380 385 390
Met Ala Ala Leu Met Ser Ser Leu Thr Ser Ile Phe Asn Ser Ser
395 400 405
Ser Thr Leu Phe Thr Ile Asp Val Trp Gln Arg Phe Arg Arg Lys
410 415 420
Ser Thr Glu Gln Glu Leu Met Val Val Gly Arg Val Phe Val Val
425 430 435
Phe Leu Val Val Ile Ser Ile Leu Trp Ile Pro Ile Ile Gln Ser
440 445 450
Ser Asn Ser Gly Gln Leu Phe Asp Tyr Ile Gln Ala Val Thr Ser
455 460 ' 465
Tyr Leu Ala Pro Pro Ile Thr Ala Leu Phe Leu Leu Ala Ile Phe
470 475 480
Cys Lys Arg Val Thr Glu Pro Gly Ala Phe Trp Gly Leu Val Phe
485 490 495
Gly Leu Gly Val Gly Leu Leu Arg Met Ile Leu Glu Phe Ser Tyr
500 505 510
Pro Ala Pro Ala Cys Gly Glu Val Asp Arg Arg Pro Ala Val Leu
515 520 525
Lys Asp Phe His Tyr Leu Tyr Phe Ala Ile Leu Leu Cys Gly Leu
530 535 540
Thr Ala Ile Val Ile Va1 Ile Val Ser Leu Cys Thr Thr Pro Ile
545 550 555
Pro Glu Glu Gln Leu Thr Arg Leu Thr Trp Trp Thr Arg Asn Cys
560 565 570
Pro Leu Ser Glu Leu Glu Lys Glu Ala His Glu Ser Thr Pro Glu
575 580 585
Ile Ser Glu Arg Pro Ala Gly Glu Cys Pro Ala Gly Gly Gly Ala
590 595 600
Ala Glu Asn Ser Ser Leu Gly Gln Glu Gln Pro Glu Ala Pro Ser
605 610 615
Arg Ser Trp Gly Lys Leu Leu Trp Ser Trp Phe Cys Gly Leu Ser
620 625 630
Gly Thr Pro Glu Gln Ala Leu Ser Pro Ala Glu Lys Ala Ala Leu
635 640 645
Glu G1n Lys Leu Thr Ser Ile Glu Glu Glu Pro Leu Trp Arg His
650 655 660
VaI Cys Asn Ile Asn Ala Val Leu Leu Leu Ala IIe Asn Ile Phe
665 670 675
Leu Trp Gly Tyr Phe Ala
680
<210> 20
<211> 1096
<212> PRT
<223> Homo Sapiens
31/87
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<220>
<221> misc_feature
<223> Incyte ID No: 7477249CD1
<400> 20
Met Trp Arg Trp Ile Arg Gln Gln Leu Gly Phe Asp Pro Pro His
1 5 10 15
G1n Ser Asp Thr Arg Thr Ile Tyr Val Ala Asn Arg Phe Pro Gln
20 25 30
Asn Gly Leu Tyr Thr Pro Gln Lys Phe Ile Asp Asn Arg Ile Ile
35 40 45
Ser Ser Lys Tyr Thr Val Trp Asn Phe Va1 Pro Lys Asn Leu Phe
50 55 60
Glu Gln Phe Arg Arg Val Ala Asn Phe Tyr Phe Leu Ile Ile Phe
65 70 75
Leu VaI Gln Leu Met Ile Asp Thr Pro Thr Ser Pro Val Thr Ser
80 85 90
Gly Leu Pro Leu Phe Phe Val Ile Thr Val Thr Ala Ile Lys Gln
95 100 105
Gly Tyr Glu Asp Trp Leu Arg His Asn Ser Asp Asn Glu Va1 Asn
120 115 120
Gly Ala Pro Val Tyr Val Val Arg Ser Gly Gly Leu Val Lys Thr
12 5 13 0 13 5
Arg Ser Lys Asn Ile Arg Val Gly Asp Ile Val Arg Ile Ala Lys
140 145 150
Asp G1u Ile Phe Pro Ala Asp Leu Val Leu Leu Ser Ser Asp Arg
155 160 165
Leu Asp Gly Ser Cys His Val Thr Thr Ala Ser Leu Asp Gly Glu
170 175 180
Thr Asn Leu Lys Thr His Val Ala Val Pro Glu Thr Ala Leu Leu
185 190 195
Gln Thr Va1 Ala Asn Leu Asp Thr Leu Va1 Ala Val Ile Glu Cys
200 205 210
Gln Gln Pro Glu Ala Asp Leu Tyr Arg Phe Met Gly Arg Met I1e
215 220 225
Ile Thr Gln Gln Met Glu Glu Ile Val Arg Pro Leu Gly Pro Glu
230 235 240
Ser Leu Leu Leu Arg Gly Ala Arg Leu Lys Asn Thr Lys Glu Ile
245 250 255
Phe Gly Val Ala Val Tyr Thr Gly Met Glu Thr Lys Met A1a Leu
260 265 270
Asn Tyr Lys Ser Lys Ser Gln Lys Arg Ser Ala Val Glu Lys Ser
275 280 285
Met Asn Thr Phe Leu Ile Ile Tyr Leu Val Ile Leu Ile Ser Glu
290 295 300
Ala Val Ile Ser Thr Ile Leu Lys Tyr Thr Trp Gln Ala Glu Glu
305 310 315
Lys Trp Asp Glu Pro Trp Tyr Asn Gln Lys Thr Glu His Gln Arg
320 325 330
Asn Ser Ser Lys Val Glu Tyr Val Phe Thr Asp Lys Thr Gly Thr
335 340 345
Leu Thr Glu Asn Glu Met Gln Phe Arg Glu Cys Ser Ile Asn Gly
350 355 360
Met Lys Tyr Gln Glu Ile Asn Gly Arg Leu Val Pro Glu Gly Pro
365 370 375
Thr Pro Asp Ser Ser Glu Gly Asn Leu Ser Tyr Leu Ser Ser Leu
380 385 390
Ser His Leu Asn Asn Leu Ser His Leu Thr Thr Ser Ser Ser Phe
395 400 405
Arg Thr Ser Pro Glu Asn Glu Thr Glu Leu Ile Lys GIu His Asp
410 415 ~ 420
Leu Phe Phe Lys Ala Val Ser Leu Cys His Thr Val Gln Ile Ser
425 43 0 43 5
Asn Val Gln Thr Asp Cys Thr Gly Asp Gly Pro Trp Gln Ser Asn
440 445 450
Leu Ala Pro Ser Gln Leu Glu Tyr Tyr Ala Ser Ser Pro Asp G1u
455 460 465
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Lys Ala Leu Val Glu Ala Ala Ala Arg Tle Gly Ile Val Phe Ile
470 475 480
Gly Asn Ser Glu Glu Thr Met Glu Val Lys Thr Leu Gly Lys Leu
485 490 495
Glu Arg Tyr Lys Leu Leu His Ile Leu G1u Phe Asp Ser Asp Arg
500 505 510
Arg Arg Met Ser Val Ile Val Gln Ala Pro Ser Gly Glu Lys Leu
515 520 525
Leu Phe Ala Lys Gly Ala Glu Ser Ser Ile Leu Pro Lys Cys Ile
530 535 540
Gly Gly Glu I1e Glu Lys Thr Arg Ile His Val Asp Glu Phe Ala
545 550 555
Leu Lys Gly Leu Arg Thr Leu Cys Ile Ala Tyr Arg Lys Phe Thr
560 565 570
Ser Lys Glu Tyr G1u Glu I1e Asp Lys Arg Ile Phe Glu Ala Arg
575 580 585
Thr Ala Leu Gln Gln Arg Glu Glu Lys Leu Ala Ala Val Phe Gln
590 595 600
Phe Ile Glu Lys Asp Leu Ile Leu Leu Gly Ala Thr Ala Val Glu
605 610 615
Asp Arg Leu Gln Asp Lys Val Arg Glu Thr Ile Glu Ala Leu Arg
620 625 630
Met Ala Gly Ile Lys Val Trp Val Leu Thr Gly Asp Lys His Glu
635 640 645
Thr Ala Val Ser Va1 Ser Leu Ser Cys Gly His Phe His Arg Thr
650 655 660
Met Asn Ile Leu Glu Leu Ile Asn Gln Lys Ser Asp Ser Glu Cys
665 670 675
Ala Glu Gln Leu Arg Gln Leu Ala Arg Arg Ile Thr Glu Asp His
680 685 690
Val I1e Gln His Gly Leu Val Val Asp Gly Thr Ser Leu Ser Leu
695 700 705
Ala Leu Arg Glu His Glu Lys Leu Phe Met Glu Val Cys Arg Asn
710 715 720
Cys Ser Ala Val Leu Cys Cys Arg Met Ala Pro Leu Gln Lys A1a
725 730 735
Lys Val Ile Arg Leu Ile Lys Ile Ser Pro Glu Lys Pro Ile Thr
740 745 750
Leu Ala Val Gly Asp Gly A1a Asn Asp Val Ser Met Ile Gln Glu
755 760 765
Ala His Val Gly Ile Gly Ile Met Gly Lys Glu Gly Arg Gln Ala
770 775 780
Ala Arg Asn Ser Asp Tyr Ala Ile Ala Arg Phe Lys Phe Leu Ser
785 790 795
Lys Leu Leu Phe Val His Gly His Phe Tyr Tyr Ile Arg Ile Ala
800 805 810
Thr Leu Val Gln Tyr Phe Phe Tyr Lys Asn Val Cys Phe Ile Thr
815 820 825
Pro Gln Phe Leu Tyr Gln Phe Tyr Cys Leu Phe Ser Gln Gln Thr
830 835 840
Leu Tyr Asp Ser Val Tyr Leu Thr Leu Tyr Asn Ile Cys Phe Thr
845 850 855
Ser Leu Pro Ile Leu Ile Tyr Ser Leu Leu Glu Gln His Va1 Asp
860 . 865 870
Pro His Val Leu Gln Asn Lys Pro Thr Leu Tyr Arg Asp Ile Ser
875 880 885
Lys Asn Arg Leu Leu Ser Tle Lys Thr Phe Leu Tyr Trp Thr I1e
890 895 900
Leu Gly Phe Ser His Ala Phe Ile Phe Phe Phe Gly Ser Tyr Leu
905 910 915
Leu Ile Gly Lys Asp Thr Ser Leu Leu G1y Asn Gly Gln Met Phe
920 925 930
Gly Asn Trp Thr Phe Gly Thr Leu Val Phe Thr Val Met Val Ile
935 940 945
Thr Val Thr Val Lys Met Ala Leu Glu Thr His Phe Trp Thr Trp
950 955 960
Ile Asn His Leu Val Thr Trp Gly Ser Ile Ile Phe Tyr Phe Val
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965 970 975
Phe Ser Leu Phe Tyr Gly Gly Ile Leu Trp Pro Phe Leu Gly Ser
980 985 990
Gln Asn Met Tyr Phe Val Phe Ile Gln Leu Leu Ser Ser Gly Ser
995 1000 1005
Ala Trp Phe Ala Ile Ile Leu Met Val Val Thr Cys Leu Phe Leu
1010 1015 1020
Asp Ile Ile Lys Lys Val Phe Asp Arg His Leu His Pro Thr Ser
1025 103 0 1035
Thr Glu Lys A1a Gln Leu Thr Glu Thr Asn Ala Gly Ile Lys Cys
1040 1045 1050
Leu Asp Ser Met Cys Cys Phe Pro Glu Gly Glu Ala Ala Cys Ala
1055 1060 1065
Ser Val Gly Arg Met Leu Glu Arg Val Ile Gly Arg Cys Ser Pro
1070 1075 1080
Thr His Ile Ser Arg Cys Glu Ile Ser Leu Ser Ser Leu Cys Cys
1085 1090 1095
Arg
<210> 21
<211> 707
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7477720CD1
<400> 21
Met Ala Leu G1n Met Phe Va1 Thr Tyr Ser Pro Trp Asn Cys Leu
1 5 10 15
Leu Leu Leu Val Ala Leu Glu Cys Ser Glu Ala Ser Ser Asp Leu
20 25 30
Asn Glu Ser Ala Asn Ser Thr Ala Gln Tyr Ala Ser Asn Ala Trp
35 40 45
Phe Ala Ala Ala Ser Ser Glu Pro Glu Glu Gly Ile Ser Va1 Phe
50 55 60
Glu Leu Asp Tyr Asp Tyr Val Gln Ile Pro Tyr Glu Val Thr Leu
65 70 75
Trp Ile Leu Leu Ala Ser Leu Ala Lys I1e Gly Phe His Leu Tyr
80 85 90
His Arg Leu Pro Gly Leu Met Pro Glu Ser Cys Leu Leu Ile Leu
95 100 105
Val Gly Ala Leu Val Gly Gly Ile Ile Phe Gly Thr Asp His Lys
110 115 120
Ser Pro Pro Val Met Asp Ser Ser Ile Tyr Phe Leu Tyr Leu Leu
125 130 135
Pro Pro Ile Val Leu Glu Gly Gly Tyr Phe Met Pro Thr Arg Pro
140 145 150
Phe Phe Glu Asn Ile Gly Ser Ile Leu Trp Trp Ala Val Leu Gly
155 160 165
Ala Leu Ile Asn Ala Leu Gly Ile Gly Leu Ser Leu Tyr Leu Ile
170 175 180
Cys Gln Val Lys Ala Phe Gly Leu Gly Asp Val Asn Leu Leu Gln
185 190 195
Asn Leu Leu Phe Gly Ser Leu Ile Ser Ala Val Asp Pro Val Ala
200 205 210
Val Leu Ala Val Phe Glu Glu Ala Arg Val Asn Glu Gln Leu Tyr
215 220 225
Met Met Ile Phe Gly Glu Ala Leu Leu Asn Asp Gly Ile Thr Val
230 235 240
Val Leu Tyr Asn Met Leu Ile Ala Phe Thr Lys Met His Lys Phe
245 250 255
Glu Asp Ile Glu Thr Val Asp Ile Leu Ala Gly Cys Ala Arg Phe
260 265 270
Ile Val Val Gly Leu Gly Gly Val Leu Phe Gly Ile Val Phe Gly
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275 280 285
Phe Ile Ser Ala Phe Ile Thr Arg Phe Thr Gln Asn Ile Ser Ala
290 295 300
Ile Glu Pro Leu Ile Val Phe Met Phe Ser Tyr Leu Ser Tyr Leu
305 310 315
Ala Ala Glu Thr Leu Tyr Leu Ser Gly Ile Leu Ala Ile Thr Ala
320 325 330
Cys Ala Val Thr Met Lys Lys Tyr Val Glu Glu Asn Val Ser Gln
335 340 345
Thr Ser Tyr Thr Thr Ile Lys Tyr Phe Met Lys Met Leu Ser Ser
350 355 360
Val Ser Glu Thr Leu Ile Phe Ile Phe Met Gly Val Ser Thr Val
365 370 375
Gly Lys Asn His Glu Trp Asn Trp Ala Phe Ile Cys Phe Thr Leu
380 385 390
Ala Phe Cys Gln Ile Trp Arg Ala Ile Ser Val Phe Ala Leu Phe
395 400 405
Tyr Ile Ser Asn Gln Phe Arg Thr Phe Pro Phe Ser Ile Lys Asp
410 415 420
Gln Cys Ile Ile Phe Tyr Ser Gly Val Arg G1y Ala Gly Ser Phe
425 430 435
Ser Leu Ala Phe Leu Leu Pro Leu Ser Leu Phe Pro Arg Lys Lys
440 445 450
Met Phe Val Thr Ala Thr Leu Val Val Ile Tyr Phe Thr Val Phe
455 460 465
Ile Gln Gly Ile Thr Val Gly Pro Leu Val Arg Tyr Leu Asp Val
470 475 480
Lys Lys Thr Asn Lys Lys Glu Ser Ile Asn Glu Glu Leu His Ile
485 490 495
Arg Leu Met Asp His Leu Lys Ala G1y Ile Glu Asp Val Cys Gly
500 505 510
His Trp Ser His Tyr Gln Val Arg Asp Lys Phe Lys Lys Phe Asp
515 520 525
His Arg Tyr Leu Arg Lys Ile Leu Ile Arg Lys Asn Leu Pro Lys
530 535 540
Ser Ser Ile Val Ser Leu Tyr Lys Lys Leu Glu Met Lys Gln Ala
545 550 555
Ile Glu Met Val Glu Thr Gly Ile Leu Ser Ser Thr Ala Phe Ser
560 565 570
Ile Pro His Gln Ala Gln Arg Ile Gln Gly Ile Lys Arg Leu Ser
575 580 585
Pro Glu Asp Val Glu Ser Ile Arg Asp Ile Leu Thr Ser Asn Met
590 595 600
Tyr Gln Val Arg Gln Arg Thr Leu Ser Tyr Asn Lys Tyr Asn Leu
605 610 615
Lys Pro Gln Thr Ser Glu Lys Gln Ala Lys Glu Ile Leu Ile Arg
620 625 63 0
Arg Gln Asn Thr Leu Arg Glu Ser Met Arg Lys Gly His Ser Leu
635 640 645
Pro Trp Gly Lys Pro Ala Gly Thr Lys Asn Ile Arg Tyr Leu Ser
650 655 660
Tyr Pro Tyr Gly Asn Pro Gln Ser Ala Gly Arg Asp Thr Arg Ala
665 670 675
Ala Gly Phe Ser Gly Lys Leu Pro Thr Trp Leu Leu Cys Cys Phe
680 685 690
Ser Va1 Glu Ser Gly Gly Lys Tyr Leu Gly Val Trp Ala Lys Arg
695 700 705
Gln His
<210> 22
<211> 729
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
35/87
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<223> Incyte ID No: 7477852CD1
<400> 22
Met Gly Gly Phe Leu Pro Lys Ala Glu Gly pro Gly Ser Gln Leu
1 5 10 15
Gln Lys Leu Leu Pro Ser Phe Leu Val Arg Glu Gln Asp Trp Asp
20 25 30
Gln His Leu Asp Lys Leu His Met Leu Gln Gln Lys Arg Ile Leu
35 40 45
Glu Ser Pro Leu Leu Arg Ala Ser Lys Glu Asn Asp Leu Ser Val
50 55 60
Leu Arg Gln Leu Leu Leu Asp Cys Thr Cys Asp Val Arg Gln Arg
65 70 75
Gly Ala Leu Gly Glu Thr Ala Leu His Ile Ala Ala Leu Tyr Asp
80 85 90
Asn Leu Glu Ala Ala Leu Val Leu Met Glu Ala Ala Pro Glu Leu
95 100 105
Val Phe Glu Pro Thr Thr Cys Glu Ala Phe A1a Gly Gln Thr Ala
110 115 120
Leu His Tle Ala Va1 Val Asn Gln Asn Val Asn Leu Val Arg Ala
125 13 0 135
Leu Leu Thr Arg Arg Ala Ser Val Ser Ala Arg Ala Thr Gly Thr
140 145 150
Ala Phe Arg Arg Ser Pro Arg Asn Leu Ile Tyr Phe Gly Glu His
155 160 165
Pro Leu Ser Phe Al.a Ala Cys Val Asn Ser G1u Glu Ile Val Arg
170 175 180
Leu Leu Ile Glu His Gly Ala Asp I1e Arg Ala G1n Asp Ser Leu
185 190 195
Gly Asn Thr Va1 Leu His Ile Leu Ile Leu Gln Pro Asn Lys Thr
200 205 210
Phe Ala Cys Gln Met Tyr Asn Leu Leu Leu Ser Tyr Asp Gly His
215 220 225
Gly Asp His Leu Gln Pro Leu Asp Leu Val Pro Asn His Gln Gly
230 235 240
Leu Thr Pro Phe Lys Leu Ala Gly Val Glu Gly Asn Thr Val Met
245 . 250 255
Phe Gln His Leu Met Gln Lys Arg Arg His Ile Gln Trp Thr Tyr
260 265 270
Gly Pro Leu Thr Ser Ile Leu Tyr Asp Leu Thr Glu Ile Asp Ser
275 280 285
Trp Gly Glu Glu Leu Ser Phe Leu Glu Leu Val Val Ser Ser Asp
290 295 300
Lys Arg Glu Ala Arg Gln Ile Leu Glu Gln Thr Pro Va1 Lys Glu
305 310 315
Leu Val Ser Phe Lys Trp Asn Lys Tyr Gly Arg Pro Tyr Phe Cys
320 325 330
Ile Leu Ala Ala Leu Tyr Leu Leu Tyr Met Ile Cys Phe Thr Thr
335 340 345
Cys Cys Val Tyr Arg Pro Leu Lys Phe Arg Gly Gly Asn Arg Thr
350 355 360
His Ser Arg Asp Ile Thr Ile Leu Gln Gln Lys Leu Leu Gln Glu
365 370 375
Ala Tyr Glu Thr Arg Glu Asp Ile Ile Arg Leu Val Gly Glu Leu
380 385 390
Val Ser I1e Val Gly Ala Val Ile Ile Leu Leu Leu Glu Ile Pro
395 - 400 405
Asp Ile Phe Arg Val Gly Ala Ser Arg Tyr Phe Gly Lys Thr Ile
410 415 420
Leu Gly G1y Pro Phe His Val Ile Met Ile Thr Tyr Ala Ser Leu
425 430 435
Val Leu Val Thr Met Val Met Arg Leu Thr Asn Thr Asn G1y Glu
440 445 450
Val Val Pro Met Ser Phe Ala Leu Val Leu Gly Trp Cys Ser Val
455 4&0 465
Met Tyr Phe Thr Arg Gly Phe Gln Met Leu Gly Pro Phe Thr Ile
470 475 480
36187
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Met Ile G1n Lys Met Ile Phe Gly Asp Leu Met Arg Phe Cys Trp
485 490 495
Leu Met Ala Val Val Ile Leu Gly Phe Ala Ser Ala Phe Tyr Tle
500 505 510
Ile Phe Gln Thr Glu Asp Pro Thr Ser Leu Gly Gln Phe Tyr Asp
515 520 525
Tyr Pro Met Ala Leu Phe Thr Thr Phe Glu Leu Phe Leu Thr Val
530 535 540
Ile Asp Ala Pro Ala Asn Tyr Asp Val Asp Leu Pro Phe Met Phe
545 550 555
Ser Ile Va1 Asn Phe Ala Phe Ala Ile Ile Ala Thr Leu Leu Met
560 565 570
Leu Asn Leu Phe Ile Ala Met Met Gly Asp Thr His Trp Arg Val
575 580 585
Ala Gln Glu Arg Asp Glu Leu Trp Arg Ala Gln Val Val Ala Thr
590 595 600
Thr Val Met Leu Glu Arg Lys Leu Pro Arg Cys Leu Trp Pro Arg
605 610 615
Ser Gly Ile Cys Gly Cys Glu Phe Gly Leu Gly Asp Arg Trp Phe
620 625 630
Leu Arg Val Glu Asn His Asn Asp Gln Asn Pro Leu Arg Val Leu
635 640 645
Arg Tyr Val Glu Val Phe Lys Asn Ser Asp Lys Glu Asp Asp Gln
650 655 660
Glu His Pro Ser Glu Lys Gln Pro Ser Gly Ala Glu Ser Gly Thr
665 670 675
Leu Ala Arg Ala Ser Leu Ala Leu Pro Thr Ser Ser Leu Ser Arg
680 685 690
Thr Ala Ser Gln Ser Ser Ser His Arg Gly Trp Glu Ile Leu Arg
695 700 705
Gln Asn Thr Leu Gly His Leu Asn Leu Gly Leu Asn Leu Ser Glu
710 715 720
Gly Asp Gly Glu Glu Val Tyr His Phe
725
<210> 23
<211> 492
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1471717CD1
<400> 23
Met Ala Thr Lys Pro Thr Glu Pro Val Thr Ile Leu Ser Leu Arg
1 5 10 15
Lys Leu Ser Leu Gly Thr Ala Glu Pro Gln Val Lys Glu Pro Lys
20 25 30
Thr Phe Thr Val Glu Asp Ala Val Glu Thr I1e Gly Phe Gly Arg
35 40 45
Phe His Ile Ala Leu Phe Leu Tle Met Gly Ser Thr Gly Val Val
50 55 60
Glu Ala Met Glu Ile Met Leu Ile Ala Val Val Ser Pro Val Ile
65 70 75
Arg Cys Glu Trp Gln Leu Glu Asn Trp Gln Val Ala Leu Val Thr
80 85 90
Thr Met Val Phe Phe Gly Tyr Met Val Phe Ser Ile Leu Phe Gly
95 100 105
Leu Leu Ala Asp Arg Tyr Gly Arg Trp Lys I1e Leu Leu Ile Ser
110 115 120
Phe Leu Trp Gly Ala Tyr Phe Ser Leu Leu Thr Ser Phe Ala Pro
125 130 135
Ser Tyr Ile Trp Phe Val Phe Leu Arg Thr Met Val Gly Cys Gly
140 145 150
Val Ser Gly His Ser Gln Gly Leu Ile Ile Lys Thr Glu Phe Leu
155 160 165
37/87
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Pro Thr Lys Tyr Arg Gly Tyr Met Leu Pro Leu Ser Gln Val Phe
170 175 180
Trp Leu A1a Gly Ser Leu Leu Ile Ile Gly Leu Ala Ser Val Ile
185 190 195
Ile Pro Thr Tle Gly Trp Arg Trp Leu Ile Arg Val Ala Ser I1e
200 205 210
Pro Gly Ile Ile Leu Ile Va1 Ala Phe Lys Phe Ile Pro Glu Ser
215 220 225
Ala Arg Phe Asn Val Ser Thr Gly Asn Thr Arg Ala Ala Leu Ala
230 235 240
Thr Leu Glu Arg Val Ala Lys Met Asn Arg Ser Val Met Pro G1u
245 250 255
Gly Lys Leu Val Glu Pro Val Leu G1u Lys Arg Gly Arg Phe A1a
260 265 270
Asp Leu Leu Asp Ala Lys Tyr Leu Arg Thr Thr Leu Gln Ile Trp
275 280 285
Val Ile Trp Leu Gly Ile Ser Phe Ala Tyr Tyr Gly Val Ile Leu
290 295 300
Ala Ser Ala Glu Leu Leu Glu Arg Asp Leu Val Cys Gly Ser Lys
305 310 315
Ser Asp Ser Ala Val Val Val Thr Gly Gly Asp Ser Gly Glu Ser
320 325 330
Gln Ser Pro Cys Tyr Cys His Met Phe Ala Pro Ser Asp Tyr Arg
335 340 345
Thr Met Ile Ile Ser Thr Ile Gly Glu Ile Ala Leu Asn Pro Leu
350 355 360
Asn Ile Leu Gly Ile Asn Phe Leu Gly Arg Arg Leu Ser Leu Ser
365 370 375
Ile Thr Met Gly Cys Thr Ala Leu Phe Cys Leu Leu Leu Asn Ile
380 385 390
Cys Thr Ser Ser Ala Gly Leu Ile Gly Phe Leu Phe Met Leu Arg
395 400 405
Ala Leu Val Ala Ala Asn Phe Asn Thr Val Tyr Ile Tyr Thr Ala
410 415 420
Glu Val Tyr Pro Thr Thr Met Arg Ala Leu Gly Met Gly Thr Ser
425 430 435
Gly Ser Leu Cys Arg Ile Gly Ala Met Val Ala Pro Phe Ile Ser
440 445 450
G1n Val Leu Met Ser Ala Ser Ile Leu Gly Ala Leu Cys Leu Phe
455 460 465
Ser Ser Val Cys Val Val Cys A1a I1e Ser Ala Phe Thr Leu Pro
470 475 480
Ile Glu Thr Lys Gly Arg Ala Leu Gln Gln Ile Lys
485 490
<210> 24
<211> 1494
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3874406CD1
<400> 24
Met Asn Met Lys Gln Lys Ser Val Tyr Gln Gln Thr Lys Ala Leu
1 5 10 15
Leu Cys Lys Asn Phe Leu Lys Lys Trp Arg Met Lys Arg G1u Ser
20 25 30
Leu Leu Glu Trp Gly Leu Ser Ile Leu Leu Gly Leu Cys Ile Ala
35 40 45
Leu Phe Ser Ser Ser Met Arg Asn Val Gln Phe Pro Gly Met Ala
50 55 60
Pro Gln Asn Leu G1y Arg Val Asp Lys Phe Asn Ser Ser Ser Leu
65 70 75
Met Val Va1 Tyr Thr Pro Ile Ser Asn Leu Thr Gln Gln Tle Met
80 85 90
38187
CA 02415808 2003-O1-06
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Asn Lys Thr Ala Leu Ala Pro Leu Leu Lys Gly Thr Ser Val Ile
95 100 105
GIy Ala Pro Asn Lys Thr His Met Asp Glu I1e Leu Leu Glu Asn
110 115 120
Leu Pro Tyr Ala Met Gly Ile Ile Phe Asn Glu Thr Phe Ser Tyr
125 130 135
Lys Leu Ile Phe Phe Gln Gly Tyr Asn Ser Pro Leu Trp Lys Glu
140 145 150
Asp Phe Ser Ala His Cys Trp Asp Gly Tyr Gly Glu Phe Ser Cys
155 160 165
Thr Leu Thr Lys Tyr Trp Asn Arg Gly Phe Va1 Ala Leu Gln Thr
170 175 180
Ala Ile Asn Thr Ala Ile Ile Glu Val Ala Leu Val Phe Leu Met
185 190 195
Ser Val Leu Leu Lys Lys Ala Val Leu Thr Asn Leu Val Val Phe
200 205 210
Leu Leu Thr Leu Phe Trp Gly Cys Leu Gly Phe Thr Va1 Phe Tyr
215 220 225
Glu Gln Leu Pro Ser Ser Leu Glu Trp Ile Leu Asn Ile Cys Ser
230 235 240
Pro Phe Ala Phe Thr Thr Gly Met Ile Gln Ile Ile Lys Leu Asp
245 250 255
Tyr Asn Leu Asn Gly Val Ile Phe Pro Asp Pro Ser Gly Asp Ser
260 265 270
Tyr Thr Met I1e Ala Thr Phe Ser Met Leu Leu Leu Asp Gly Leu
275 280 285
Ile Tyr Leu Leu Leu Ala Leu Tyr Phe Asp Lys Ile Leu Pro Tyr
290 295 300
Gly Asp Glu Arg His Tyr Ser Pro Leu Phe Phe Leu Asn Ser Ser
305 310 315
Ser Cys Phe Gln His Gln Arg Thr Asn Ala Lys Val Ile G1u Lys
320 325 330
Glu Ile Asp Ala Glu His Pro Ser,Asp Asp Tyr Phe Glu Pro Val
335 340 345
Ala Pro Glu Phe Gln Gly Lys G1u Ala IIe Arg Ile Arg Asn Val
350 355 360
Lys Lys Glu Tyr Lys Gly Lys Ser Gly Lys Va1 Glu Ala Leu Lys
365 370 375
Gly Leu Leu Phe Asp Ile Tyr Glu Gly Gln Ile Thr Ala Ile Leu
380 385 390
Gly His Ser Gly Ala Gly Lys Ser Ser Leu Leu Asn Ile Leu Asn
395 400 405
Gly Leu Ser Val Pro Thr Glu Gly Ser Val Thr Ile Tyr Asn Lys
410 415 420
Asn Leu Ser Glu Met Gln Asp Leu Glu G1u Ile Arg Lys Ile Thr
425 430 435
Gly Val Cys Pro Gln Phe Asn Val Gln Phe Asp Ile Leu Thr Val
440 445 450
Lys Glu Asn Leu Ser Leu Phe Ala Lys Ile Lys Gly Ile His Leu
455 460 465
Lys G1u Val Glu Gln Glu Val Gln Arg Ile Leu Leu Glu Leu Asp
470 475 480
Met Gln Asn Ile Gln Asp Asn Leu Ala Lys His Leu Ser Glu Gly
485 490 495
Gln Lys Arg Lys Leu Thr Phe Gly Ile Thr Ile Leu Gly Asp Pro
500 505 510
Gln Ile Leu Leu Leu Asp Glu Pro Thr Thr Gly Leu Asp Pro Phe
515 520 525
Ser Arg Asp Gln Val Trp Ser Leu Leu Arg Glu Arg Arg Ala Asp
530 535 540
His Val Ile Leu Phe Ser Thr Gln Ser Met Asp Glu Ala Asp Ile
545 550 555
Leu Ala Asp Arg Lys Val Ile Met Ser Asn Gly Arg Leu Lys Cys
560 565 570
Ala Gly Ser Ser Ile Phe Leu Lys Arg Arg Trp Gly Leu Gly Tyr
575 580 585
His Leu Ser Leu His Arg Asn G1u Ile Cys Asn Pro Glu Gln I1e
39!87
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590 595 600
Thr Ser Phe Ile Thr His His Ile Pro Asp Ala Lys Leu Lys Thr
605 610 615
Glu Asn Lys Glu Lys Leu Val Tyr Thr Leu Pro Leu Glu Arg Thr
620 625 630
Asn Thr Phe Pro Asp Leu Phe Ser Asp Leu Asp Lys Cys Ser Asp
635 640 645
G1n Gly Val Thr Gly Tyr Asp Ile Ser Met Ser Thr Leu Asn Glu
650 655 660
Val Phe Met Lys Leu Glu Gly Gln Ser Thr Ile Glu Gln Asp Phe
665 670 675
Glu Gln Val Glu Met Ile Arg Asp Ser Glu Ser Leu Asn Glu Met
680 685 690
Glu Leu Ala His Ser Ser Phe Ser Glu Met Gln Thr Ala Val Ser
695 700 705
Asp Met Gly Leu Trp Arg Met G1n Val Phe Ala Met Ala Arg Leu
710 715 720
Arg Phe Leu Lys Leu Lys Arg Gln Thr Lys Va1 Leu Leu Thr Leu
725 730 735
Leu Leu Va1 Phe Gly Ile Ala Ile Phe Pro Leu Ile Val Glu Asn
740 745 750
Ile Ile Tyr Ala Met Leu Asn Glu Lys Ile Asp Trp Glu Phe Lys
755 760 765
Asn Glu Leu Tyr Phe Leu Ser Pro Gly Gln Leu Pro Gln Glu Pro
770 775 780
Arg Thr Ser Leu Leu Ile Ile Asn Asn Thr Glu Ser Asn Ile Glu
785 790 795
Asp Phe Ile Lys Ser Leu Lys His Gln Asn Ile Leu Leu Glu Val
800 805 810
Asp Asp Phe Glu Asn Arg Asn Gly Thr Asp Gly Leu Ser Tyr Asn
815 820 825
G1y Ala Ile Ile Val Ser Gly Lys Gln Lys Asp Tyr Arg Phe Ser
830 835 840
Val Val Cys Asn Thr Lys Arg Leu His Cys Phe Pro Ile Leu Met
845 ' 850 855
Asn Ile Ile Ser Asn Gly Leu Leu Gln Met Phe Asn His Thr Gln
860 865 870
His Ile Arg Ile Glu Ser Ser Pro Phe Pro Leu Ser His Ile Gly
875 880 885
Leu Trp Thr Gly Leu Pro Asp Gly Ser Phe Phe Leu Phe Leu Val
890 895 900
Leu Cys Ser Ile Ser Pro Tyr Ile Thr Met Gly Ser Ile Ser Asp
905 910 915
Tyr Lys Lys Asn Ala Lys Ser Gln Leu Trp Ile Ser Gly Leu Tyr
920 925 930
Thr Ser Ala Tyr Trp Cys Gly Gln Ala Leu Val Asp Val Ser Phe
935 940 945
Phe Ile Leu Ile Leu Leu Leu Met Tyr Leu Ile Phe Tyr I1e Glu
950 955 960
Asn Met Gln Tyr Leu Leu Ile Thr Ser Gln Ile Val Phe Ala Leu
965 970 975
Val Ile Val Thr Pro Gly Tyr Ala Ala Ser Leu Val Phe Phe Ile
980 985 990
Tyr Met Ile Ser Phe Ile Phe Arg Lys Arg Arg Lys Asn Ser Gly
995 1000 1005
Leu Trp Ser Phe Tyr Phe Phe Phe Ala Ser Thr Ile Met Phe Ser
1010 1015 1020
Ile Thr Leu Tle Asn His Phe Asp Leu Ser Tle Leu Ile Thr Thr
1025 1030 1035
Met Val Leu Val Pro Ser Tyr Thr Leu Leu Gly Phe Lys Thr Phe
1040 1045 1050
Leu Glu Val Arg Asp Gln Glu His Tyr Arg Glu Phe Pro Glu Ala
1055 1060 1065
Asn Phe Glu Leu Ser Ala Thr Asp Phe Leu Val Cys Phe Ile Pro
1070 1075 1080
Tyr Phe Gln Thr Leu Leu Phe Val Phe Val Leu Arg Cys Met Glu
1085 1090 1095
40/87
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Leu Lys Cys Gly Lys Lys Arg Met Arg Lys Asp Pro Val Phe Arg
1100 1105 1110
Ile Ser Pro Gln Ser Arg Asp Ala Lys Pro Asn Pro G1u Glu Pro
1115 1120 1125
Ile Asp Glu Asp G1u Asp Ile Gln Thr Glu Arg Ile Arg Thr Val
1130 1135 2240
Thr Ala Leu Thr Thr Ser Ile Leu Asp Glu Lys Pro Val Ile Ile
1145 1150 1155
Ala Ser Cys Leu His Lys Glu Tyr Ala Gly Gln Lys Lys Ser Cys
1160 1165 1170
Phe Ser Lys Arg Lys Lys Lys Ile Ala Ala Arg Asn Ile Ser Phe
1175 1280 1185
Cys Val G1n Glu Gly Glu Ile Leu Gly Leu Leu Gly Pro Ser Gly
1190 1195 1200
Ala G1y Lys Ser Ser Ser Ile Arg Met Ile Ser Gly Ile Thr Lys
1205 1210 1215
Pro Thr Ala G1y Glu Val Glu Leu Lys Gly Cys Ser Ser Val Leu
1220 1225 1230
Gly His Leu Gly Tyr Cys Pro Gln Glu Asn Val Leu Trp Pro Met
1235 1240 1245
Leu Thr Leu Arg Glu His Leu Glu Val Tyr Ala Ala Val Lys Gly
1250 1255 1260
Leu Arg Glu Ala Asp Ala Arg Leu Ala Ile Ala Arg Leu Val Ser
1265 1270 1275
Ala Phe Lys Leu His Glu G1n Leu Asn Val Pro Val Gln Lys Leu
1280 1285 1290
Thr Ala Gly Ile Thr Arg Lys Leu Cys Phe Val Leu Ser Leu Leu
1295 1300 1305
Gly Asn Ser Pro Val Leu Leu Leu Asp G1u Pro Ser Thr Gly Ile
1310 1315 1320
Asp Pro Thr Gly Gln Gln Gln Met Trp Gln Ala Ile Gln Ala Val
1325 1330 1335
Val Lys Asn Thr Glu Arg Gly Val Leu Leu Thr Thr His Asn Leu
1340 1345 1350
Ala Glu Ala Glu Ala Leu Cys Asp Arg Val Ala Ile Met Val Ser
1355 1360 1365
Gly Arg Leu Arg Cys Ile Gly Ser Ile Gln His Leu Lys Asn Lys
1370 1375 1380
Leu Gly Lys Asp Tyr Ile Leu Glu Leu Lys Val Lys G1u Thr Ser
1385 1390 1395
Gln Val Thr Leu Val His Thr Glu I1e Leu Lys Leu Phe Pro Gln
2400 1405 1410
Ala Ala Gly Gln Gln Arg Tyr Ser Ser Leu Leu Thr Tyr Lys Leu
1415 1420 1425
Pro Val Ala Asp Val Tyr Pro Leu Ser Gln Thr Phe His Lys Leu
1430 1435 1440
G1u Ala Val Lys His Asn Phe Asn Leu G1u Glu Tyr Ser Leu Ser
1445 1450 1455
Gln Cys Thr Leu Glu Lys Val Phe Leu G1u Leu Ser Lys Glu Gln
1460 1465 ~ 1470
Glu Val Gly Asn Phe Asp Glu Glu Ile Asp Thr Thr Met Arg Trp
1475 1480 1485
Lys Leu Leu Pro His Ser Asp Glu Pro
1490
<210> 25
<211> 774
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4599654CD1
<400> 25
Met Glu Ala Glu Gln Arg Pro Ala Ala Gly Ala Ser Glu Gly Ala
1 5 10 15
41/87
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Thr Pro Gly Leu Glu Ala Val Pro Pro Val Ala Pro Pro Pro Ala
20 25 30
Thr Ala Ala Ser Gly Pro Ile Pro Lys Ser Gly Pro Glu Pro Lys
35 40 45
Arg Arg His Leu Gly Thr Leu Leu Gln Pro Thr Val Asn Lys Phe
50 55 60
Ser Leu Arg Val Phe Gly Ser His Lys Ala Val Glu Ile Glu Gln
65 70 75
Glu Arg Val Lys Ser Ala Gly Ala Trp Ile Ile His Pro Tyr Ser
80 85 90
Asp Phe Arg Phe Tyr Trp Asp Leu Ile Met Leu Leu Leu Met Val
95 100 105
Gly Asn Leu Ile Val Leu Pro Val Gly Ile Thr Phe Phe Lys Glu
110 115 120
Glu Asn Ser Pro Pro Trp Ile Val Phe Asn Val Leu Ser Asp Thr
125 130 135
Phe Phe Leu Leu Asp Leu Val Leu Asn Phe Arg Thr Gly Ile Val
140 145 150
Val Glu Glu Gly A1a Glu Ile Leu Leu Ala Pro Arg Ala Ile Arg
155 160 165
Thr Arg Tyr Leu Arg Thr Trp Phe Leu Val Asp Leu Ile Ser Ser
170 175 180
Ile Pro Val Asp Tyr Ile Phe Leu Val Val Glu Leu Glu Pro Arg
185 190 195
Leu Asp Ala Glu Val Tyr Lys Thr A1a Arg Ala Leu Arg Ile Val
200 205 210
Arg Phe Thr Lys Ile Leu Ser Leu Leu Arg Leu Leu Arg Leu Ser
215 220 225
Arg Leu Ile Arg Tyr Ile His Gln Trp Glu Glu Ile Phe His Met
230 235 240
Thr Tyr Asp Leu Ala Ser Ala Val Val Arg I1e Phe Asn Leu I1e
245 250 255
Gly Met Met Leu Leu Leu Cys His Trp Asp Gly Cys Leu Gln Phe
260 265 270
Leu Val Pro Met Leu Gln Asp Phe Pro Pro Asp Cys Trp Val Ser
275 280 285
Tle Asn His Met Val Asn His Ser Trp Gly Arg Gln Tyr Ser His
290 295 300
Ala Leu Phe Lys Ala Met Ser His Met Leu Cys Ile Gly Tyr Gly
305 310 315
G1n Gln Ala Pro Val Gly Met Pro Asp Val Trp Leu Thr Met Leu
320 325 330
Ser Met Ile Val Gly Ala Thr Cys Tyr Ala Met Phe Ile Gly His
335 340 345
Ala Thr Ala Leu Ile Gln Ser Leu Asp Ser Ser Arg Arg Gln Tyr
350 355 360
Gln Glu Lys Tyr Lys Gln Val Glu Gln Tyr Met Ser Phe His Lys
365 370 375
Leu Pro Ala Asp Thr Arg Gln Arg Ile His Glu Tyr Tyr Glu His
380 385 390
Arg Tyr Gln Gly Lys Met Phe Asp Glu Glu Ser Ile Leu Gly Glu
395 400 405
Leu Ser Glu Pro Leu Arg Glu Glu Ile Ile Asn Phe Thr Cys Arg
410 415 420
Gly Leu Val Ala His Met Pro Leu Phe Ala His Ala Asp Pro Ser
425 430 435
Phe Val Thr Ala Va1 Leu Thr Lys Leu Arg Phe Glu Val Phe Gln
440 445 450
Pro Gly Asp Leu Val Val Arg Glu Gly Ser Val Gly Arg Lys Met
455 460 465
Tyr Phe Ile Gln His Gly Leu Leu Ser Val Leu Ala Arg Gly Ala
470 475 480
Arg Asp Thr Arg Leu Thr Asp Gly Ser Tyr Phe Gly Glu Ile Cys
485 490 495
Leu Leu Thr Arg Gly Arg Arg Thr Ala Ser Val Arg Ala Asp Thr
500 505 510
Tyr Cys Arg Leu Tyr Ser Leu Ser Val Asp His Phe Asn Ala Val
42/87
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515 520 525
Leu Glu Glu Phe Pro Met Met Arg Arg Ala Phe Glu Thr Val Ala
530 535 540
Met Asp Arg Leu Leu Arg I1e Gly Lys Lys Asn Ser Tle Leu Gln
545 550 555
Arg Lys Arg Ser Glu Pro Ser Pro Gly Ser Ser Gly Gly Ile Met
560 565 570
Glu Gln His Leu Val Gln His Asp Arg Asp Met Ala Arg Gly Va1
575 580 585
Arg Gly Arg Ala Pro Ser Thr Gly Ala Gln Leu Ser Gly Lys Pro
590 595 600
Val Leu Trp Glu Pro Leu Val His Ala Pro Leu Gln Ala A1a Ala
605 610 615
Val Thr Ser Asn Va1 Ala Ile Ala Leu Thr His Gln Arg Gly Pro
620 625 630
Leu Pro Leu Ser Pro Asp Ser Pro Ala Thr Leu Leu Ala Arg Ser
635 640 645
Ala Trp Arg Ser Ala Gly Ser Pro Ala Ser Pro Leu Val Pro Val
650 655 660
Arg Ala Gly Pro Trp Ala Ser Thr Ser Arg Leu Pro Ala Pro Pro
665 670 675
Ala Arg Thr Leu His Ala Ser Leu Ser Arg Ala Gly Arg Ser Gln
680 685 690
Val Ser Leu Leu Gly Pro Pro Pro Gly Gly Gly Gly Arg Arg Leu
695 700 705
Gly Pro Arg Gly Arg Pro Leu Ser Ala Ser G1n Pro Ser Leu Pro
710 7l5 720
Gln Arg Ala Thr Gly Asp Gly Ser Pro Gly Arg Lys Gly Ser Gly
725 730 735
Ser Glu Arg Leu Pro Pro Ser Gly Leu Leu Ala Lys Pro Pro Arg
740 745 750
Thr Ala Gln Pro Pro Arg Pro Pro Val Pro Glu Pro Ala Thr Pro
755 760 765
Arg Gly Leu Gln Leu Ser Ala Asn Met
770
<210> 26
<211> 614
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5047435CD1
<400> 26
Met A1a Glu Gly Glu Arg Gly Ala Asp Val Pro His Gly Leu Gly
1 5 10 15
Ala Trp Leu Ala Asp Val Ala Leu Ala Ala Leu Arg Ala G1y Gly
20 25 30
Gln Gly Arg Arg Asp Arg Gly Gly Gly Gly Pro Glu Ser Leu Ser
35 40 45
Gly Gly Ser Gly Val Gly Asp Ser Gly Gly Gly Cys Ala Pro Gly
50 55 60
Pro Ser Ala Pro Pro Ala Arg Arg Arg Val Pro Leu Ala Met Gly
65 70 75
His Ser Pro Pro Val Leu Pro Leu Cys Ala Ser Val Ser Leu Leu
80 85 90
Gly Gly Leu Thr Phe Gly Tyr Glu Leu Ala Val Ile Ser Gly Ala
95 100 105
Leu Leu Pro Leu Gln Leu Asp Phe G1y Leu Ser Cys Leu Glu Gln
1l0 115 120
Glu Phe Leu Val Gly Ser Leu Leu Leu Gly Ala Leu Leu Ala Ser
125 130 135
Leu Va1 Gly Gly Phe Leu Ile Asp Cys Tyr Gly Arg Lys Gln Ala
140 145 150
Ile Leu Gly Ser Asn Leu Val Leu Leu Ala Gly Ser Leu Thr Leu
43/87
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155 160 165
Gly Leu Ala Gly Ser Leu Ala Trp Leu Val Leu Gly Arg Ala Val
170 175 180
Val G1y Phe Ala Tle Ser Leu Ser Ser Met Ala Cys Cys Ile Tyr
185 190 195
Val Ser Glu Leu Val Gly Pro Arg Gln Arg Gly Val Leu Val Ser
200 205 210
Leu Tyr Glu Ala Gly Ile Thr Val G1y Ile Leu Leu Ser Tyr Ala
215 220 225
Leu Asn Tyr Ala Leu Ala Gly Thr Pro Trp Gly Trp Arg His Met
230 235 240
Phe Gly Trp Ala Thr Ala Pro Ala Val Leu Gln Ser Leu Ser Leu
245 250 255
Leu Phe Leu Pro Ala Gly Thr Asp Glu Thr A1a Thr His Lys Asp
260 265 270
Leu Ile Pro Leu Gln Gly Gly GIu Ala Pro Lys Leu Gly Pro Gly
275 280 285
Arg Pro Arg Tyr Ser Phe Leu Asp Leu Phe Arg Ala Arg Asp Asn
290 295 300
Met Arg Gly Arg Thr Thr Val Gly Leu Gly Leu Val Leu Phe Gln
305 310 315
Gln Leu Thr Gly Gln Pro Asn Val Leu Cys Tyr Ala Ser Thr Ile
320 325 330
Phe Ser Ser Val Gly Phe His Gly Gly Ser Ser Ala Val Leu Ala
335 340 345
Ser Val Gly Leu Gly Ala Val Lys Val Ala Ala Thr Leu Thr Ala
350 355 360
Met Gly Leu Val Asp Arg Ala Gly Arg Arg Ala Leu Leu Leu Ala
365 370 375
Gly Cys Ala Leu Met Ala Leu Ser Val Ser Gly Ile Gly Leu Va1
380 385 390
Ser Phe AIa Val Pro Met Asp Ser Gly Pro Ser Cys Leu Ala Val
395 400 405
Pro Asn Ala Thr Gly Gln Thr Gly Leu Pro Gly Asp Ser Gly Leu
410 415 420
Leu Gln Asp Ser Ser Leu Pro Pro Ile Pro Arg Thr Asn Glu Asp
425 430 435
Gln Arg Glu Pro Ile Leu Ser Thr Ala Lys Lys Thr Lys Pro His
440 445 450
Pro Arg Ser Gly Asp Pro Ser Ala Pro Pro Arg Leu Ala Leu Ser
455 460 465
Ser Ala Leu Pro Gly Pro Pro Leu Pro Ala Arg Gly His A1a Leu
470 475 480
Leu Arg Trp Thr Ala Leu Leu Cys Leu Met Val Phe Val Ser Ala
485 490 495
Phe Ser Phe Gly Phe Gly Pro Val Thr Trp Leu Val Leu Ser Glu
500 505 510
Ile Tyr Pro Val Glu Ile Arg Gly Arg Ala Phe Ala Phe Cys Asn
515 520 525
Ser Phe Asn Trp Ala A1a Asn Leu Phe Ile Ser Leu Ser Phe Leu
530 535 540
Asp Leu Ile Gly Thr Ile Gly Leu Ser Trp Thr Phe Leu Leu Tyr
545 550 555
Gly Leu Thr Ala Val Leu Gly Leu Gly Phe Ile Tyr Leu Phe Val
560 565 570
Pro Glu Thr Lys Gly Gln Ser Leu Ala Glu Ile Asp Gln Gln Phe
575 580 585
G1n Lys Arg Arg Phe Thr Leu Ser Phe Gly His Arg G1n Asn Ser
590 595 600
Thr Gly Ile Pro Tyr Ser Arg Ile Glu Ile Ser Ala Ala Ser
605 610
<210> 27
<211> 2180
<212> PRT
<213> Homo Sapiens
44/87
CA 02415808 2003-O1-06
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<220>
<221> misc_feature
<223> Incyte ID No: 7475603CD1
<400> 27
Met Arg Phe Arg Lys Gly Gln Glu Leu Pro Ala Ala Ala Pro His
1 5 10 15
Val Phe Ser Pro Thr Val Val Leu Thr Ser Leu Ser Arg Pro Leu
20 25 30
Pro Ser Leu Thr Met Ala Phe Trp Thr Gln Leu Met Leu Leu Leu
35 40 45
Trp Lys Asn Phe Met Tyr Arg Arg Arg Gln Pro Val Gln Leu Leu
50 55 60
Val Glu Leu Leu Trp Pro Leu Phe Leu Phe Phe Ile Leu Val Ala
65 70 75
Val Arg His Ser His Pro Pro Leu Glu His His Glu Cys His Phe
80 85 90
Pro Asn Lys Pro Leu Pro Ser Ala Gly Thr Val Pro Trp Leu Gln
95 100 105
Gly Leu Ile Cys Asn Val Asn Asn Thr Cys Phe Pro Gln Leu Thr
110 115 120
Pro Gly Glu Glu Pro Gly Arg Leu Ser Asn Phe Asn Asp Ser Leu
125 130 135
Val Ser Arg Leu Leu Ala Asp Ala Arg Thr Val Leu Gly Gly Ala
140 145 150
Ser Ala His Arg Thr Leu Ala Gly Leu Gly Lys Leu Ile Ala Thr
155 160 165
Leu Arg Ala Ala Arg Ser Thr Ala Gln Pro Gln Pro Thr Lys Gln
170 175 180
Ser Pro Leu Glu Pro Pro Met Leu Asp Val Ala Glu Leu Leu Thr
185 190 195
Ser Leu Leu Arg Thr G1u Ser Leu Gly Leu A1a Leu Gly Gln Ala
200 205 2l0
Gln Glu Pro Leu His Ser Leu Leu Glu Ala Ala Glu Asp Leu Ala
215 220 225
G1n G1u Leu Leu Ala Leu Arg Ser Leu Val Glu Leu Arg A1a Leu
230 235 240
Leu Gln Arg Pro Arg Gly Thr Ser Gly Pro Leu Glu Leu Leu Ser
245 250 . 255
Glu Ala Leu Cys Ser Val Arg Gly Pro Ser Ser Thr Val Gly Pro
260 265 270
Ser Leu Asn Trp Tyr Glu Ala Ser Asp Leu Met Glu Leu Val Gly
275 280 285
Gln Glu Pro-Glu Ser Ala Leu Pro Asp Ser Ser Leu Ser Pro Ala
290 295 300
Cys Ser G1u Leu Ile Gly Ala Leu Asp Ser His Pro Leu Ser Arg
305 310 315
Leu Leu Trp Arg Arg Leu Lys Pro Leu Ile Leu Gly Lys Leu Leu
320 325 330
Phe Ala Pro Asp Thr Pro Phe Thr Arg Lys Leu Met Ala G1n Va1
335 340 345
Asn Arg Thr Phe Glu Glu Leu Thr Leu Leu Arg Asp Val Arg Glu
350 355 360
Val Trp Glu Met Leu Gly Pro Arg Ile Phe Thr Phe Met Asn Asp
365 370 375
Ser Ser Asn Val Ala Met Leu Gln Arg Leu Leu Gln Met Gln Asp
380 385 390
Glu Gly Arg Arg Gln Pro Arg Pro G1y Gly Arg Asp His Met Glu
395 400 405
Ala Leu Arg Ser Phe Leu Asp Pro Gly Ser Gly Gly Tyr Ser Trp
410 415 420
Gln Asp Ala His A1a Asp Val Gly His Leu Val Gly Thr Leu Gly
425 430 435
Arg Val Thr Glu Cys Leu Ser Leu Asp Lys Leu Glu Ala Ala Pro
440 445 450
Ser Glu Ala Ala Leu Val Ser Arg Ala Leu Gln Leu Leu Ala Glu
455 460 465
45/7
CA 02415808 2003-O1-06
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His Arg Phe Trp Ala Gly Val Val Phe Leu Gly Pro Glu Asp Ser
470 475 480
Ser Asp Pro Thr Glu His Pro Thr Pro Asp Leu Gly Pro G1y His
485 490 495
Val Arg Ile Lys I1e Arg Met Asp Ile Asp Val Val Thr Arg Thr
500 505 510
Asn Lys I1e Arg Asp Arg Phe Trp Asp Pro Gly Pro Ala Ala Asp
515 520 525
Pro Leu Thr Asp Leu Arg Tyr Val Trp G1y Gly Phe Val Tyr Leu
530 535 540
Gln Asp Leu Val Glu Arg Ala Ala Val Arg Val Leu Ser Gly Ala
545 550 555
Asn Pro Arg Ala Gly Leu Tyr Leu Gln Gln Met Pro Tyr Pro Cys
560 565 570
Tyr Val Asp Asp Val Phe Leu Arg Val Leu Ser Arg Ser Leu Pro
575 580 585
Leu Phe Leu Thr Leu Ala Trp Ile Tyr Ser Val Thr Leu Thr Val
590 595 600
Lys Ala Val Val Arg Glu Lys Glu Thr Arg Leu Arg Asp Thr Met
605 610 615
Arg A1a Met Gly Leu Ser Arg Ala Val Leu Trp Leu Gly Trp Phe
620 625 630
Leu Ser Cys Leu Gly Pro Phe Leu Leu Ser Ala Ala Leu Leu Val
635 640 645
Leu Va1 Leu Lys Leu Gly Asp Ile Leu Pro Tyr Ser His Pro Gly
650 655 660
Val Val Phe Leu Phe Leu Ala Ala Phe Ala Val Ala Thr Val Thr
665 670 675
Gln Ser Phe Leu Leu Ser Ala Phe Phe Ser Arg Ala Asn Leu Ala
680 685 690
Ala Ala Cys Gly Gly Leu Ala Tyr Phe Ser Leu Tyr Leu Pro Tyr
695 700 705
Val Leu Cys Val Ala Trp Arg Asp Arg Leu Pro Ala Gly Gly Arg
710 715 720
Val Ala Ala Ser Leu Leu Ser Pro Val Ala Phe Gly Phe Gly Cys
725 730 735
Glu Sex Leu Ala Leu Leu Glu Glu Gln G1y Glu Gly Ala Gln Trp
740 745 750
His Asn Val Gly Thr Arg Pro Thr Ala Asp Val Phe Ser Leu Ala
755 760 765
Gln Val Ser Gly Leu Leu Leu Leu Asp Ala Ala Leu Tyr Gly Leu
770 775 780
Ala Thr Trp Tyr Leu Glu Ala Val Cys Pro Gly Gln Tyr Gly Ile
785 790 795
Pro Glu Pro Trp Asn Phe Pro Phe Arg Arg Ser Tyr Trp Cys Gly
800 805 810
Pro Arg Pro Pro Lys Ser Pro Ala Pro Cys Pro Thr Pro Leu Asp
815 820 825
Pro Lys Val Leu Val Glu Glu Ala Pro Pro Gly Leu Ser Pro Gly
830 835 840
Val Ser Val Arg Ser Leu Glu Lys Arg Phe Pro G1y Ser Pro Gln
845 850 855
Pro Ala Leu Arg Gly Leu Ser Leu Asp Phe Tyr Gln Gly His Ile
860 865 870
Thr Ala Phe Leu Gly His Asn Gly Ala Gly Lys Thr Thr Thr Leu
875 880 885
Ser Ile Leu Ser Gly Leu Phe Pro Pro Ser Gly Gly Ser Ala Phe
890 895 900
Ile Leu Gly His Asp Val Arg Ser Ser Met Ala Ala Ile Arg Pro
905 910 915
His Leu Gly Val Cys Pro Gln Tyr Asn Val Leu Phe Asp Met Leu
920 925 930
Thr Val Asp Glu His Val Trp Phe Tyr Gly Arg Leu Lys Gly Leu
935 940 945
Ser Ala Ala Val Val Gly Pro Glu Gln Asp Arg Leu Leu Gln Asp
950 955 960
Val Gly Leu Val Ser Lys Gln Ser Val Gln Thr Arg His Leu Ser
46/87
Val Ser Glu Leu Val Gly Pro Arg Gln Arg Gly Val Leu Val Ser
200
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
965 970 975
Gly Gly Met Gln Arg Lys Leu Ser Val Ala Ile Ala Phe Val Gly
980 985 990
Gly Ser Gln Val Val Ile Leu Asp Glu Pro Thr Ala Gly Val Asp
995 1000 1005
Pro Ala Ser Arg Arg Gly I1e Trp Glu Leu Leu Leu Lys Tyr Arg
1010 1015 1020
G1u Gly Arg Thr Leu Ile Leu Ser Thr His His Leu Asp Glu Ala
1025 1030 1035
Glu Leu Leu Gly Asp Arg Va1 Ala Val Val Ala Gly Gly Arg Leu
1040 1045 1050
Cys Cys Cys Gly Ser Pro Leu Phe Leu Arg Arg His Leu Gly Ser
1055 2060 1065
Gly Tyr Tyr Leu Thr Leu Val Lys Ala Arg Leu Pro Leu Thr Thr
1070 1075 2080
Asn Glu Lys Ala Asp Thr Asp Met Glu Gly Ser Val Asp Thr Arg
1085 1090 1095
Gln Glu Lys Lys Asn Gly Ser Gln Gly Ser Arg Val Gly Thr Pro
1100 1105 1110
Gln Leu Leu Ala Leu Val Gln His Trp Val Pro Gly Ala Arg Leu
1115 1120 1125
Val Glu Glu Leu Pro His Glu Leu Val Leu Val Leu Pro Tyr Thr
1130 2135 1140
Gly Ala His Asp Gly Ser Phe Ala Thr Leu Phe Arg Glu Leu Asp
1145 1150 1155
Thr Arg Leu Ala Glu Leu Arg Leu Thr Gly Tyr Gly Ile Ser Asp
2160 1165 2270
Thr Ser Leu Glu Glu Ile Phe Leu Lys Val Val G1u Glu Cys Ala
1175 1180 1185
Ala Asp Thr Asp Met Glu Asp Gly Ser Cys Gly Gln His Leu Cys
1190 1195 1200
Thr Gly Ile Ala G1y Leu Asp Val Thr Leu Arg Leu Lys Met Pro
1205 1210 1215
Pro Gln Glu Thr Ala Leu Glu Asn Gly Glu Pro Ala Gly Ser A1a
1220 1225 1230
Pro Glu Thr Asp Gln Gly Ser Gly Pro Asp Ala Val Gly Arg Val
1235 1240 1245
Gln Gly Trp Ala Leu Thr Arg Gln Gln Leu Gln Ala Leu Leu Leu
1250 1255 1260
Lys Arg Phe Leu Leu Ala Arg Arg Sex Arg Arg Gly Leu Phe Ala
1265 1270 1275
Gln Ile Val Leu Pro Ala Leu Phe Val G1y Leu Ala Leu Val Phe
1280 2285 1290
Ser Leu Ile Val Pro Pro Phe Gly His Tyr Pro Ala Leu Arg Leu
1295 1300 1305
Ser Pro Thr Met Tyr Gly Ala G1n Val Ser Phe Phe Ser Glu Asp
2310 1315 1320
Ala Pro Gly Asp Pro Gly Arg Ala Arg Leu Leu Glu Ala Leu Leu
1325 1330 1335
Gln Glu Ala Gly Leu Glu Glu Pro Pro Val Gln His Ser Ser His
2340 1345 1350
Arg Phe Ser Ala Pro Glu Val Pro Ala Glu Val Ala Lys Val Leu
2355 1360 1365
Ala Ser Gly Asn Trp Thr Pro Glu Ser Pro Ser Pro Ala Cys Gln
2370 1375 1380
Cys Ser Arg Pro Gly Ala Arg Arg Leu Leu Pro Asp Cys Pro Ala
1385 1390 1395
Ala Ala Gly Gly Pro Pro Pro Pro Gln Ala VaI Thr Gly Ser Gly
2400 1405 1420
Glu Val Val Gln Asn Gln Thr Gly Arg Asn Leu Ser Asp Phe Leu
1415 1420 1425
Va1 Lys Thr Tyr Pro Arg Leu Val Arg Gln Gly Leu Lys Thr Lys
1430 1435 1440
Lys Trp Val Asn Glu Val Arg Tyr Gly Gly Phe Ser Leu Gly Gly
1445 1450 1455
Arg Asp Pro Gly Leu Pro Ser Gly Gln Glu Leu Gly Arg Ser Val
1460 1465 1470
47/87
CA 02415808 2003-O1-06
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Glu Glu Leu Trp Ala Leu Leu Ser Pro Leu Pro Gly Gly Ala Leu
1475 1480 1485
Asp Arg Val Leu Lys Asn Leu Thr Ala Trp Ala His Ser Leu Asp
1490 1495 1500
Ala Gln Asp Ser Leu Lys Ile Trp Phe Asn Asn Lys Gly Trp His
1505 1510 1515
Ser Met Val Ala Phe Val Asn Arg Ala Ser Asn Ala Ile Leu Arg
1520 1525 1530
Ala His Leu Pro Pro Gly Pro Ala Arg His Ala His Ser I1e Thr
1535 1540 1545
Thr Leu Asn His Pro Leu Asn Leu Thr Lys Glu Gln Leu Ser Glu
1550 1555 1560
Ala Ala Leu Met Ala Ser Ser Val Asp Val Leu Val Ser Ile Cys
1565 1570 1575
Val Val Phe Ala Met Ser Phe Val Pro Ala Ser.Phe Thr Leu Val
1580 1585 1590
Leu Ile Glu Glu Arg Val Thr Arg Ala Lys His Leu Gln Leu Met
1595 1600 1605
Gly Gly Leu Ser Pro Thr Leu Tyr Trp Leu Gly Asn Phe Leu Trp
1610 1615 1620
Asp Met Cys Asn Tyr Leu Val Pro Ala Cys Ile Val Val Leu Ile
1625 1630 1635
Phe Leu Ala Phe Gln Gln Arg Ala Tyr Val Ala Pro Ala Asn Leu
1640 1645 1650
Pro Ala Leu Leu Leu Leu Leu Leu Leu Tyr Gly Trp Ser Ile Thr
1655 1660 1665
Pro Leu Met Tyr Pro Ala Ser Phe Phe Phe Ser Val Pro Ser Thr
1670 1675 1680
Ala Tyr Val Val Leu Thr Cys Ile Asn Leu Phe Ile Gly Ile Asn
1685 . 1690 1695
G1y Ser Met A1a Thr Phe Val Leu Glu Leu Phe Ser Asp Gln Lys
1700 1705 1710
Leu Gln Glu Val Ser Arg Ile Leu Lys Gln Val Phe Leu Ile Phe
1715 1720 1725
Pro His Phe Cys Leu Gly Arg Gly Leu Ile Asp Met Val Arg Asn
1730 1735 1740
Gln Ala Met Ala Asp Ala Phe Glu Arg Leu Gly Asp Arg Gln Phe
1745 1750 1755
Gln Ser Pro Leu Arg Trp Glu Val Val Gly Lys Asn Leu Leu Ala
1760 1765 1770
Met Val Ile Gln Gly Pro Leu Phe Leu Leu Phe Thr Leu Leu Leu
1775 1780 1785
Gln His Arg Ser Gln Leu Leu Pro Gln Pro Arg Val Arg Ser Leu
1790 1795 1800
Pro Leu Leu Gly Glu Glu Asp Glu Asp Val Ala Arg Glu Arg Glu
1805 1810 1815
Arg Val Val G1n Gly Ala Thr Gln Gly Asp Val Leu Val Leu Arg
1820 1825 1830
Asn Leu Thr Lys Val Tyr Arg Gly Gln Arg Met Pro Ala Val Asp
1835 1840 1845
Arg Leu Cys Leu Gly Ile Pro Pro Gly Glu Cys Phe G1y Leu Leu
1850 1855 1860
Gly Val Asn Gly Ala Gly Lys Thr Ser Thr Phe Arg Met Va1 Thr
1865 1870 1875
Gly Asp Thr Leu A1a Ser Arg Gly Glu Ala Val Leu Ala Gly His
1880 1885 1890
Ser Val Ala Arg Glu Pro Ser Ala Ala His Leu Ser Met Gly Tyr
1895 1900 1905
Cys Pro Gln Ser Asp Ala Ile Phe Glu Leu Leu Thr Gly Arg Glu
1910 1915 1920
His Leu G1u Leu Leu Ala Arg Leu Arg Gly Val Pro Glu Ala Gln
1925 1930 1935
Val Ala Gln Thr A1a Gly Ser Gly Leu Ala Arg Leu Gly Leu Ser
1940 1945 1950
Trp Tyr Ala Asp Arg Pro Ala Gly Thr Tyr Ser Gly Gly Asn Lys
1955 1960 1965
Arg Lys Leu Ala Thr Ala Leu Ala Leu Val Gly Asp Pro Ala Val
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
1970 1975 1980
Val Phe Leu Asp Glu Pro Thr Thr Gly Met Asp Pro Ser Ala Arg
1985 1990 1995
Arg Phe Leu Trp Asn Ser Leu Leu Ala Val Val Arg Glu Gly Arg
2000 2005 2010
Ser Val Met Leu Thr Ser His Ser Met Glu Glu Cys Glu Ala Leu
2015 2020 2025
Cys Ser Arg Leu Ala Ile Met Val Asn G1y Arg Phe Arg Cys Leu
2030 2035 2040
Gly Ser Pro Gln His Leu Lys Gly Arg Phe Ala Ala Gly His Thr
2045 2050 2055
Leu Thr Leu Arg Val Pro Ala Ala Arg Ser Gln Pro Ala Ala Ala
2060 2065 2070
Phe Val Ala Ala Glu Phe Pro Gly Ala Glu Leu Arg Glu Ala His
2075 2080 2085
G1y Gly Arg Leu Arg Phe Gln Leu Pro Pro Gly Gly Arg Cys Ala
2090 2095 2100
Leu Ala Arg Val Phe Gly Glu Leu Ala Val His G1y Ala Glu His
2105 2110 2115
Gly Val Glu Asp Phe Ser Va1 Ser Gln Thr Met Leu Glu Glu Val
2120 2125 2130
Phe Leu Tyr Phe Ser Lys Asp Gln Gly Lys Asp Glu Asp Thr G1u
2135 2140 2145
Glu Gln Lys Glu Ala Gly Val Gly Val Asp Pro Ala Pro Gly Leu
2150 2155 21&0
Gln His Pro Lys Arg Val Ser G1n Phe Leu Asp Asp Pro Ser Thr
21&5 2170 2175
Ala Glu Thr Val Leu
2180
<210> 28
<211> 1737
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7477845CD1
<400> 28
Met Leu Lys Arg Lys Gln Ser Ser Arg Va1 Glu Ala G1n Pro Val
1 5 10 15
Thr Asp Phe Gly Pro Asp Glu Ser Leu Ser Asp Asn Ala Asp Ile
20 25 30
Leu Trp Ile Asn Lys Pro~Trp Val His Ser Leu Leu Arg Ile Cys
35 40 45
Ala Ile Ile Ser Val I1e Ser Val Cys Met Asn Thr Pro Met Thr
50 55 60
Phe Glu His Tyr Pro Pro Leu Gln Tyr Val Thr Phe Thr Leu Asp
65 70 75
Thr Leu Leu Met Phe Leu Tyr Thr Ala Glu Met Ile Ala Lys Met
80 85 90
His Ile Arg Gly Ile Val Lys Gly Asp Ser Ser Tyr Val Lys Asp
95 100 105
Arg Trp Cys Val Phe Asp Gly Phe Met Val Phe Cys Leu Trp Val
110 115 120
Ser Leu Val Leu Gln Val Phe Glu Ile Ala Asp Ile Val Asp Gln
125 13 0 13 5
Met Ser Pro Trp Gly Met Leu Arg Ile Pro Arg Pro Leu I1e Met
140 145 150
Ile Arg Ala Phe Arg Ile Tyr Phe Arg Phe Glu Leu Pro Arg Thr
155 160 165
Arg Ile Thr Asn Ile Leu Lys Arg Ser Gly Glu Gln I1e Trp Ser
170 175 180
Val Ser Ile Phe Leu Leu Phe Phe Leu Leu Leu Tyr Gly Ile Leu
185 190 195
Gly Val Gln Met Phe Gly Thr Phe Thr Tyr His Cys Val Val Asn
49/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
200 205 210
Asp Thr Lys Pro Gly Asn Val Thr Trp Asn Ser Leu Ala Ile Pro
215 220 225
Asp Thr His Cys Ser Pro Glu Leu Glu Glu Gly Tyr Gln Cys Pro
230 235 240
Pro Gly Phe Lys Cys Met Asp Leu Glu Asp Leu Gly Leu Ser Arg
245 250 255
Gln Glu Leu Gly Tyr Ser Gly Phe Asn Glu Ile Gly Thr Ser Ile
260 265 270
Phe Thr Val Tyr Glu Ala Ala Ser Gln Glu Gly Trp Val Phe Leu
275 280 285
Met Tyr Arg Ala Ile Asp Ser Phe Pro Arg Trp Arg Ser Tyr Phe
290 295 300
Tyr Phe Ile Thr Leu Ile Phe Phe Leu Ala Trp Leu Val Lys Asn
305 310 315
Val Phe Ile Ala Val Ile Ile Glu Thr Phe Ala Glu Ile Arg Val
320 325 330
G1n Phe Gln G1n Met Trp Gly Ser Arg Ser Ser Thr Thr Ser Thr
335 340 345
Ala Thr Thr Gln Met Phe His Glu Asp Ala Ala Gly Gly Trp Gln
350 355 360
Leu Val Ala Val Asp Val Asn Lys Pro Gln Gly Arg Ala Pro Ala
365 370 375
Cys Leu Gln Lys Met Met Arg Ser Ser Val Phe His Met Phe Ile
380 385 390
Leu Ser Met Val Thr Val Asp Val Ile Val Ala Ala Ser Asn Tyr
395 400 405
Tyr Lys Gly Glu Asn Phe Arg Arg Gln Tyr Asp Glu Phe Tyr Leu
410 415 420
Ala Glu Val Ala Phe Thr Val Leu Phe Asp Leu Glu Ala Leu Leu
425 430 435
Lys Tle Trp Cys Leu Gly Phe Thr Gly Tyr Ile Ser Ser Ser Leu
440 445 450
His Lys Phe Glu Leu Leu Leu Val Ile Gly Thr Thr Leu His Val
455 460 465
Tyr Pro Asp Leu Tyr His Ser Gln Phe Thr Tyr Phe Gln Val Leu
470 475 480
Arg Val Val Arg Leu Ile Lys Ile Ser Pro Ala Leu Glu Asp Phe
485 490 495
Val Tyr Lys Ile Phe Gly Pro Gly Lys Lys Leu Gly Ser Leu Val
500 505 510
Val Phe Thr Ala Ser Leu Leu Ile Val Met Ser A1a Ile Ser Leu
515 520 525
Gln Met Phe Cys Phe Val Glu Glu Leu Asp Arg Phe Thr Thr Phe
530 535 540
Pro Arg Ala Phe Met Ser Met Phe Gln Ile Leu Thr Gln Glu Gly
545 550 555
Trp Val Asp Val Met Asp Gln Thr Leu Asn Ala Val Gly His Met
560 565 570
Trp Ala Pro Val Val Ala Ile Tyr Phe Ile Leu Tyr His Leu Phe
575 580 585
Ala Thr Leu Ile Leu Leu Ser Leu Phe Val Ala Val Ile Leu Asp
590 595 600
Asn Leu Glu Leu Asp Glu Asp Leu Lys Lys Leu Lys Gln Leu Lys
605 610 615
G1n Ser Glu Ala Asn Ala Asp Thr Lys Glu Lys Leu Pro Leu Arg
620 625 630
Leu Arg Ile Phe Glu Lys Phe Pro Asn Arg Pro Gln Met Val Lys
635 640 645
Ile Ser Lys Leu Pro Ser Asp Phe Thr Val Pro Lys Ile Arg Glu
650 655 660
Ser Phe Met Lys G1n Phe Ile Asp Arg G1n Gln Gln Asp Thr Cys
665 670 675
Cys Leu Leu Arg Ser Leu Pro Thr Thr Ser Ser Ser Ser Cys Asp
680 685 690
His Ser Lys Arg Ser Ala Ile Glu Asp Asn Lys Tyr Ile Asp G1n
695 700 705
5~/g~
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
Lys Leu Arg Lys Ser Val Phe Ser Ile Arg Ala Arg Asn Leu Leu
710 715 720
Glu Lys Glu Thr Ala Val Thr Lys Ile Leu Arg Ala Cys Thr Arg
725 730 735
Gln Arg Met Leu Ser Gly Ser Phe Glu Gly Gln Pro Ala Lys Glu
740 745 750
Arg Ser Ile Leu Ser Val Gln His His Ile Arg Gln Glu Arg Arg
755 760 765
Ser Leu Arg His Gly Sex Asn Ser Gln Arg Ile Ser Arg G1y Lys
770 775 780
Ser Leu Glu Thr Leu Thr Gln Asp His Cys Asn Thr Val Ile Tyr
785 790 795
Arg Asn Ala Gln Arg Glu Val Ser Glu Ile Lys Met Ile Gln Glu
800 805 810
Lys Lys Glu Leu Ala Glu Met Leu Gln Gly Lys Cys Lys Lys Glu
815 820 825
Leu Arg Glu Ser His Pro Tyr Phe Asp Lys Pro Leu Phe Ile Val
830 835 840
Gly Arg Glu His Arg Phe Arg Asn Phe Cys Arg Val Val Val Arg
845 850 855
Ala Arg Phe Asn Ala Ser Lys Thr Asp Pro Val Thr Gly Ala Val
860 865 870
Lys Asn Thr Lys Tyr His Leu Leu Tyr Asp Leu Leu Gly Leu Val
875 880 885
Thr Tyr Leu Asp Trp Val Met Ile Ile Val Thr Ser Asp Ser Cys
890 895 900
Ile Ser Met Met Phe Glu Ser Pro Phe Arg Arg Val Met His Ala
905 910 915
Pro Thr Leu Gln Ile Ala Glu Tyr Val Phe Va1 Ile Phe Met Ser
920 925 930
Ile Glu Leu Asn Leu Lys Ile Met Ala Asp Gly Leu Phe Phe Thr
935 940 945
Pro Thr Ala Val Ile Arg Asp Phe Gly Gly Val Met Asp Ile Phe
950 955 960
Ile Tyr Leu Val Ser Leu Ile Phe Leu Cys Trp Met Pro Gln Asn
965 970 975
Val Pro Ala G1u Ser Gly Ala Gln Leu Leu Met Val Leu Arg Cys
980 985 990
Leu Arg Pro Leu Arg Ile Phe Lys Leu Val Pro Gln Met Arg Lys
995 1000 1005
Val Val Arg Glu Leu Phe Ser Gly Phe Lys Glu I1e Phe Leu Va1
1010 1015 1020
Ser Ile Leu Leu Leu Thr Leu Met Leu Val Phe A1a Ser Phe Gly
1025 1030 1035
Val G1n Leu Phe Ala Gly Lys Leu Ala Lys Cys Asn Asp Pro Asn
1040 1045 1050
Ile Ile Arg Arg Glu Asp Cys Asn Gly Ile Phe Arg Ile Asn Val
1055 1060 1065
Ser Val Ser Lys Asn Leu Asn Leu Lys Leu Arg Pro Gly Glu Lys
1070 1075 1080
Lys Pro Gly Phe Trp Val Pro Arg Val Trp Ala Asn Pro Arg Asn
1085 1090 1095
Phe Asn Phe Asp Asn Val Gly Asn Ala Met Leu Ala Leu Phe Glu
1100 1105 1110
Val Leu Ser Leu Lys Gly Trp Val Glu Val Arg Asp Val Ile Ile
1115 1120 1125
His Arg Val Gly Pro Ile His Gly Ile Tyr Ile His Val Phe Val
1130 1135 1140
Phe Leu Gly Cys Met Ile Gly Leu Thr Leu Phe Val Gly Val Val
1145 1150 1155
Ile Ala Asn Phe Asn Glu Asn Lys Gly Thr Ala Leu Leu Thr Va1
1160 1165 1170
Asp Gln Arg Arg Trp Glu Asp Leu Lys Ser Arg Leu Lys Ile Ala
1175 1180 1185
Gln Pro Leu His Leu Pro Pro Arg Pro Asp Asn Asp Gly Phe Arg
1190 1195 1200
Ala Lys Met Tyr Asp Ile Thr Gln His Pro Phe Phe Lys Arg Thr
51/87
CA 02415808 2003-O1-06
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1205 1210 1215
Ile Ala Leu Leu Val Leu Ala Gln Ser Val Leu Leu Ser Val Lys
1220 1225 1230
Trp Asp Val Glu Asp Pro Val Thr Val Pro Leu Ala Thr Met Ser
1235 1240 1245
Val Val Phe Thr Phe Ile Phe Val Leu Glu Val Thr Met Lys Ile
1250 2255 1260
Ile Ala Met Ser Pro Ala Gly Phe Trp Gln Ser Arg Arg Asn Arg
1265 1270 1275
Tyr Asp Leu Leu Val Thr Ser Leu Gly Val Val Trp Val Val Leu
1280 1285 1290
His Phe Ala Leu Leu Asn Ala Tyr Thr Tyr Met Met Gly Ala Cys
1295 1300 1305
Val Ile Val Phe Arg Phe Phe Ser Ile Cys Gly Lys His Val Thr
1310 1315 1320
Leu Lys Met Leu Leu Leu Thr Val Val Val Ser Met Tyr Lys Ser
1325 1330 1335
Phe Phe Ile Ile Val Gly Met Phe Leu Leu Leu Leu Cys Tyr Ala
1340 1345 1350
Phe Ala Gly Val Val Leu Phe Gly Thr Val Lys Tyr Gly Glu Asn
1355 1360 1365
Ile Asn Arg His Ala Asn Phe Ser Ser Ala Gly Lys Ala Ile Thr
1370 1375 1380
Val Leu Phe Arg Ile Val Thr Gly Glu Asp Trp Asn Lys Ile Met
1385 1390 1395
His Asp Cys Met Val Gln Pro Pro Phe Cys Thr Pro Asp Glu Phe
1400 1405 1410
Thr Tyr Trp Ala Thr Asp Cys Gly Asn Tyr A1a Gly Ala Leu Met
1415 1420 1425
Tyr Phe Cys Ser Phe Tyr Val Ile Ile Ala Tyr Ile Met Leu Asn
1430 1435 1440
Leu Leu Val Ala Ile Ile Val Glu Asn Phe Ser Leu Ile Tyr Ser
1445 1450 1455
Thr Glu Glu Asp Gln Leu Leu Ser Tyr Asn Asp Leu Arg His Phe
1460 1465 1470
G1n Ile I1e Trp Asn Met Val Asp Asp Lys Arg Glu Val Phe Pro
1475 1480 1485
Thr Phe Arg Val Lys Phe Leu Leu Arg Leu Leu Arg Gly Arg Leu
1490 1495 1500
Glu Val Asp Leu Asp Lys Asp Lys Leu Leu Phe Lys His Met Cys
1505 1510 1515
Tyr Glu Met Glu Arg Leu His Asn Gly Gly Asp Val Thr Phe His
1520 1525 1530
Asp Val Leu Ser Met Leu Ser Tyr Arg Ser Val Asp Ile Arg Lys
1535 1540 1545
Ser Leu Gln Leu Glu Glu Leu Leu Ala Arg Glu Gln Leu Glu Tyr
1550 1555 1560
Thr Ile Glu G1u Glu Val Ala Lys Gln Thr Ile Arg Met Trp Leu
1565 1570 1575
Lys Lys Cys Leu Lys Arg Ile Arg A1a Lys Gln Gln Gln Ser Cys
1580 1585 1590
Ser I1e Ile His Ser Leu Arg Glu Ser Gln Gln Gln Glu Leu Ser
1595 1600 1605
Arg Phe Leu Asn Pro Pro Ser Ile Glu Thr Thr Gln Pro Ser Glu
1610 1615 1620
Asp Thr Asn Ala Asn Ser Gln Asp Asn Ser Met Gln Pro Glu Thr
1625 1630 1635
Ser Ser Gln Gln Gln Leu Leu Ser Pro Thr Leu Ser Asp Arg Gly
1640 1645 1650
Gly Ser Arg Gln Asp Ala Ala Asp Ala Gly Lys Pro Gln Arg Lys
1655 1660 1665
Phe Gly G1n Trp Arg Leu Pro Ser Ala Pro Lys Pro Ile Ser His
1670 2675 1680
Ser Val Ser Ser Val Asn Leu Arg Phe Gly Gly Arg Thr Thr Met
1685 1690 1695
Lys Ser Val Val Cys Lys Met Asn Pro Met Thr Asp Ala Ala Ser
1700 1705 1710
52/87
CA 02415808 2003-O1-06
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Cys Gly Sex Glu Val Lys Lys Trp Trp Thr Arg Gln Leu Thr Val
1715 1720 1725
Glu Ser Asp Glu Ser Gly Asp Asp Leu Leu Asp Ile
1730 1735
<210> 29
<211> 547
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 168827CD1
<400> 29
Met A1a Phe Gln Asp Leu Leu Asp Gln Val Gly Gly Leu Gly Arg
1 5 10 15
Phe Gln Ile Leu Gln Met VaI Phe Leu Ile Met Phe Asn Val Ile
20 25 30
Val Tyr His Gln Thr Gln Leu Glu Asn Phe Ala Ala Phe Ile Leu
35 40 45
Asp His Arg Cys Trp Val His Ile Leu Asp Asn Asp Thr Ile Pro
50 55 60
Asp Asn Asp Pro Gly Thr Leu Ser Gln Asp Ala Leu Leu Arg Ile
65 70 75
Ser Ile Pro Phe Asp Sex Asn Leu Arg Pro Glu Lys Cys Arg Arg
80 85 90
Phe Val His Pro Gln Trp Lys Leu Ile His Leu Asn Gly Thr Phe
95 100 105
Pro Asn Thr Ser Glu Pro Asp Thr Glu Pro Cys Val Asp Gly Trp
110 115 120
Val Tyr Asp Gln Ser Ser Phe Pro Ser Thr Ile Val Thr Lys Trp
125 130 135
Asp Leu Val Cys Glu Ser Gln Pro Leu Asn Ser Val Ala Lys Phe
140 145 150
Leu Phe Met Ala Gly Met Met Val Gly Gly Asn Leu Tyr Gly His
155 160 165
Leu Ser Asp Arg Phe Gly Arg Lys Phe Va1 Leu Arg Trp Ser Tyr
170 175 180
Leu Gln Leu Ala Ile Val Gly Thr Cys Ala Ala Phe Ala Pro Thr
185 190 195
Ile Leu Val Tyr Cys Ser Leu Arg Phe Leu Ala Gly Ala Ala Thr
200 205 210
Phe Ser Ile Ile Val Asn Thr Val Leu Leu Ile Val Glu Trp Ile
215 220 225
Thr His Gln Phe Cys Ala Met Ala Leu Thr Leu Thr Leu Cys Ala
230 235 240
Ala Ser Ile Gly His Ile Thr Leu Gly Ser Leu Ala Phe Val Ile
245 250 255
Arg Asp Gln Cys Ile Leu Gln Leu Val Met Ser Ala Pro Cys Phe
260 265 270
Val Phe Phe Leu Phe Ser Arg Trp Leu Ala Glu Ser Ala Arg Trp
275 280 285
Leu Ile Ile Asn Asn Lys Pro Glu Glu Gly Leu Lys Glu Leu Thr
290 295 300
Lys Ala Ala His Arg Asn Gly Met Lys Asn Ala Glu Asp Ile Leu
305 310 315
Thr Met Glu Val Leu Lys Ser Thr Met Lys Gln Glu Leu G1u Ala
320 325 330
Ala Gln Lys Lys His Ser Leu Cys Glu Leu Leu Arg Ile Pro Asn
335 340 345
Ile Cys Lys Arg Ile Cys Phe Leu Ser Phe Val Arg Phe Ala Ser
350 355 360
Thr Ile Pro Phe Trp Gly Leu Thr Leu His Leu Gln His Leu Gly
365 370 375
Asn Asn Va1 Phe Leu Leu Gln Thr Leu Phe Gly Ala Va1 Thr Leu
380 38S 390
53/87
CA 02415808 2003-O1-06
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Leu Ala Asn Cys Val Ala Pro Trp A1a Leu Asn His Met Ser Arg
395 400 405
Arg Leu Ser Gln Met Leu Leu Met Phe Leu Leu Ala Thr Cys Leu
410 415 420
Leu A1a Ile Ile Phe Val Pro Gln Glu Met Gln Thr Leu Arg Val
425 430 435
Val Leu Ala Thr Leu Gly Val Gly Ala Ala Ser Leu Gly Ile Thr
440 445 450
Cys Ser Thr Ala Gln Glu Asn Glu Leu Ile Pro Ser Ile Ile Arg
455 460 465
Gly Arg Ala Thr Gly Ile Thr Gly Asn Phe Ala Asn Ile Gly Gly
470 475 480
Ala Leu Ala Ser Leu Met Met Ile Leu Ser Ile Tyr Ser Arg Pro
485 490 495
Leu Pro Trp Ile Ile Tyr Gly Val Phe Ala Ile Leu Ser Gly Leu
500 505 510
Val Val Leu Leu Leu Pro Glu Thr Arg Asn Gln Pro Leu Leu Asp
515 520 525
Ser Ile Gln Asp Val Glu Asn Glu Gly Val Asn Ser Leu Ala Ala
530 535 540
Pro Gln Arg Ser Ser Val Leu
545
<210> 30
<211> 547
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472734CD1
<400> 30
Met G1y Phe Asp Val Leu Leu Asp Gln Val Gly Gly Met Gly Arg
1 5 10 15
Phe Gln I1e Cys Leu Ile Ala Phe Phe Cys Ile Thr Asn Ile Leu
20 25 30
Leu Phe Pro Asn Ile Val Leu Glu Asn Phe Thr Ala Phe Thr Pro
35 40 45
Ser His Arg Cys Trp Val Pro Leu Leu Asp Asn Asp Thr Val Ser
50 55 60
Asp Asn Asp Thr Gly Thr Leu Ser Lys Asp Asp Leu Leu Arg Ile
65 70 75
Ser Ile Pro Leu Asp Ser Asn Leu Arg Pro Gln Lys Cys Gln Arg
80 85 90
Phe Ile His Pro Gln Trp Gln Leu Leu His Leu Asn Gly Thr Phe
95 100 105
Pro Asn Thr Asn Glu Pro Asp Thr Glu Pro Cys Val Asp Gly Trp
110 115 120
Va1 Tyr Asp Arg Ser Ser Phe Leu Ser Thr Ile Val Thr Glu Trp
12 5 13 0 13 5
Asp Leu Val Cys Glu Ser G1n Ser Leu Lys Ser Met Val Gln Ser
140 145 150
Leu Phe Met Ala Gly Ser Leu Leu Gly Gly Leu Ile Tyr Gly His
155 160 165
Leu Ser Asp Arg Phe GIy Arg Lys Phe Val Leu Arg Trp Ser Tyr
170 175 180
Leu Gln Leu Ala Ile Val Gly Thr Cys Ala Ala Phe A1a Pro Thr
185 190 195
Ile Leu Val Tyr Cys Ser Leu Arg Phe Leu Ala Gly Ala Ala Thr
200 205 210
Phe Ser Ile Ile Val Asn Thr Val Leu Leu Ile Val Glu Trp Ile
215 220 225
Thr His Gln Phe Cys Ala Met Ala Leu Thr Leu Thr Leu Cys Ala
230 235 240
Ala Ser Ile Gly His Ile Thr Leu Gly Ser Leu Ala Phe Val Ile
245 250 255
54/7
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
Arg Asp Gln Cys Ile Leu G1n Leu Val Met Ser Ala Pro Cys Phe
260 265 270
Val Phe Phe Leu Phe Ser Arg Trp Leu Ala Glu Ser Ala Arg Trp
275 280 285
Leu Ile Ile Asn Asn Lys Pro Glu Glu Gly Leu Lys Glu Leu Arg
290 295 300
Lys Ala Ala His Arg Asn Gly Met Lys Asn Ala Glu Asp Ile Leu
305 310 315
Thr Met Glu Val Leu Lys Ser Thr Met Lys Gln Glu Leu Glu Ala
320 325 330
Ala Gln Lys Lys His Ser Leu Cys Glu Leu Leu Arg Ile Pro Asn
335 340 345
Ile Cys Lys Arg I1e Cys Phe Leu Ser Phe Val Arg Phe Ala Ser
350 355 360
Thr Ile Pro Phe Trp Gly Leu Thr Leu His Leu Gln His Leu Gly
365 370 375
Asn Asn Val Phe Leu Leu Gln Thr Leu Phe Gly Ala Val Thr Leu
380 385 ~ 390
Leu Ala Asn Cys Val Ala Pro Trp Ala Leu Asn His Met Ser Arg
395 400 405
Arg Leu Ser Gln Met Leu Leu Met Phe Leu Leu Ala Thr Cys Leu
410 415 420
Leu Ala Ile Ile Phe Val Pro Gln Glu Met Gln Thr Leu Arg Val
425 430 435
Val Leu Ala Thr Leu Gly Val Gly Ala Ala Ser Leu Gly Ile Thr
440 445 ' 450
Cys Ser Thr Ala Gln Glu Asn Glu Leu Ile Pro Ser Ile I1e Arg
455 460 465
Gly Arg Ala Thr Gly Ile Thr Gly Asn Phe Ala Asn Ile Gly Gly
470 475 480
Ala Leu Ala Ser Leu Met Met Ile Leu Ser Ile Tyr Ser Arg Pro
485 490 495
Leu Pro Trp Ile Ile Tyr Gly Val Phe Ala Ile Leu Ser Gly Leu
500 505 510
Val Val Leu Leu Leu Pro Glu Thr Arg Asn Gln Pro Leu Leu Asp
515 520 525
Ser Ile Gln Asp Val Glu Asn Glu Gly Val Asn Ser Leu Ala Ala
530 535 540
Pro Gln Arg Ser Ser Val Leu
545
<210> 31
<211> 988
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473473CD1
<400> 31
Met Pro Gly Gly Lys Arg Gly Leu Val Ala Pro Gln Asn Thr Phe
1 5 10 15
Leu Glu Asn Ile Val Arg Arg Ser Ser Glu Ser Ser Phe Leu Leu
20 25 30
Gly Asn Ala Gln Ile Val Asp Trp Pro Val Val Tyr Ser Asn Asp
35 40 45
Gly Phe Cys Lys Leu Ser Gly Tyr His Arg Ala Asp Va1 Met Gln
50 55 60
Lys Ser Ser Thr Cys Ser Phe Met Tyr Gly Glu Leu Thr Asp Lys
65 70 75
Lys Thr Ile Glu Lys Val Arg Gln Thr Phe Asp Asn Tyr Glu Ser
80 85 90
Asn Cys Phe Glu Val Leu Leu Tyr Lys Lys Asn Arg Thr Pro Val
95 100 105
Trp Phe Tyr Met Gln Ile Ala Pro Ile Arg Asn Glu His Glu Lys
110 115 120
55/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
Val Val Leu Phe Leu Cys Thr Phe Lys Asp Ile Thr Leu Phe Lys
125 230 235
Gln Pro Ile Glu Asp Asp Ser Thr Lys Gly Trp Thr Lys Phe Ala
140 145 150
Arg Leu Thr Arg Ala Leu Thr Asn Ser Arg Ser Val Leu Gln Gln
155 160 165
Leu Thr Pro Met Asn Lys Thr Glu Val Val His Lys His Sex Arg
170 175 180
Leu Ala Glu Val Leu Gln Leu Gly Ser Asp Ile Leu Pro Gln Tyr
185 190 195
Lys Gln Glu Ala Pro Lys Thr Pro Pro His Ile Ile Leu His Tyr
200 205 210
Cys Ala Phe Lys Thr Thr Trp Asp Trp Val Ile Leu Ile Leu Thr
215 220 225
Phe Tyr Thr Ala Ile Met Val Pro Tyr Asn Val Ser Phe Lys Thr
230 235 240
Lys Gln Asn Asn Ile Ala Trp Leu Val Leu Asp Ser Val Val Asp
245 250 255
Val Ile Phe Leu Val Asp Ile Val Leu Asn Phe His Thr Thr Phe
260 265 270
Val Gly Pro Gly Gly Glu Val Ile Ser Asp Pro Lys Leu Ile Arg
275 280 285
Met Asn Tyr Leu Lys Thr Trp Phe Val Ile Asp Leu Leu Ser Cys
290 295 300
Leu Pro Tyr Asp Ile Ile Asn Ala Phe Glu Asn Val Asp Glu Gly
305 310 315
Ile Ser Ser Leu Phe Ser Ser Leu Lys Val Val Arg Leu Leu Arg
320 325 330
Leu Gly Arg Val Ala Arg Lys Leu Asp His Tyr Leu Glu Tyr Gly
335 340 345
Ala Ala Val Leu Val Leu Leu Val Cys Va1 Phe Gly Leu Val Ala
350 355 360
His Trp Leu Ala Cys Ile Trp Tyr Ser I1e Gly Asp Tyr Glu Val
365 370 375
Ile Asp Glu Val Thr Asn Thr Ile Gln Ile Asp Ser Trp Leu Tyr
380 385 390
Gln Leu Ala Leu Ser Ile Gly Thr Pro Tyr Arg Tyr Asn Thr Ser
395 400 405
Ala Gly Ile Trp Glu G1y Gly Pro Ser Lys Asp Ser Leu Tyr Val
410 415 420
Ser Ser Leu Tyr Phe Thr Met Thr Ser Leu Thr Thr Ile Gly Phe
425 430 435
Gly Asn Ile Ala Pro Thr Thr Asp Val Glu Lys Met Phe Ser Val
440 445 450
Ala Met Met Met Val Gly Ala Leu Leu Tyr Ala Thr Ile Phe Gly
455 460 465
Asn Val Thr Thr Ile Phe Gln Gln Met Tyr Ala Asn Thr Asn Arg
470 475 480
Tyr His Glu Met Leu Asn Asn Val Arg Asp Phe Leu Lys Leu Tyr
485 490 495
Gln Val Pro Lys Gly Leu Ser Glu Arg Val Met Asp Tyr Ile Val
500 505 510
Ser Thr Trp Ser Met Ser Lys Gly Ile Asp Thr Glu Lys Val Leu
515 520 525
Ser I1e Cys Pro Lys Asp Met Arg Ala Asp Ile Cys Val His Leu
530 535 540
Asn Arg Lys Val Phe Asn Glu His Pro Ala Phe Arg Leu Ala Ser
545 550 555
Asp Gly Cys Leu Arg Ala Leu Ala Val Glu Phe Gln Thr Ile His
560 565 570
Cys Ala Pro Gly Asp Leu Ile Tyr His A1a Gly Glu Ser Val Asp
575 580 585
Ala Leu Cys Phe Val Val Ser Gly Ser Leu Glu Va1 Ile Gln Asp
590 595 600
Asp Glu Val Val A1a Ile Leu Gly Lys Gly Asp Val Phe Gly Asp
605 610 615
Ile Phe Trp Lys Glu Thr Thr Leu A1a His Ala Cys Ala Asn Val
56!87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
620 625 630
Arg Ala Leu Thr Tyr Cys Asp Leu His Ile Ile Lys Arg G1u Ala
635 640 645
Leu Leu Lys Val Leu Asp Phe Tyr Thr Ala Phe Ala Asn Ser Phe
650 655 660
Ser Arg Asn Leu Thr Leu Thr Cys Asn Leu Arg Lys Arg Ile Ile
665 670 675
Phe Arg Lys Ile Ser Asp Val Lys Lys Glu Glu Glu Glu Arg Leu
680 685 690
Arg Gln Lys Asn Glu Val Thr Leu Ser I1e Pro Val Asp His Pro
695 700 705
Val Arg Lys Leu Phe Gln Lys Phe Lys Gln Gln Lys Glu Leu Arg
710 715 720
Asn Gln Gly Ser Thr Gln G1y Asp Pro Glu Arg Asn Gln,Leu Gln
725 730 735
Val Glu Ser Arg Ser Leu Gln Asn Gly Ala Ser Ile Thr Gly Thr
740 745 750
Ser Val Val Thr Val Ser Gln Ile Thr Pro Ile Gln Thr Ser Leu
755 760 765
Ala Tyr Val Lys Thr Ser Glu Ser Leu Lys Gln Asn Asn Arg Asp
770 775 780
Ala Met Glu Leu Lys Pro Asn Gly Gly Ala Asp Gln Lys Cys Leu
785 790 795
Lys Val Asn Ser Pro Ile Arg Met Lys Asn Gly Asn Gly Lys Gly
800 805 810
Trp Leu Arg Leu Lys Asn Asn Met Gly Ala His Glu Glu Lys Lys
815 820 825
Glu Asp Trp Asn Asn Val Thr Lys Ala Glu Ser Met Gly Leu Leu
830 835 840
Ser Glu Asp Pro Lys Ser Ser Asp Ser Glu Asn Ser Va1 Thr Lys
845 850 855
Asn Pro Leu Arg Lys Thr Asp Ser Cys Asp Ser Gly Tle Thr Lys
860 865 870
Ser Asp Leu Arg Leu Asp Lys Ala Gly Glu Ala Arg Ser Pro Leu
875 880 885
Glu His Ser Pro Ile Gln Ala Asp Ala Lys His Pro Phe Tyr Pro
890 895 900
Ile Pro Glu Gln Ala Leu Gln Thr Thr Leu Gln Glu Val Lys His
905 910 915
Glu Leu Lys Glu Asp Ile Gln Leu Leu Ser Cys Arg Met Thr Ala
920 925 930
Leu G1u Lys Gln Val Ala Glu Ile Leu Lys Ile Leu Ser Glu Lys
935 940 945
Ser Val Pro Gln Ala Ser Ser Pro Lys Ser Gln Met Pro Leu Gln
950 955 960
Val Pro Pro Gln Ile Pro Cys Gln Asp Ile Phe Ser Val Ser Arg
965 970 975
Pro Glu Ser Pro Glu Ser Asp Lys Asp Glu Ile His Phe
980 985
<210> 32
<211> 533
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7477725CD1
<400> 32
Met Ala Phe Glu Glu Leu Leu Ser Gln Val Gly Gly Leu Gly Arg
1 5 10 15
Phe Gln Met Leu His Leu Va1 Phe Ile Leu Pro Ser Leu Met Leu
20 25 30
Leu Ile Pro His I1e Leu Leu Glu Asn Phe Ala Ala Ala Ile Pro
35 40 45
Gly His Arg Cys Trp Val His Met Leu Asp Asn Asn Thr Gly Ser
57/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
50 55 60
Gly Asn Glu Thr Gly Ile Leu Ser Glu Asp Ala Leu Leu Arg Ile
65 70 75
Ser I1e Pro Leu Asp Ser Asn Leu Arg Pro Glu Lys Cys Arg Arg
80 85 90
Phe Val His Pro Gln Trp Gln Leu Leu His Leu Asn Gly Thr Ile
95 100 105
His Ser Thr Ser Glu Ala Asp Thr Glu Pro Cys Val Asp Gly Trp
110 115 120
Val Tyr Asp Gln Ser Tyr Phe Pro Ser Thr Ile Val Thr Lys Trp
125 130 135
Asp Leu Val Cys Asp Tyr Gln Ser Leu Lys Ser Val Val Gln Phe
140 145 150
Leu Leu Leu Thr Gly Met Leu Val Gly Gly Ile Ile Gly Gly His
155 160 165
Val Ser Asp Arg Phe Gly Arg Arg Phe Ile Leu Arg Trp Cys Leu
170 175 180
Leu Gln Leu Ala Ile Thr Asp Thr Cys Ala Ala Phe Ala Pro Thr
185 190 195
Phe Pro Val Tyr Cys Val Leu Arg Phe Leu Ala Gly Phe Ser Ser
200 205 210
Met Ile Ile Ile Ser Asn Asn Ser Leu Pro Ile Thr Glu Trp I1e
215 220 225
Arg Pro Asn Ser Lys Ala Leu Val Val Ile Leu Ser Ser Gly Ala
230 235 240
Leu Ser Ile Gly Gln Ile Ile Leu Gly Gly Leu Ala Tyr Val Phe
245 250 255
Arg Asp Trp Gln Thr Leu His Val Val Ala Ser Val Pro Phe Phe
260 265 270
Val Phe Phe Leu Leu Ser Arg Trp Leu Val G1u Ser Ala Arg Trp
275 280 285
Leu Ile Ile Thr Asn Lys Leu Asp Glu Gly Leu Lys Ala Leu Arg
290 295 300
Lys Val Ala Arg Thr Asn Gly Ile Lys Asn Ala Glu Glu Thr Leu
305 310 31S
Asn Ile Glu Val Val Arg Ser Thr Met Gln G1u Glu Leu Asp Ala
320 325 330
Ala Gln Thr Lys Thr Thr Val Cys Asp Leu Phe Arg Asn Pro Ser
335 340 345
Met Arg Lys Arg Ile Cys I1e Leu Val Phe Leu Arg Phe Ala Asn
350 355 360
Thr Ile Pro Phe Tyr Gly Thr Met Val Asn Leu Gln His Val Gly
365 370 375
Ser Asn Ile Phe Leu Leu Gln Val Leu Tyr Gly Ala Val Ala Leu
380 385 390
Tle Va1 Arg Cys Leu Ala Leu Leu Thr Leu Asn His Met Gly Arg
395 400 405
Arg Ile Ser Gln Ile Leu Phe Met Phe Leu Val Gly Leu Ser Ile
410 415 420
Leu Ala Asn Thr Phe Val Pro Lys Glu Met G1n Thr Leu Arg Va1
425 430 435
Ala Leu~AIa Cys Leu Gly Tle Gly Cys Ser Ala Ala Thr Phe Ser
440 445 450
Ser Val Ala Val His Phe Ile Glu Leu Ile Pro Thr Val Leu Arg
455 460 465
Ala Arg Ala Ser Gly .Ile Asp Leu Thr Ala Ser Arg Ile Gly Ala
470 475 480
Ala Leu Ala Pro Leu Leu Met Thr Leu Thr Val Phe Phe Thr Thr
485 490 495
Leu Pro Trp Ile Ile Tyr Gly Ile Phe Pro Ile Ile Gly Gly Leu
500 505 510
Ile Val Phe Leu Leu Pro Glu Thr Lys Asn Leu Pro Leu Pro Asp
515 520 525
Thr Ile Lys Asp Val Glu Asn Gln
53 0
<210> 33
58/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
<211> 1775
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3474673CB1
<400> 33
atttcaggaa atgtgagggg gctctgggcc ccttccctca gcgcctgcgg tcacccagca 60
gcttcctcct tctccctggc ctaggcctag caggtgggca ccccgcacac atttgaggcg 120
gggccagatg cccacagttc agagcctctt tttgtcccgg ggattggatc ccagggctgg 180
gtggggccag gctgtcccat tccccaacac tcctcctccc cggcgaaacc gggcaccagc 240
aggcgtttgc gagaggagat acgagctgga cgcctggccc ttccctccca ccgggtccta 300
gtccaccgct cccggcgccg gctccccgcc tctcccgcta tgtaccgacc gcgagcccgg 360
gcggctcccg agggcagggt ccggggctgc gcggtgccca gcaccgtgct cctgctgctc 420
gcctacctgg cttacctggc gctgggcacc ggcgtgttct ggacgctgga gggccgcgcg 480
gcgcaggact ccagccgcag cttccagcgc gacaagtggg agctgttgca gaacttcacg 540
tgtctggacc gcccggcgct ggactcgctg atccgggatg tcgtccaagc atacaaaaac 600
ggagccagcc tcctcagcaa caccaccagc atggggcgct gggagctcgt gggctccttc 660
ttcttttctg tgtccaccat caccaccatt ggctatggca acctgagccc caacacgatg 720
gctgcccgcc tcttctgcat cttctttgcc cttgtgggga tcccactcaa cctcgtggtg 780
ctcaaccgac tggggcatct catgcagcag ggagtaaacc actgggccag caggctgggg 840
ggcacctggc aggatcctga caaggcgcgg tggctggcgg gctctggcgc cctcctctcg 900
ggcctcctgc tcttcctgct gctgccaccg ctgctcttct cccacatgga gggctggagc 960
tacacagagg gcttctactt cgccttcatc accctcagca ccgtgggctt cggcgactac 1020
gtgattggaa tgaacccctc ccagaggtac ccactgtggt acaagaacat ggtgtccctg 1080
tggatcctct ttgggatggc atggctggcc ttgatcatca aactcatcct ctcccagctg 1140
gagacgccag ggagggtatg ttcctgctgc caccacagct ctaaggaaga cttcaagtcc 1200
caaagctgga gacagggacc tgaccgggag ccagagtccc actccccaca gcaaggatgc 1260
tatccagagg gacccatggg aatcatacag catctggaac cttctgctca cgctgcaggc 1320
tgtggcaagg acagctagtt atactccatt ctttggtcgt cgtcctcggt agcaagaccc 1380
ctgattttaa gctttgcaca tgtccaccca aactaaagac tacattttcc atccacccta 1440
gaggctgggt gcagctatat gattaattct gcccaatagg gtatacagag acatgtcctg 1500
ggtgacatgg gatgtgactt tcgggtgtcg gggcagcatg cccttctccc ccacttcctt 1560
actttagcgg gctgcaatgc cgccgatatg atggctggga gctctggcag ccatacggca 1620
ccatgaagta gcggcaatgt ttgagcggca caataagata ggaagagtct ggatctctga 1680
tgatcacaga gccatcctaa caaacggaat atcacccgac ctcctttatg tgagagagaa 1740
ataaacatct tatgtaaaat accaaaaaaa aaaaa 1775
<210> 34
<211> 1545
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4588877CB1
<400> 34
aatgagggcc ctgggggttg ggcccaggag tggggctgtg gtggtgagtg gacagggctg 60
ggctggaaat gtcccctgag tgccccctct cacctcaggc tatggcaacc tgagccccaa 120
cacgatggct gcccgcctct tctgcatctt ctttgccctt gtggggatcc cactcaacct 180
cgtggtgctc aaccgactgg ggcatctcat gcagcaggga gtaaaccact gggccagcag 240
gctggggggc acctggcagg tgagggggct gctggacggg gtggggatgg gtcacttcta 300
gaatgagggg ctgtggtggg aattggggtt actaatgaca agaggtggga gcaagtgtta 360
ctggtgaggt tgtgttggga ttgggggtca ctgctcagaa tagggtcctt agtgaaaaag 420
ggcattaatg gtggagatgg ggtgggactg ggcagacagg aaggacatga ggcacaggct 480
ccaggcaggg aacctggaga acacagacca ggtgaagagc ccccttctta ctggggacag 540
ctctggcctg cctccagctc cctcggctcc cacgcatggg gtgaaggcct caggaggcct 600
ggggacaata ttgcacccac aggatcctga caaggcgcgg tggctggegg gctctggcgc 660
cctcctctcg ggcctcctgc tcttcctgct gctgccaccg ctgctcttct cccacatgga 720
gggctggagc tacacagagg gcttctactt cgccttcatc accctcagca ccgtgggctt 780
cggcgactac gtgattggaa tgaacccctc ccagaggtac ccactgtggt acaagaacat 840
ggtgtccctg tggatcctct ttgggatggc atggctggcc ttgatcatca aactcatcct 900
ctcccagctg gagacgccag ggagggtatg ttcctgctgc caccacagct ctaaggaaga 960
cttcaagtcc caaagctgga gacagggacc tgaccgggag ccagagtccc actccccaca 1020
59/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
gcaaggatgc tatccagagg gacccatggg aatcatacag catctggaac cttctgctca 1080
cgctgcaggc tgtggcaagg acagctagtt atactccatt ctttggtcgt cgtcctcggt 1140
agcaagaccc ctgattttaa gctttgcaca tgtccaccca aactaaagac tacattttcc 1200
atccacccta gaggctgggt gcagctatat gattaattct gcccaatagg gtatacagag 1260
acatgtcctg ggtgacatgg gatgtgactt tcgggtgtcg gggcagcatg cccttctccc 1320
ccacttcctt actttagcgg gctgcaatgc cgccgatatg atggctggga gctctggcag 1380
ccatacggca ccatgaagta gcggcaatgt ttgagcggca caataagata ggaagagtct 1440
ggatctctga tgatcacaga gccatcctaa caaacggaat atcacccgac ctcctttatg 1500
tgagagagaa ataaacatct tatgtaaaat accaaaaaaa aaaaa 2545
<210> 35
<211> 1941
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472214CB1
<400> 35
atggcggaga aggcgctgga ggccgtgggc tgtggactag ggccgggggc tgtggccatg 60
gccgtgacgc tggaggacgg ggcggaaccc cctgtgctga ccacgcacct gaagaaggtg 120
gagaaccaca tcactgaagc ccagcgcttc tcccacctac ccaagcgctc agccgtggac 180
atcgagttcg tggagctgtc ctattccgtg cgggaggggc cctgctggcg caaaaggggt 240
tataagaccc ttctcaagtg cctctcaggt aaattctgcc gccgggagct gattggcatc 300
atgggcccct caggggctgg caagtctaca ttcatgaaca tcttggcagg atacagggag 360
tctggaatga aggggcagat cctggttaat ggaaggccac gggagctgag gaccttccgc 420
aagatgtcct gctacatcat gcaagatgac atgctgctgc cgcacctcac ggtgttggaa 480
gccatgatgg tgtctgctaa cctgaatctt actgagaatc ccgatgtgaa aaacgatctc 540
gtgacagaga tcctgacggc actgggcctg atgtcgtgct cccacacgag gacagccctg 600
ctctctggcg ggcagaggaa gcgtctggcc atcgccctgg agctggtcaa caacccgcct 660
gtcatgttct ttgatgagcc caccagtggt ctggatagcg cctcttgttt ccaagtggtg 720
tccctcatga agtccctggc acaggggggc cgtaccatca tctgcaccat ccaccagccc 780
agtgccaagc tctttgagat gtttgacaag ctctacatcc tgagccaggg tcagtgcatc 840
ttcaaaggcg tggtcaccaa cctgatcccc tatctaaagg gactcggctt gcattgcccc 900
acctaccaca acccggctga cttcgtcatc gaggtggcct ctggcgagta tggagacctg 960
aaccccatgt tgttcagggc tgtgcagaat gggctgtgcg ctatggctga gaagaagagc 1020
agccctgaga agaacgaggt ccctgcccca tgccctcctt gtcctccgga agtggatccc 1080
attgaaagcc acacctttgc caccagcacc ctcacacagt tctgcatcct cttcaagagg 1140
accttcctgt ccatcctcag ggacacggtg ctgacccacc tacggttcat gtcccacgtg 1200
gttattggcg tgctcatcgg cctcctctac ctgcatattg gcgacgatgc cagcaaggtc 1260
ttcaacaaca ccggctgcct cttcttctcc atgctgttcc tcatgttcgc cgccctcatg 1320
ccaactgtgc tcaccgtccc cttagagatg gcggtcttca tgagggagca cctcaactac 1380
tggtacagcc tcaaagcgta ttacctggcc aagaccatgg ctgacgtgcc ctttcaggtg 1440
gtgtgtccgg tggtctactg cagcattgtg tactggatga cgggccagcc cgctgagacc 1500
agccgcttcc tgctcttctc agccctggcc accgccaccg ccttggtggc ccaatctttg 1560
gggctgctga tcggagctgc ttccaactcc ctacaggtgg ccacttttgt gggcccagtt 1620
accgccatcc ctgtcctctt gttctccggc ttctttgtca gcttcaagac catccccact 1680
tacctgcaat ggagctccta tctctcctat gtcaggtatg gctttgaggg tgtgatcctg 1740
acgatctatg gcatggagcg aggagacctg acatgtttag aggaacgctg cccgttccgg 1800
gagccacaga gcatcctccg agcgctggat gtggaggatg ccaagctcta catggacttc 1860
ctggtcttgg gcatcttctt cctagccctg cggctgctgg cctaccttgt gctgcgttac 1920
cgggtcaagt cagagagata g 1941
<210> 36
<211> 4971
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473053CB1
<400> 36
caaagtagcg ggccgaggcc cgggggagcg gggccgcagc tgggggggcg ggagcccgtg 60
gggagccgag ccgagcgccc cccgccccag cccccggcat gggcagtacg gggccgccgg 120
ggcgggcgcc gagcgctgag cgctgagggt ctcccatggg attgctggga tcttgctggg 180
60187
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
tgagatggca gtgtgtgcaa aaaagcgccc cccagaagaa gaaaggaggg cgcgggctaa 240
tgaccgagaa tacaatgaga aattccagta tgcgagtaac tgcatcaaga cctccaagta 300
caatattctc accttcctgc ctgtcaacct ctttgagcag ttccaggaag ttgccaacac 360
ttacttcctg ttcctcctca ttctgcagtt gatcccccag atctcttccc tgtcctggtt 420
caccaccatt gtgcctttgg ttcttgtcct caccatcaca gctgttaaag atgccactga 480
tgactatttc cgccacaaga gcgataacca ggtgaataac cgccagtctc aggtgctgat 540
caacggaatc ctccagcagg agcagtggat gaatgtctgt gttggtgata ttatcaagct 600
agaaaataac cagtttgtgg cggcggatct cctcctcctt tccagcagtg agccccatgg 660
gctgtgttac atagagacag cagaacttga tggcgagacc aacatgaaag tacgtcaggc 720
gattccagtc acctcagaat tgggagacat cagtaagctt gccaagtttg acggtgaagt 780
gatctgtgaa cctcccaaca acaaactgga caaattcagc ggaaccctct actggaagga 840
aaataagttc cctctgagca accagaacat gctgctgcgg ggctgtgtgc tgcgaaacac 900
cgagtggtgc ttcgggctgg tcatctttgc aggtcccgac actaagctga tgcaaaacag 960
cggcagaaca aagttcaaaa gaacgagtat cgatcgccta atgaataccc tggtgctctg 1020
gatttttgga ttcctggttt gcatgggggt gatcctggcc attggcaatg ccatctggga 1080
gcacgaggtg gggatgcgtt tccaggtcta cctgccgtgg gatgaggcag tggacagtgc 1140
cttcttctct ggcttcctct ccttctggtc ctacatcatc atcctcaaca ccgttgtgcc 1200
catttcactc tatgtcagtg tggaggtcat ccgtctgggc cacagctact tcatcaactg 1260
ggataagaag atgttctgca tgaagaagcg gacgcctgca gaagcccgca ccaccaccct 1320
aaacgaggag ctgggccagg tggagtacat cttctccgac aagacgggca ccctcaccca 1380
gaacatcatg gttttcaaca agtgctccat caatggccac agctatggtg atgtgtttga 1440
cgtcctggga cacaaagctg aattgggaga gaggcctgaa cctgttgact tctccttcaa 1500
tcctctggct gacaagaagt tcttattttg ggaccccagc ctgctggagg ctgtcaagat 1560
cggggacccc cacacgcatg agttcttccg cctcctttcc ctgtgtcata ctgtcatgtc 1620
agaagaaaag aacgaaggag agctgtacta caaagctcag tccccagatg agggggccct 1680
ggtcaccgca gccaggaact ttggttttgt tttccgctct cgcaccccca aaacaatcac 1740
cgtccatgag atgggcacag ccatcaccta ccagctgctg gccatcctgg acttcaacaa 1800
catccgcaag cggatgtcgg tcatagtgcg gaatccagag gggaagatcc gactctactg 1860
caaaggggct gacactatcc tactggacag actgcaccac tccactcaag agctgctcaa 1920
caccaccatg gaccacctta atgagtacgc aggggaaggg.ctgaggaccc tggtgctggc 1980
ctacaaggat ctggatgaag agtactatga ggagtgggct gagcgacgcc tccaggccag 2040
cctggcccag gacagccggg aggacaggct ggctagcatc tatgaggagg ttgagaacaa 2100
catgatgctg ctgggtgcaa cggccattga ggacaaactt cagcaagggg ttccagagac 2160
cattgccctc ctgacactgg ccaacatcaa gatttgggtg ctaaccggag acaagcaaga 2220
gacggctgtg aacatcggct attcctgcaa gatgctgacg gatgacatga ctgaggtttt 2280
catagtcact ggccatactg tcctggaggt gcgggaggag ctcaggaaag cccgggagaa 2340
gatgatggac tcatcccgct ctgtaggcaa cggcttcacc tatcaggaca agctttcttc 2400
ttccaagcta acttctgtcc tggaggccgt tgctggggag tacgccctgg tcataaatgg 2460
tcacagcctg gcccacgcac tggaggcaga catggagctg gagtttctgg agacagcgtg 2520
tgcctgcaaa gctgtcatct gctgccgggt gacccccttg cagaaggcac aggtggtaga 2580
actggtcaag aagtacaaga aggctgtgac gcttgccatt ggagacggag ccaatgatgt 2640
cagcatgatc aaaacggctc acattggtgt ggggatcagt gggcaggaag ggatccaggc 2700
tgtcttggcc tccgattact ccttctccca gttcaagttc ctgcagcgcc tcctgctggt 2760
gcatgggcgc tggtcctacc tgcgaatgtg caagtttctt tgctatttct tctacaaaaa 2820
ctttgctttc accatggtcc acttctggtt tggcttcttc tgtggcttct cagcccagac 2880
cgtctatgac cagtatttca tcaccctgta taacatcgtg tacacctccc tgccagtcct 2940
ggctatgggg gtctttgatc aggatgtccc cgagcagcgg agcatggagt accctaagct 3000
gtatgagccg ggccagctga accttctctt caacaagcgg gagttcttca tctgcatcgc 3060
ccagggcatc tacacctccg tgctcatgtt cttcattccc tatggggtgt ttgctgatgc 3120
cacccgggat gatggcactc agctggctga ctaccagtcc tttgcagtca ctgtggccac 3180
atccttggtc attgtggtta gcgtgcagat tgggctcgac acaggctact ggacggccat 3240
caaccacttc ttcatctggg gaagccttgc tgtttacttt gccatcctct ttgccatgca 3300
cagcaatggg ctcttcgaca tgtttcccaa ccagttccgg tttgtgggga atgcccagaa 3360
caccttggcc cagcccacgg tgtggctgac cattgtgctc accacagtcg tctgcatcat 3420
gcccgtggtt gccttccgat tcctcaggct caacctgaag ccggatctct ccgacacggt 3480
ccgctacaca cagctcgtga ggaagaagca gaaggcccag caccgctgca tgcggcgggt 3540
tggccgcact ggctcccggc gctccggcta tgccttctcc catcaggagg gcttcgggga 3600
gctcatcatg tctggcaaga acatgcggct gagctctctc gcgctctcca gcttcaccac 3660
ccgctccagc tccagctgga ttgagagcct gcgcaggaag aagagtgaca gtgccagtag 3720
ccccagtggc ggtgccgaca agcccctcaa gggctgaagg ccgaggatgg atgccctgtg 3780
ccagtgacca gagcacccag ggctggccag tcactgaggg aacagcgtct cggaactgct 3840
ggtcctcatt ccttgcttcc cgtccccccg gtagactctg tcctgctggt cccaccacac 3900
atggctggga catctgttcc cagctgtagg cccttccacc agctggggag ctagagggag 3960
caggcccaag ggcagagcag aggctgaggc acggggagcc agccccactc ggggaccaga 4020
agtggaacca aaaacaagaa aaaactgtga gagattgtgt ctgcccctgc cctgcctggg 4080
acccacaggg agactataat ctccttattt ttttactcct actccccaga ggggccctag 4140
tgcctctgtt cctgaattac ataagaatgt accatgccgg gaagccagag acctgcaggg 4200
61/7
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
gcctcggccc ctcacatcgt gtatgtctct ccttgatttg tgttgtgtcc agtttggttt 4260
tgtctttttt tatttggcaa gtggaggagg cttttatgtg acttttatgt tgtggttggt 4320
gtcttaactc tcctgggaaa aggaggctgg cacacactgg gatgccgcag cctggccggc 4380
tgtggggtgg tttgggagga tccatgtcgg ctctgcctgc agtgaccagt gctctgtggg 4440
gcagaggagc tgaccaggga gggaggtacc catgagcaga gggtagtggg agagtgtaaa 4500
ggagggtttg gtcctgtctg cttcctcacc ttgagagtaa agtgctgccc tctgccccca 4560
acacacacac atatcaattc ctggattcct tagtcctgct ggccttgggc tggagcctag 4620
gaaagtggcc cccaaatcct tagtgagcta aagctgggtc tgaaatttgg tcagtgggga 4680
ggggtagttt tcttttcttt tttctttttc tttttttctt tttttttttg agatggagtc 4740
tcactcttgt cacctaggca agagtgcaat ggcacaatct cagctcactg caacctccac 4800
ctcctgggtt caagcgattc tcctgcctcc ccggacccaa ccactggact taatctcact 4860
ttcttaaatt cttctattct cagacacggg tctagtacca ttccttcctc ttagccccag 4920
ggagcaaatt aaagaggtta cgagttaaaa tcctaaaaaa aaaaaaaaaa a 4971
<210> 37
<211> 1404
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473347CB1
<400> 37
atggtcctgg ctttccagtt agtctccttc acctacatct ggatcatatt gaaaccaaat 60
gtttgtgctg cttctaacat caagatgaca caccagcggt gctcctcttc aatgaaacaa 120
acctgcaaac aagaaactag aatgaagaaa gatgacagta ccaaagcgcg gcctcagaaa 180
tatgagcaac ttctccatat agaggacaac gatttcgcaa tgagacctgg atttggaggg 240
tctccagtgc cagtaggtat agatgtccat gttgaaagca ttgacagcat ttcagagact 300
aacatggact ttacaatgac tttttatctc aggcattact ggaaagacga gaggctctcc 360
tttcctagca cagcaaacaa aagcatgaca tttgatcata gattgaccag aaagatctgg 420
gtgcctgata tcttttttgt ccactctaaa agatccttca tccatgatac aactatggag 480
aatatcatgc tgcgcgtaca ccctgatgga aacgtcctcc taagtctcag gataacggtt 540
tcggccatgt gctttatgga tttcagcagg tttcctcttg acactcaaaa ttgttctctt 600
gaactggaaa gctatgccta caatgaggat gacctaatgc tatactggaa acacggaaac 660
aagtccttaa atactgaaga acatatgtcc ctttctcagt tcttcattga agacttcagt 720
gcatctagtg gattagcttt ctatagcagc acaggctggt acaataggct tttcatcatc 780
tctgtgctaa ggaggcatgt tttcttcttt gtgctgccaa cctattaccc agccatattg 840
atggtgatgc tttcatgggt ttcattttgg attgaccgaa gagctgttcc tgcaagagtt 900
tccctgggaa tcaccacagt gctgaccatg tccacaatca tcactgctgt gagcgcctcc 960
atgccccagg tgtcctacct caaggctgtg gatgtgtacc tgtgggtcag ctccctcttt 1020
gtgttcctgt cagtcattga gtatgcagct gtgaactacc tcaccacagt ggaagagcgg 1080
aaacaattca agaagacagg aaagatttct aggatgtaca atattgatgc agttcaagct 1140
atggcctttg atggttgtta ccatgacagc gagattgaca tggaccagac ttccctctct 1200
ctaaactcag aagacttcat gagaagaaaa tcgatatgca gccccagcac cgattcatct 1260
cggataaaga gaagaaaatc cctaggagga catgttggta gaatcattct ggaaaacaac 1320
catgtcattg acacctattc taggatttta ttccccattg tgtatatttt atttaatttg 1380
ttttactggg gtgtatatgt atga
1404
<220> 38
<211> 4048
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7474240CB1
<400> 38
cttccatccc ccctcagcca ttccttactg ctctgggcaa ccgccaggtt aagcccattt 60
gcactgggaa attggcgctg tttgggagaa gagaaacaga tcgattgccc ttgtgactcc 120
ccgccccctt cccatcccca cccccaccgc tctctccctc tttccctccc ccgccacctc 180
ccctcacccc gcctccttcc cgttccccac ccccaaaccc tctcacccgc ggcagtccgg 240
tgcgaggccc cctccggaag gtgaggggaa tggattggac tccggtggag aaagcgggtg 300
tctagaagtg gtgctaatgg gaagagaatt ctggtttcaa aagaggatgc tctgccacaa 360
agagcggctc gcgcgctggc ctgggctcta gccgaggaga gatcccggga ggactccaga 420
gctccggggg agcgctcctc ggaagaccgg ggccaacatg cctgtgcgca gggggcatgt 480
62/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
ggcaccacaa aatacatttc tggggaccat cattcggaaa tttgaagggc aaaataaaaa 540
atttatcatt gcaaatgcca gagtgcagaa ctgtgccatc atttattgca acgatgggtt 600
ctgtgagatg actggtttct ccaggccaga tgtcatgcaa aagccatgca cctgcgactt 660
tctccatgga cccgagacca agaggcatga tattgcccaa attgcccagg cattgctggg 720
gtcagaagag aggaaagtgg aggtcaccta ctatcacaaa aatgggtcca cttttatttg 780
taacactcac ataattccag tgaaaaacca agagggcgtg gctatgatgt tcatcattaa 840
ttttgaatat gtgacggata atgaaaacgc tgccacccca gagagggtaa acccaatatt 900
accaatcaaa actgtaaacc ggaaattttt tgggttcaaa ttccctggtc tgagagttct 960
cacttacaga aagcagtcct taccacaaga agaccccgat gtggtggtca tcgattcatc 1020
taaacacagt gatgattcag tagccatgaa gcattttaag tctcctacaa aagaaagctg 1080
cagcccctct gaagcagatg acacaaaagc tttgatacag cccagcaaat gttctccctt 1140
ggtgaatata tccggacctc ttgaccattc ctctcccaaa aggcaatggg accgactcta 1200
ccctgacatg ctgcagtcaa gttcccagct gtcccattcc agatcaaggg aaagcttatg 1260
tagtatacgg agagcatctt cggtccatga tatagaagga ttcggcgtcc accccaagaa 1320
catatttaga gaccgacatg ccagcgaaga caatggtcgc aatgtcaaag ggccttttaa 1380
tcatatcaag tcaagcctcc tgggatccac atcagattca aacctcaaca aatacagcac 2440
cattaacaag attccacagc tcactctgaa tttttcagag gtcaaaactg agaaaaagaa 1500
ttcatcacct ccttcttcag ataaaaccat tattgcaccc aaggttaaag atcgaacaca 1560
caatgtgact gagaaagtga cccaggttct ctctttagga gcagatgtcc tacctgaata 1620
caaactgcag acaccacgca tcaacaagtt tacgatattg cactacagcc ctttcaaggc 1680
agtctgggac tggcttatcc tgctgttggt catatacact gctatattta ctccctactc 1740
tgcagccttc ctcctcaatg acagagaaga acagaaaaga cgagaatgtg gctattcttg 1800
tagccctttg aatgtggtag acttgattgt ggatattatg tttatcatag atattttaat 1860
aaacttcaga acaacatatg taaatcagaa tgaagaagtg gtaagtgatc ccgccaaaat 1920
agcaatacac tacttcaaag gctggttcct gattgacatg gttgcagcaa ttccttttga 1980
cttgctgatt tttggatcag gttctgatga gacaacaaca ttaattggtc ttttgaagac 2040
tgcccgactc ctccgtcttg tgcgcgtggc caggaaactg gatcgatatt cagaatatgg 2100
cgctgctgtt ctaatgctct taatgtgcat ctttgccctg attgctcact ggctggcttg 2160
catttggtat gcgattggga atgtagaaag gccttacctg actgacaaaa tcggatggtt 2220
ggattcctta ggacagcaaa ttgggaaacg ttacaatgac agtgactcaa gttctggacc 2280
atccattaaa gacaaatacg tcacagcact ttattttacc ttcagcagtt taaccagtgt 2340
aggattcggg aatgtgtctc ctaacacgaa ttcggagaaa atcttttcaa tttgtgtcat 2400
gttgattggc tcactaatgt atgcaagcat ttttgggaat gtatctgcaa ttatccaaag 2460
actatactcg ggaactgcca ggtaccacat gcagatgctg cgagtaaaag agttcattcg 2520
ctttcaccaa atccccaacc ctctgaggca acgtcttgaa gaatatttcc agcacgcatg 2580
gacttacacc aatggcattg acatgaacat ggtcctaaag ggtttcccag aatgcttaca 2640
agcagacatt tgtctacatc tcaaccagac attgctgcaa aactgcaaag cctttcgggg 2700
ggcaagtaaa ggttgcctta gagctttggc aatgaagttc aaaaccaccc atgcacctcc 2760
aggagacacc ctcgttcact gtggggatgt cctcactgca ctttatttct tatccagagg 2820
ctccattgaa attctcaaag atgacattgt ggtggctatt ctgggaaaaa atgatatatt 2880
tggagaaatg gttcatcttt atgccaaacc tggaaagtct aatgcagatg taagagccct 2940
cacatactgt gacttgcata agattcagcg agaagacttg ttagaggttt tggatatgta 3000
tcctgagttt tctgatcact ttctaacaaa cctagagttg actttcaacc taaggcatga 3060
gagcgcaaag gctgatctcc tacgatcaca atccatgaat gattcagaag gagacaactg 3120
taaactaaga agaaggaaat tgtcatttga aagtgaagga gagaaagaaa acagtacaaa 3180
tgatcctgaa gactctgcag ataccataag acattatcag agttccaaga gacactttga 3240
agagaaaaaa agcagatcct catctttcat ctcctccatt gatgatgaac aaaagccgct 3300
cttctcagga atagtagact cttctccagg aatagggaaa gcatctgggc tcgattttga 3360
agaaacagtg cccacctcag gaagaatgca catagataaa agaagtcact cttgcaaaga 3420
tatcactgac atgcgaagct gggaacgaga aaatgcacat ccccagcctg aagactccag 3480
tccatctgca cttcagcgag ctgcctgggg tatctctgaa accgaaagcg acctcaccta 3540
cggggaagtg gaacaaagat tagatctgct ccaggagcaa cttaacaggc ttgaatccca 3600
aatgaccact gacatccaga ccatcttaca gttgctgcag aaacaaacca ctgtggtccc 3660
cccagcctac agtatggtaa cagcaggatc agaatatcag agacccatca tccagctgat 3720
gagaaccagt caaccggaag catccatcaa aactgaccga agtttcagcc cttcctcaca 3780
atgtcctgaa tttctagacc ttgaaaaatc taaacttaaa tccaaagaat ccctttcaag 3840
tggggtgcat ctgaacacag cttcagaaga caacttgact tcacttttaa aacaagacag 3900
tgatctctct ttagagcttc acctgcggca aagaaaaact tacgttcatc caattaggca 3960
tccttctttg ccagattcat ccctaagcac tgtaggaatc gtgggtcttc ataggcatgt 4020
ttctgatcct ggtcttccag ggaaataa 4048
<210> 39
<211> 1539
<212> DNA
<213> Homo sapiens
<220>
63187
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
<221> misc_feature
<223> Incyte TD No: 7475338CB1
<400> 39
atggagaaca aagaggcggg aacccctcca cccattccat ccagggaggg gcggctccag 60
ccgacgctgt tgctggcgac actgagcgcg gcctttggct cagccttcca gtacggctac 120
aacctctctg tggtcaacac gccgcacaag gtgttcaagt cattttacaa cgaaacctac 180
tttgagcgac acgcaacatt catggacggg aagctcatgc tgcttctatg gtcttgcacc 240
gtctccatgt ttcctctggg cggcctgttg gggtcattgc tcgtgggcct gctggttgat 300
agctgcggca ggaaggggac cctgctgatc aacaacatct ttgccatcat ccccgccatc 360
ctgatgggag tcagcaaagt ggccaaggct tttgagctga tcgtcttttc ccgagtggtg 420
ctgggagtct gtgcaggtat ctcctacagc gcccttccca tgtacctggg agaactggcc 480
cccaagaacc tgagaggcat ggtgggaaca atgaccgagg ttttcgtcat cgttggagtc 540
ttcctagcac agatcttcag cctccaggcc atcttgggca acccggcagg ttggccggtg 600
cttctggcgc tcacaggggt gcccgccctg ctgcagctgc tgaccctgcc cttcttcccc 660
gaaagccccc gctactccct gattcagaaa ggagatgaag ccacagcgcg acaagctctg 720
aggaggctga gaggccacac ggacatggag gccgagctgg aggacatgcg tgcggaggcc 780
cgggccgagc gcgccgaggg ccacctgtct gtgctgcacc tctgtgccct gcggtccctg 840
cgctggcagc tcctctccat catcgtgctc atggccggcc agcagctgtc gggcatcaat 900
gcgatcaact actatgcgga caccatctac acatctgcgg gcgtggaggc cgctcactcc 960
caatatgtaa cggtgggctc tggcgtcgtc aacatagtga tgaccatcac ctcggctgtc 1020
cttgtggagc ggctgggacg gcggcacctc ctgctggccg gctacggcat ctgcggctct 1080
gcctgcctgg tgctgacggt ggtgctccta ttccagaaca gggtccccga gctgtcctac 2140
ctcggcatca tctgtgtctt tgcctacatc gcgggacatt ccattgggcc cagtcctgtc 1200
ccctcggtgg tgaggaccga gatcttcctg cagtcctccc ggcgggcagc tttcatggtg 1260
gacggggcag tgcactggct caccaacttc atcataggct tcctgttccc atccatccag 1320
gaggccatcg gtgcctacag tttcatcatc tttgccggaa tctgcctcct cactgcgatt 1380
tacatctacg tggttattcc ggagaccaag ggcaaaacat ttgtggagat aaaccgcatt 1440
tttgccaaga gaaacagggt gaagcttcca gaggagaaag aagaaaccat tgatgctggg 1500
cctcccacag cctctcctgc caaggaaact tccttttag 1539
<210> 40
<221> 3114
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7476747CB1
<400> 40
ccaagcagtg cctcacttct gccttgtcta gctgtactct ggaaaattaa gaaatttatg 60
agtgtagcac caagtatacc aatgggaagg atgggagtca gaagtcaagt gaactcagcc 120
cgcctctgtg tactttgcac ttttccattt cccttggtac caggcacttt catacttaat 180
ccatagtgga gctgtcacag tgagcaactc tgacaatgac agcttctacc ccagaggcga 240
ccccaaacat ggagctaaag gctccagctg caggaggtct taatgctggc cctgtccccc 300
cagctgccat gtccacgcag agacttcgga atgaagacta ccacgactac agctccacgg 360
acgtgagccc tgaggagagc ccgtcggaag gcctcaacaa cctctcctcc ccgggctcct 420
accagcgctt tggtcaaagc aatagcacaa catggttcca gaccttgatc cacctgttaa 480
aaggcaacat tggcacagga ctcctgggac tccctctggc ggtgaaaaat gcaggcatcg 540
tgatgggtcc catcagcctg ctgatcatag gcatcgtggc cgtgcactgc atgggtatcc 600
tggtgaaatg tgctcaccac ttctgccgca ggctgaataa atcctttgtg gattatggtg 660
atactgtgat gtatggacta gaatccagcc cctgctcctg gctccggaac cacgcacact 720
ggggaagacg tgttgtggac ttcttcctga ttgtcaccca gctgggattc tgctgtgtct 780
attttgtgtt tctggctgac aactttaaac aggtgataga agcggccaat gggaccacca 840
ataactgcca caacaatgag acggtgattc tgacgcctac catggactcg cgactctaca 900
tgctctcctt cctgcccttc ctggtgctgc tggttttcat caggaacctc cgagccctgt 960
ccatcttctc cctgttggcc aacatcacca tgctggtcag cttggtcatg atctaccagt 1020
tcattgttca gaggatccca gaccccagcc acctcccctt ggtggcccct tggaagacct 1080
accctctctt ctttggcaca gcgatttttt catttgaagg cattggaatg gttctgcccc 1140
tggaaaacaa aatgaaggat cctcggaagt tcccactcat cctgtacctg ggcatggtca 1200
tcgtcaccat cctctacatc agcctggggt gtctggggta cctgcaattt ggagctaata 1260
tccaaggcag cataaccctc aacctgccca actgctggtt gtaccagtca gttaagctgc 2320
tgtactccat cgggatcttt ttcacctacg cactccagtt ctacgtcccg gctgagatca 1380
tcatcccctt ctttgtgtcc cgagcgcccg agccctgtga gttagtggtg gacctgtttg 1440
tgcgcccagt gctggtctgc ctgacatcac tgtctggcag tgttgacaat ggctggtatg 1500
gcacggaagc cgatggcacc tcctgcggca gtgcaccatt ggtcttcgtc agttcctcct 1560
64/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
tcctggctca cccgtggctg agtttcagat gtgagagcca gtgggtgtcc tgtcacagag 1620
atacggtcgt cgtgtggggc ttcgccaggg gcatcttggc catcctcatc ccccgcctgg 1680
acctggtcat ctccctggtg ggctccgtga gcagcagcgc cctggccctc atcatcccac 1740
cgctcctgga ggtcaccacc ttctactcag agggcatgag ccccctcacc atctttaagg 1800
acgccctgat cagcatcctg ggcttcgtgg gctttgtggt ggggacctat gaggctctct 1860
atgagctgat ccagccaagc aatgctccca tcttcatcaa ttccacctgt gccttcatat 1920
agggatctgg gttcgtctct gcagctgcct acccctgccc catgtgtccc ccgttacctg 1980
tcctcagagc ctcaggtatg gtccaggctc tgaggaaagt cagggttgct gtgtgggaac 2040
ccctctgcct ggcacctgga taccctgggc caggtaacct gagggcagtg gagaggtggg 2100
gtggcagaca cgcagaagtg ctactagtga cagggctgcc atcgctcacc tgtacctatt 2160
tacacccaga actttccagc tccccctcat catgcctcct ccttcctacc tgcctcccct 2220
ctgctggtgc acctcgccca actcattctt actgcacagt tcactttatt taacaatttt 2280
catgtccccc acctcatgtt ttcacctttt ctgggccagg catagattaa gtaactggga 2340
acgccccctc tttataaagc tgggcttctt tctcatctct ctcccaaatg ttgtatctca 2400
gtattcttcc tattcgagtc tccagggggt ggctggacct acctggtcat ttgaaacagg 2460
cccccaagct ggagttttta atctggactc tctggcttgc tgtgacccct aaggcaatgc 2520
ttctcttccc tggattcctt agtgtgggtc acagtactgt gttcttagtt gctttagctc 2580
ttaaaacata cgaagtgttg cctaaactga aaatatttat cttttattta aaatcagatt 2640
tttgttttta gactgtctta gatctggggc tattacgaat cacttcttct tcagtaaact 2700
ttgactcaac ttctcctgct gaaaagaagc tcgctccaga tgtctgcatg ggtcctcggc 2760
actcttggct gaggactcaa aggttttaat caggatcgtc taaaaatgta cctcggtgag 2820
gaggcacaga ttttgcctcc tgttgaccag cctggtttca taccgaaaag acattgaagg 2880
actgcagaaa tgtatgggtg caccgggccg agggaagggt ggctgagtga gaggcgtata 2940
aaatggggct gtgtgcatgc aggcccatgt ttcagcctca gcccacgcca ggtgaaagga 3000
tcagcaatgc tctgttgcca tcgtgctggg acgacaccag ctctattgcc accgatgagt 3060
agctgaggtc agtgtgcaca gagtttgaaa ttaagttaat agactttaca gcag 3114
<210> 41
<211> 2877
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7477898CB1
<400> 41
atgccggtcc gcaggggcca cgtcgctccc caaaacactt acctggacac catcatccgc 60
aagttcgagg gccaaagtcg gaagttcctg attgccaatg ctcagatgga gaactgcgcc 120
atcatttact gcaacgacgg cttctgcgaa ctcttcggct actcccgagt ggaggtgatg 180
cagcaaccct gcacctgcga cttcctcaca ggccccaaca caccaagcag cgccgtgtcc 240
cgcctagcgc aggccctgct gggggctgag gagtgcaagg tggacatcct ctactaccgc 300
aaggatgcct ccagcttccg ctgcctggta gatgtggtgc ccgtgaagaa cgaggacggg 360
gctgtcatca tgttcattct caacttcgag gacctggccc agctcctggc caagtgcagc 420
agccgcagct tgtcccagcg cctgttgtcc cagagcttcc tgggctccga gggctctcat 480
ggcaggccag gcggaccagg gccaggcaca ggcaggggca agtacaggac catcagccag 540
atcccacagt tcacgctcaa cttcgtggag ttcaacttgg agaagcaccg ctccagctcc 600
accacggaga ttgagatcat cgcgccccat aaggtggtgg agcggacaca gaacgtcact 660
gagaaggtca cccaggtcct gtccctgggc gcggatgtgc tgccggagta caagctgcag 720
gcgccgcgca tccaccgctg gaccatcctg cactacagcc ccttcaaggc cgtgtgggac 780
tggctcatcc tgctgctggt catctacacg gctgtcttca cgccctactc agccgccttc 840
ctgctcagcg atcaggacga atcacggcgt ggggcctgca gctatacctg cagtcccctc 900
actgtggtgg atctcatcgt ggacatcatg ttcgtcgtgg acatcgtcat caacttccgc 960
accacctatg tcaacaccaa tgatgaggtg gtcagccacc cccgccgcat cgccgtccac 1020
tacttcaagg gctggttcct cattgacatg gtggccgcca tccctttcga cctcctgatc 1080
ttccgcactg gctccgatga gaccacaacc ctgattgggc tattgaagac agcgcggctg 1140
ctgcggctgg tgcgcgtagc acggaagctg gaccgctact ctgagtatgg ggcggctgtg 1200
ctcttcttgc tcatgtgcac ctttccgctc atagcgcact ggctggcctg catctggtac 1260
gccatcggca atgtggagcg gccctaccta gaacacaaga tcggctggct ggacagcctg 1320
ggtgtgcagc ttggcaagcg ctacaacggc agcgacccag cctcgggccc ctcggtgcag 1380
gacaagtatg tcacagccct ctacttcacc ttcagcagcc tcaccagcgt gggcttcggc 1440
aatgtctcgc ccaacaccaa ctccgagaag gtcttctcca tctgcgtcat gctcatcggc 1500
tccctgatgt acgccagcat cttcgggaac gtgtccgcga tcatccagcg cctgtactcg 1560
ggcaccgcgc gctaccacac gcagatgctg cgtgtcaagg agttcatccg cttccaccag 1620
atceccaacc cactgcgcca gcgcctggag gagtatttcc agcacgcctg gtcctacacc 1680
aatggcattg acatgaacgc ggtgctgaag ggcttccccg agtgcctgca ggctgacatc 1740
tgcctgcacc tgcaccgcgc actgctgcag cactgcccag ctttcagcgg cgccggcaag 1800
65187
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
ggctgcctgc gcgcgctagc cgtcaagttc aagaccaccc acgcgccgcc tggggacacg 1860
ctggtgcacc tcggcgacgt gctctccacc ctctacttca tctcccgagg ctccatcgag 1920
atcctgcgcg acgacgtggt cgtggccatc ctaggaaaga atgacatctt tggggaaccc 2980
gtcagcctcc atgcccagcc aggcaagtcc agtgcagacg tgcgggctct gacctactgc 2040
gacctgcaca agatccagcg ggcagatctg ctggaggtgc tggacatgta cccggccttt 2100
gcggagagct tctggagtaa gctggaggtc accttcaacc tgcgggacgt aaccgggggt 2160
ctccactcat ccccccgaca ggctcctggc agccaagacc accaaggttt ctttctcagt 2220
gacaaccagt cagatgcagc ccctcccctg agcatctcag atgcattctg gctctggcct 2280
gagctactgc aggaaatgcc cccaaagcac agcccccaaa gccctcagga agacccagat 2340
tgctggcctc tgaagctggg ctccaggcta gagcagctcc aggcccagat gaacaggctg 2400
gagtcccgcg tgtcctcaga cctcagccgc atcttgcagc tcctccagaa gcccatgccc 2460
cagggccacg ccagctacat tctggaagcc cctgcctcca atgacctggc cttggttcct 2520
atagcctcgg agacgacgag tccagggccc aggctgcccc agggctttct gcctcctgca 2580
cagaccccaa gctatggaga cttggatgac tgtagtccaa agcacaggaa ctcctccccc 2640
aggatgcctc acctggctgt ggcaatggac aaaactctgg caccatcctc agaacaggaa 2700
cagcctgagg ggctctggcc acccctagcc tcacctctac atcccctgga agtacaagga 2760
ctcatctgtg gtccctgctt ctcctccctc cctgaacacc ttggctctgt tcccaagcag 2820
ctggacttcc agagacatgg ctcagatcct ggatttgcag ggagttgggg ccactga 2877
<210> 42
<211> 2820
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472728CB1
<400> 42
atggggcatc aagggccatt tgaagaagga aatggtggac tgagagtgat agcgacctgg 60
aggaggaagg aggcttggag aagggactgt cttttaggag ccctgcccag tgtttcctgt 120
ggagggtggg gccatcgtgg aagacagacc tatggtaggg cttgtggggt gaaagaaaag 180
ccctttagtc ttttgggtcc tcaaatcaca gtttatgcag tttggcccca gtcagaggga 240
ccccaggaag gcagactcag ggtaaattct gcctgtcttc caccagagag gggactcacc 300
aacgcttgta caaaccatga agaactctct ttggactgtt tgctttttga gaatgttaac 360
accttgactc tggatttctg cctatgggaa aaaaccacaa tagtgccagg ggtgcttcca 420
tatgcaggat taactctgca gtcaaagttt ctgttgggca gagcattgtt agcaggggtc 480
catgtgatca cactgacacc tgagagagtg acacaccatg tacatggctg gtatatggag 540
gatggattta agggggacag gactgaaggc tgtcgcagtg attcagtggc cgttcccgca 600
gcagcaccgg tgtgccagcc caagagcgcc actaacgggc aacccccggc tccggctccg 660
actccaactc cgcgcctgtc catttcctcc cgagccacag tggtagccag gatggaaggc 720
acctcccaag ggggcttgca gaccgtcatg aagtggaaga cggtggttgc catctttgtg 780
gttgtggtgg tctaccttgt cactggcggt cttgtcttcc gggcattgga gcagcccttt 840
gagagcagcc agaagaatac catcgccttg gagaaggcgg aattcctgcg ggatcatgtc 900
tgtgtgagcc cccaggagct ggagacgttg atccagcatg ctcttgatgc tgacaatgcg 960
ggagtcagtc caataggaaa ctcttccaac aacagcagcc actgggacct cggcagtgcc 1020
tttttctttg ctggaactgt cattacgacc atgtatggga atattgctcc gagcactgaa 1080
ggaggcaaaa tcttttgtat tttatatgcc atctttggaa ttccactctt tggtttctta 1140
ttggctggaa ttggagacca acttggaacc atctttggga aaagcattgc aagagtggag 1200
aaggtctttc gaaaaaagca agtgagtcag accaagatcc gggtcatctc aaccatcctg 1260
ttcatcttgg ccggctgcat tgtgtttgtg acgatccctg ctgtcatctt taagtacatc 1320
gagggctgga cggccttgga gtccatttac tttgtggtgg tcactctgac cacggtgggc 1380
tttggtgatt ttgtggcagt ggttgttttc aggggaaacg ctggcatcaa ttatcgggag 1440
tggtataagc ccctagtgtg gttttggatc cttgttggcc ttgcctactt tgcagctgtc 1500
ctcagtatga tcggagattg gctacgggtt ctgtccaaaa agacaaaaga agaggtgggt 1560
gaaatcaagg cccatgcggc agagtggaag gccaatgtca cggctgagtt ccgggagaca 1620
cggcgaaggc tcagcgtgga gatccacgat aagctgcagc gggcagccac catccgcagc 1680
atggagcgcc ggcggctggg cctggaccag cgggcccact cactggacat gctgtccccc 2740
gagaagcgct ctgtctttgc tgccctggac accggccgct tcaaggcctc atcccaggag 1800
agcatcaaca accggcccaa caacctgcgc ctgaaggggc cggagcagct gaacaagcat 1860
gggcagggtg cgtccgagga caacatcatc aacaagttcg ggtccacctc cagactcacc 1920
aagaggaaaa acaaggacct caaaaagacc ttgcccgagg acgttcagaa aatctacaag 1980
accttccgga attactccct ggacgaggag aagaaagagg aggagacgga aaagatgtgt 2040
aactcagaca actccagcac agccatgctg acggactgta tccagcagca cgctgagttg 2100
gagaacggaa tgatacccac ggacaccaaa gaccgggagc cggagaacaa ctcattactt 2160
gaagacagaa actaaatgtg aaggacattg gtcttggact gagcgttgtg tgtgtgtgtg 2220
tgtgtgtgtt tttaatattc acactgagac atgtgcctta aacagacttt ttagtccaaa 2280
66/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
attacatagc attgaagaat atatttcact gtgccataaa caactgaaag cttgctctgc 2340
caaaaggaat cagagaacaa gaacttcatt tcagatagca aacgcaggac acaccaagag 2400
tgtccgtgca cgtagccggt tctggccgta catgttaagg gcatttcagt ggcagtgctg 2460
tacccctggg cagtgctacc tgggcacaca cgtagacaag ggcagctatt ccttagacca 2520
gcctcctgaa agaaacaggt gtgtcttttt agtggagtcg tagtaatatg tgcacacaca 2580
gaaggggacc tgattgggtg ggagctggtt atgtgtaact agcgttggag ttgacatttt 2640
ggcatgtgct ctgagcttga attttgatac caaccattca gtgcatcata cctagtcttt 2700
ctatgctcca aatgaatgtc tgtggggacc tgagagcacc tggaatttgt tggaagcaga 2760
tcagagcaca cgtacgaaaa ggtgcaattc cttttctcat gacaaaaggg aaaaaaataa 2820
<210> 43
<211> 1440
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7474322CB1
<220>
<221> unsure
<222> 757
<223> a, t, c, g, or other
<400> 43
atgtacaatg agattctgat gctgggggcc aaactgcacc cgacgctgaa gctggaggag 60
ctcaccaaca agaagggaat gacgccgctg gctctggcag ctgggaccgg gaagatcggg 120
aatcgccacg acatgctctt ggtggagccg ctgaaccgac tcctgcagga caagtgggac 180
agattcgtca agcgcatctt ctacttcaac ttcctggtct actgcctgta catgatcatc 240
ttcaccatgg ctgcctacta caggcccgtg gatggcttgc ctccctttaa gatggaaaaa 300
actggagact atttccgagt tactggagag atcctgtctg tgttaggagg agtctacttc 360
tttttccgag ggattcagta tttcctgcag aggcggccgt cgatgaagac cctgtttgtg 420
gacagctaca gtgagatgct tttgtttctg cagtcactgt tcatgctggc caccgtggtg 480
ctgtacttca gccacctcaa ggagtatgtg gcttccatgg tattctccct ggccttgggc 540
tggaccaaca tgctctacta cacccgcggt ttccagcaga tgggcatcta tgccgtcatg &00
atagagaaga tgatcctgag agacctgtgc cgtttcatgt ttgtctacat cgtcttcttg 660
ttcgggtttt ccacagcggt ggtgacgctg attgaagacg ggaagaatga ctccctgccg 720
tctgagtcca cgtcgcacag gtggcggggg cctgctngca ggcccaatag ctcctacaac 780
agcctgtact ccacctgcct ggagctgttc aagttcacca tcggcatggg cgacctggag 840
ttcactgaga actatgactt caaggctgtc ttcatcatcc tgctgctggc ctatgtaatt 900
ctcacctaca tagttctcct cctcaacatg ctcattgctc tgatgggcga gactgtggag 960
aacgtctcca aggagagcga acgcatctgg cgcctgcaga gagccatcac catcctggac 1020
acggagaaga gcttccttaa gtgcatgagg aaggccttcc gctcaggcaa gctgctgcag 1080
gtggggtaca cacctgatgg caaggacgac taccggtggt gcttcgtgga cgaggtgaac 1140
tggaccacct ggaacaccaa cgtgggcatc atcaacgaag acccgggcaa ctgtgagggc 1200
gtcaagcgca ccctgagctt ctccctgcgg tcaagcagag tttcaggcag acactggaag 1260
aactttgccc tggtccccct tttaagagag gcaagtgctc gagataggca gtctgctcag 1320
cccgaggaag tttatctgcg acagttttca gggtctctga agccagagga cgctgaggtt 1380
ttcaagagtc ctgccgcttc cggggagaag tgaggacgtc acgcagacag cactgtcaac 1440
<210> 44
<211> 2394
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5455&21CB1
<400> 44
atttcagaac acatctgaat tccttctctg tggcatatgc tttaggagag gagcagacag 60
ctcttagcta gggtcagatt tcaaattctc atctcttggt gccaatacca ccaccagatt 120
cttctttgaa gtcaactttt gagatcttca ctaagtacac gttggtgtct gaagattcac 180
acgagtgcct ctggtaatca ttttcttcag ggaatcacag tctctcctct cagcaaagca 240
tccactgtac tgaactttgc ttttggaaac atcttcttcc tgagacctcg ttgaaagaaa 300
ctctctggtg tcatactttc caatatggag gtgaagaact ttgcagtttg ggattatgtt 360
gtatttgcag ccctcttttt catttcctct ggaattgggg tgttctttgc cattaaggag 420
67/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
agaaaaaagg caacttcccg agagttcctg gttgggggaa ggcaaatgag ctttggccct 480
gtcggcttgt ctctgacagc cagcttcatg tcagctgtca cggtcctggg gaccccttct 540
gaagtctacc gctttggggc atccttccta gtcttcttca ttgcttacct atttgtcatc 600
ctcttaacat cagagctctt tctccctgtg ttctacagat ctggtatcac cagcacttat 660
gagtacttac aactacgatt caacaaacca gttcgctatg ctgccacagt catctacatt 720
gtacagacga ttctctacac aggagtggtg gtgtatgctc ctgccctggc actcaatcaa 780
gtgactgggt ttgatctctg gggctctgtg tttgcaacag gaattgtttg cacattctac 840
tgtaccctgg gaggattaaa agcagtggtg tggacagatg catttcagat ggttgtcatg 900
attgtgggct tcttaacggt tctcattcaa ggatcaactc atgctggggg attccacaat 960
gtattagagc aatcaacaaa tggatctcga ctacatatat ttgactttga tgtagatcct 1020
ctcaggcgac acactttttg gactatcaca gtgggaggaa cttttacttg gctcggaatc 1080
tatggggtca atcaatcaac tattcagcga tgcatctctt gcaaaacaga aaagcatgct 1140
aagcttgcct tgtattttaa cttgctgggt ctctggatca ttctggtgtg tgctgtcttc 1200
tctggcttaa tcatgtactc tcactttaaa gactgtgacc cttggacttc tggcatcatc 1260
tcagcaccag accagctgat gccgtacttt gtcatggaga tatttgccac aatgccagga 1320
ctgccaggac tttttgtggc ttgtgccttc agtggaactc tgagcaccgt ggcttccagc 1380
atcaatgcct tggcaacagt gacctttgag gattttgtca agagctgttt tcctcatctc 1440
tccgacaagc tgagcacctg gatcagtaaa ggcttatgtc tcttatttgg cgtgatgtgt 1500
acctctatgg ctgtggctgc atctgtcatg ggaggtgttg tgcaggcttc cctcagcatt 1560
cacggcatgt gtggaggacc aatgctgggc ttattctccc tgggaatcgt gttccctttt 1620
gtgaactgga agggtgcact aggaggtctt cttactggaa tcaccttgtc attttgggtg 1680
gccattgggg ccttcattta ccctgcacca gcctctaaga catggccttt gcctctatca 1740
acagaccaat gtatcaaatc aaatgtgaca gcaacagggc ctccagtact atccagcaga 1800
cctggaatag ctgatacctg gtactcgatc tcctaccttt actacagtgc actgggctgc 1860
ttaggatgca ttgttgctgg agtaatcatc agcctcataa caggtcgcca aagaggtgag 1920
gatattcaac cactgttaat tagaccagtt tgtaatttat tttgcttttg gtctaagaag 1980
tacaaaacac tatgctggtg tggagttcag catgacagtg ggacagagca ggaaaacctt 2040
gagaatggca gtgcccggaa acagggggct gaatctgtct tacagaacgg actcagaaga 2100
gaaagcctgg tacatgttcc aggctatgat cctaaggaca aaagctacaa caatatggca 2160
tttgagacta cccatttcta aggcaatacc tgtatgaatg cacacacaca cgtgcaatac 2220
acacacacac acacaaactc cacatacttc ttgcctactt gttagtagat atgtatagtt 2280
gccattgcta gaagacaggg atgtctggtg cctatttcta cttatttata actacatgca 2340
aaatgactgt ctctcgggat attctttgaa agactccaac tttcacagag aaaa 2394
<210> 45
<211> 2890
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7477248CB1
<400> 45
gaatactaag ccagggcaga atgcttgtga agtagcaact aaagtggcag tgtttcttct 60
gaaattctca ggcagtcaga ctgtcttagg caaatcttga taaaatagcc cttatccagg 120
tttttatcta aggaatccca agaagactgg ggaatggaga gacagtcaag ggttatgtca 180
gaaaaggatg agtatcagtt tcaacatcag ggagcggtgg agctgcttgt cttcaatttt 240
ttgctcatcc ttaccatttt gacaatctgg ttatttaaaa atcatcgatt ccgcttcttg 300
catgaaactg gaggagcaat ggtgtatggc cttataatgg gactaatttt acgatatgct 360
acagcaccaa ctgatattga aagtggaact gtctatgact gtgtaaaact aactttcagt 420
ccatcaactc tgctggttaa tatcactgac caagtttatg aatataaata caaaagagaa 480
ataagtcagc acaacatcaa tcctcatcaa ggaaatgcta tacttgaaaa gatgacattt 540
gatccagaaa tcttcttcaa tgttttactg ccaccaatta tatttcatgc aggatatagt 600
ctaaagaaga gacacttttt tcaaaactta ggatctattt taacgtatgc cttcttggga 660
actgccatct cctgcatcgt catagggtta attatgtatg gttttgtgaa ggctatgata 720
catgctggcc agctgaaaaa tggagacttt catttcactg actgtttatt ttttggttca 780
ctgatgtctg ctacagatcc agtgacagtg ctggccattt tccatgaact gcacgtcgac 840
cctgacctgt acacactctt gtttggagag agtgtgttga atgatgcagt ggccatagtc 900
cttacatatt ctatatccat ttacagtccc aaggagaatc caaatgcatt tgatgccgca 960
gcattcttcc agtctgtggg gaatttcctg ggaatcttcg ctggctcatt tgcaatgggg 1020
tctgcgtatg ccatcatcac agcactgttg accaaattta ccaagctgtg tgagttcccg 1080
atgctggaaa ccggcctgtt tttcctgctt tcttggagtg ccttcctgtc tgccgaggct 1140
gccggcctaa cagggatagt tgctgttctc ttctgtggag tcacacaagc acattatacc 1200
tacaacaatc tgtcttcgga ttccaaaata agaactaaac agttgtttga atttatgaac 1260
tttttggcgg agaacgtcat cttctgttac atgggcctgg cactgttcac gttccagaat 1320
catatcttta atgctctttt tatacttgga gcctttctag caatttttgt tgccagagcc 1380
68/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
tgcaacatat atcccctctc cttcctcctg aatctaggcc gaaaacagaa gatcccctgg 1440
aactttcagc acatgatgat gttttcaggt ttgcgaggag cgatcgcatt tgccttagct 1500
attcggaaca cagaatctca gcccaaacaa atgatgttta ccactacgct gctcctcgtg 1560
ttcttcactg tctgggtatt tggaggagga acaaccccca tgttgacttg gcttcagatc 1620
agagttggcg tggacctgga tgaaaatctg aaggaggacc cctcctcaca acaccaggaa 1680
gcaaataact tggataaaaa catgacgaaa gcagagagtg ctcggctctt cagaatgtgg 1740
tatagctttg accacaagta tctgaaacca attttaaccc actctggtcc tccgctgact 1800
acaacattac ctgaatggtg tggtccgatt tccaggctgc ttaccagtcc tcaagcctat 1860
ggggaacagc taaaagagga tgatgtggaa tgcattgtaa accaggatga actagccata 1920
aattaccagg agcaagcctc ctcaccctgc agtcctcctg caaggctagg tctggaccag 1980
aaagcttcac cccagacgcc aggcaaggaa aacatttatg agggagacct cggccctggg 2040
aggctatgaa ctcaagcttg agcaaacttt gggtcaatcc cagttgaatt aattggcatg 2100
aagagtacag atgtaatcac aagtaatgca agactcactg aggaatacaa gccaagctga 2160
tgaggcagta caggggagag gctggaaaac atattaagag cataaattgg agagaatcaa 2220
agccttgtca catggatcct ctggtgcctg aagaaatgag attttattat ccctctctat 2280
tatgcaaatg aatttagttt tttgacagca gccattctga ttactggatt ggctggggtg 2340
gggatggagg tatcaggagt ctagctgctg gaggatggga cagctgtgct gggtcttcag 2400
ggcatttctg ctgcgaatgc ggctctccag gcccttcact tctattctgg attttattcc 2460
ctccattaag gagagtttaa aaataaaaga aagcttctga gagtaaacat tttgctccta 2520
agctgaaggg aatgcccagc tatttagtaa gtgataagtt tcttattttg aggacttgac 2580
tcccatttgc tctcagtgac cccagggcag agcccagaga agtgttccgt acccactgct 2640
gatggtttcc cagagcccac actgagttga agaacctatt gttcttcttg gcatccttct 2700
tatgctactt ctcccatcgc tcaaaggggt tgcctatggc tgggtgtgcc ctgccctaaa 2760
tgcagcacca ctttcaagca gcttctagct atagctttcc accaggtatt tttaatccca 2820
tttcacctcc tcccccagca attcaccagt caggagtgat ttttactgta aagatggttg 2880
cttagtaaaa 2890
<210> 46
<211> 3926
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2944004CB1
<400> 46
ctggaccttt aatccactgt aggtatggac agggaagaaa ggaagaccat caatcagggt 60
caagaagatg aaatggagat ttatggttac aatttgagtc gctggaagct tgccatagtt 120
tctttaggag tgatttgctc tggtggggtt tctcctcctc ctctctattg gatgcctgag 180
tggcgggtga aagcgacctg tgtcagagct gcaattaaag actgtgaagt agtgctgctg 240
aggactactg atgaattcaa aatgtggttt tgtgcaaaaa ttcgcgttct ttctttggaa 300
acttacccag tttcaagtcc aaaatctatg tctaataagc tttcaaatgg ccatgcagtt 360
tgtttaattg agaatcccac tgaagaaaat aggcacagga tcagtaaata ttcacagact 420
gaatcacaac agattcgtta tttcacccac catagtgtaa aatatttctg gaatgatacc 480
attcacaatt ttgatttctt aaagggactg gatgaaggtg tttcttgtac gtcaatttat 540
gaaaagcata gtgcaggact gacaaagggg atgcatgcct acagaaaact gctttatgga 600
gtaaatgaaa ttgctgtaaa agtgccttct gtttttaagc ttctaattaa agaggttctc 660
aacccatttt acattttcca gctgttcagt gttatactgt ggagcactga tgaatactat 720
tactatgctc tagctattgt ggttatgtcc atagtatcaa tcgtaagctc actatattcc 780
attagaaagc aatatgttat gttgcatgac atggtggcaa ctcatagtac cgtaagagtt 840
tcagtttgta gagtaaatga agaaatagaa gaaatctttt ctaccgacct tgtgccagga 900
gatgtcatgg tcattccatt aaatgggaca ataatgcctt gtgatgctgt gcttattaat 960
ggtacctgca ttgtaaacga aagcatgtta acaggagaaa gtgttccagt gacaaagact 1020
aatttgccaa atccttcagt ggatgtgaaa ggaataggag atgaattata taatccagaa 1080
acacataaac gacatacttt gttttgtggg acaactgtta ttcagactcg tttctacact 1140
ggagaactcg tcaaagccat agttgttaga acaggattta gtacttccaa aggacagctt 1200
gttcgttcca tattgtatcc caaaccaact gattttaaac tctacagaga tgcctacttg 1260
tttctactat gtcttgtggc agttgctggc attgggttta tctacactat tattaatagc 1320
attttaaatg aggtacaagt tggggtcata attatcgagt ctcttgatat tatcacaatt 1380
actgtgcccc ctgcacttcc tgctgcaatg actgctggta ttgtgtatgc tcagagaaga 1440
ctgaaaaaaa tcggtatttt ctgtatcagt cctcaaagaa taaatatttg tggacagctc 1500
aatcttgttt gctttgacaa gactggaact ctaactgaag atggtttaga tctttggggg 1560
attcaacgag tggaaaatgc acgatttctt tcaccagaag aaaatgtgtg caatgagatg 1620
ttggtaaaat cccagtttgt tgcttgtatg gctacttgtc attcacttac aaaaattgaa 1680
ggagtgctct ctggtgatcc acttgatctg aaaatgtttg aggctattgg atggattctg 1740
gaagaagcaa ctgaagaaga aacagcactt cataatcgaa ttatgcccac agtggttcgt 1800
69!87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
cctcccaaac aactgcttcc tgaatctacc cctgcaggaa accaagaaat ggagctgttt 1860
gaacttccag ctacttatga gataggaatt gttcgccagt tcccattttc ttctgctttg 1920
caacgtatga gtgtggttgc cagggtgctg ggggatagga aaatggacgc ctacatgaaa 1980
ggagcgcccg aggccattgc cggtctctgt aaacctgaaa cagttcctgt cgattttcaa 2040
aacgttttgg aagacttcac taaacagggc ttccgtgtga ttgctcttgc acacagaaaa 2100
ttggagtcaa aactgacatg gcataaagta cagaatatta gcagagatgc aattgagaac 2160
aacatggatt ttatgggatt aattataatg cagaacaaat taaagcaaaa aacccctgca 2220
gtacttgaag atttgcataa agccaacatt cgcaccgtca tggtcacagg tgacagtatg 2280
ttgactgctg tctctgtggc cagagattgt ggaatgattc tacctcagga taaagtgatt 2340
attgctgaag cattacctcc aaaggatggg aaagttgcca aaataaattg gcattatgca 2400
gactccctca cgcagtgcag tcatccatca gcaattgacc cagaggctat tccggttaaa 2460
ttggtccatg atagcttaga ggatcttcaa atgactcgtt atcattttgc aatgaatgga 2520
aaatcattct cagtgatact ggagcatttt caagaccttg ttcctaagtt gatgttgcat 2580
ggcaccgtgt ttgcccgtat ggcacctgat cagaagacac agttgataga agcattgcaa 2640
aatgttgatt attttgttgg gatgtgtggt gatggcgcaa atgattgtgg tgctttgaag 2700
agggcacacg gaggcatttc cttatcggag ctcgaagctt cagtggcatc tccctttacc 2760
tctaagactc ctagtatttc ctgtgtgcca aaccttatca gggaaggccg tgctgcttta 2820
ataacttcct tctgtgtgtt taaattcatg gcattgtaca gcattatcca gtacttcagt 2880
gttactctgc tgtattctat cttaagtaac ctaggagact tccagtttct cttcattgat 2940
ctggcaatca ttttggtagt ggtatttaca atgagtttaa atcctgcctg gaaagaactt 3000
gtggcacaaa gaccaccttc gggtcttata tctggggccc ttctcttctc cgttttgtct 3060
cagattatca tctgcattgg atttcaatct ttgggttttt tttgggtcaa acagcaacct 3120
tggtatgaag tgtggcatcc aaaatcagat gcttgtaata caacaggaag cgggttttgg 3180
aattcttcac acgtagacaa tgaaaccgaa cttgatgaac ataatataca aaattatgaa 3240
aataccacag tgttttttat ttccagtttt cagtacctca tagtggcaat tgccttttca 3300
aaaggaaaac ccttcaggca accttgctac aaaaattatt tttttgtttt ttctgtgatt 3360
tttttatata tttttatatt attcatcatg ttgtatccag ttgcctctgt tgaccaggtt 3420
cttcagatag tgtgtgtacc atatcagtgg cgtgtaacta tgctcatcat tgttcttgtc 3480
aatgcctttg tgtctatcac agtggagaac ttcttccttg acatggtcct ttggaaagtt 3540
gtgttcaacc gagacaaaca aggagagtat cggttcagca ccacacagcc accgcaggag 3600
tcagtggatc ggtggggaaa atgctgctta ccctgggccc tgggctgtag aaagaagaca 3660
ccaaaggcaa agtacatgta tctggcgcag gagctcttgg ttgatccaga atggccacca 3720
aaacctcaga caaccacaga agctaaagct ttagttaagg agaatggatc atgtcaaatc 3780
atcaccataa catagcagtg aatcagtctc agtggtattg ctgatagcag tattcaggaa 3840
tatgtgattt taggagtttc tgatcctgtg tgtcagaatg gcactagttc agtttatgtc 3900
ccttctgata tagtagctta tttgac 3926
<220> 47
<211> 2135
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Tncyte ID No: 3046849CB1
<400> 47
cgctcaggcc cctctttcga atgctccacg ccctcctgcg atctagaacg attcagggca 60
ggatcctgct cctgaccatc tgcgctgccg gcattggtgg gacttttcag tttggctata 120
acctctctat catcaatgcc ccgaccttgc acattcagga attcaccaat gagacatggc 180
aggcgcgtac tggagagcca ctgcccgatc acctagtcct gcttatgtgg tccctcatcg 240
tgtctctgta tcccctggga ggcctctttg gagcactgct tgcaggtccc ttggccatca 300
cgctgggaag gaagaagtcc ctcctggtga ataacatctt tgtggtgtca gcagcaatcc 360
tgtttggatt cagccgcaaa gcaggctcct ttgagatgat catgctggga agactgctcg 420
tgggagtcaa tgcaggtgtg agcatgaaca tccagcccat gtacctgggg gagagcgccc 480
ctaaggagct ccgaggagct gtggccatga gctcagccat ctttacggct ctggggatcg 540
tgatgggaca ggtggtcgga ctcagggagc tcctaggtgg ccctcaggcc tggcccctgc 600
tgctggccag ctgcctggtg cccggggcgc tccagctcgc ctccctgcct ctgctccctg 660
aaagcccgcg ctacctcctc attgactgtg gagacaccga ggcctgcctg gcagcactac 720
ggcagctacg gggctccggg gacttggcag gggagctgga ggagctggag gaggagcgcg 780
ctgcctgcca gggctgccgt gcccggcgcc catgggagct gttccagcat cgggccctga 840
ggagacaggt gacaagcctc gtggttctgg gcagtgccat ggagctctgc gggaatgact 900
cggtgtacgc ctacgcctcc tccgtgttcc ggaaggcagg agtgccggaa gcgaagatcc 960
agtacgcgat catcgggact gggagctgcg agctgctcac ggcggttgtt agttgtgtgg 1020
taatcgagag ggtgggtcgg cgcgtgctgc tcatcggtgg gtacagcctg atgacctgct 1080
gggggagcat cttcactgtg gccctgtgcc tgcagagctc cttcccctgg acactctacc 1140
tggccatggc ctgcatcttt gccttcatcc tcagctttgg cattggccct gccggagtga 1200
7/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
cggggatcct ggccacagag ctgtttgacc agatggccag gcctgctgcc tgcatggtct 1260
gcggggcgct catgtggatc atgctcatcc tggtcggcct gggatttccc tttatcatgg 1320
aggccttgtc ccacttcctc tatgtccctt tccttggtgt ctgtgtctgt ggggccatct 1380
acactggcct gttccttcct gagaccaaag gcaagacctt ccaagagatc tccaaggaat 1440
tacacagact caacttcccc aggcgggccc agggccccac gtggaggagc ctggaggtta 1500
tccagtcaac agaactctag tcccaaaggg gtggccagag ccaaagccag ctactgtcct 1560
gtcctctgct tcctgccagg gccctggtcc tcactccctc ctgcattcct catttaagga 1620
gtgtttattg agcacccttt gtgtgcagac atggctccag gtgcttagca atcaatggtg 1680
agcgtggtat tccaggctaa aggtaattaa ctgacagaaa atcagtaaca acataattac 1740
aggctggttg tggcagctca tgactgtaat cccagcactt tgggaggcca aggtgggagg 1800
atcaattgag gccagagttt gaaaccagcc taggtaacat agtgagaccc cctatctcta 1860
caaaaaattt taaacattag ctgggcatgg tggtatgtgc taacagctct agctactcag 1920
gaggctgagg cagcaggatc acttgagtcc caagagttca aggtagcagt aagctaacaa 1980
ttcacaccac tgcatgccca gactggggtg acagagggag acttcatctc tttaaaaaca 2040
taataataat aattacggac tccggaaatg cgttgacaac gaaacatacc ggtggccccg 2100
tgaggtggtg atcccgtatc ccagccttgg gaagc 2135
<210> 48
<211> 2637
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4538363CB1
<400> 48
atgggctgga gatgccactg tccgcttggt ttaatgatca atgagctccc tgccaggaaa 60
ccctttctga cctggtttgc ccctcagtcc ctcgggctca tacctagtgc ctgcggcagg 120
acagccatgg ccgccaactc caccagcgac ctccacactc ccgggacgca gctgagcgtg 180
gctgacatca tcgtcatcac tgtgtatttt gctctgaacg tggccgtggg catatggtcc 240
tcttgtcggg ccagtaggaa cacggtgaat ggctacttcc tggcaggccg ggacatgacg 300
tggtggccga ttggagcctc cctcttcgcc agcagcgagg gctctggcct cttcattgga 360
ctggcgggct caggcgcggc aggaggtctg gccgtggcag gcttcgagtg gaatgccacg 420
tacgtgctgc tggcactggc atgggtgttc gtgcccatct acatctcctc agagatcgtc 480
accttacctg agtacattca gaagcgctac gggggccagc ggatccgcat gtacctgtct 540
gtcctgtccc tgctactgtc tgtcttcacc aagatatcgc tggacctgta cgcgggggct 600
ctgtttgtgc acatctgcct gggctggaac ttctacctct ccaccatcct cacgctcggc 660
atcacagccc tgtacaccat cgcagggggc ctggctgctg taatctacac ggacgccctg 720
cagacgctca tcatggtggt gggggctgtc atcctgacaa tcaaagcttt tgaccagatc 780
ggtggttacg ggcagctgga ggcagcctac gcccaggcca ttccctccag gaccattgcc 840
aacaccacct gccacctgcc acgtacagac gccatgcaca tgtttcgaga cccccacaca 900
ggggacctgc cgtggaccgg gatgaccttt ggcctgacca tcatggccac ctggtactgg 960
tgcaccgacc aggtcatcgt gcagcgatca ctgtcagccc gggacctgaa ccatgccaag 1020
gcgggctcca tcctggccag ctacctcaag atgctcccca tgggcctgat catcatgccg 1080
ggcatgatca gccgcgcatt gttcccagat gatgtgggct gcgtggtgcc gtccgagtgc 1140
ctgcgggcct gcggggccga ggtcggctgc tccaacatcg cctaccccaa gctggtcatg 1200
gaactgatgc ccatcggtct gcgggggctg atgatcgcag tgatgctggc ggcgctcatg 1260
tcgtcgctga cctccatctt caacagcagc agcaccctct tcactatgga catctggagg 1320
cggctgcgtc cccgctccgg cgagcgggag ctcctgctgg tgggacggct ggtcatagtg 1380
gcactcatcg gcgtgagtgt ggcctggatc cccgtcctgc aggactccaa cagcgggcaa 1440
ctcttcatct acatgcagtc agtgaccagc tccctggccc caccagtgac tgcagtcttt 1500
gtcctgggcg tcttctggcg acgtgccaac gagcaggggg ccttctgggg cctgatagca 1560
gggctggtgg tgggggccac gaggctggtc ctggaattcc tgaacccagc cccaccgtgc 1620
ggagagccag acacgcggcc agccgtcctg gggagcatcc actacctgca cttcgctgtc 1680
gccctctttg cactcagtgg tgctgttgtg gtggctggaa gcctgctgac cccaccccca 1740
cagagtgtcc agattgagaa ccttacctgg tggaccctgg ctcaggatgt gcccttggga 1800
actaaagcag gtgatggcca aacaccccag aaacacgcct tctgggcccg tgtctgtggc 1860
ttcaatgcca tcctcctcat gtgtgtcaac atattctttt atgcctactt cgcctgaaca 1920
ctgccatcct ggacagaaag gcaggagctc tgagtcctca ggtccaccca tttccctcat 1980
ggggatcccg aggccccaag aggggcagat tcccctcaca gctgcacagc agctcggtgc 2040
ccaagaactg gccaagccag caaagcggga gcctgaaaac attagggggg aaactgggac 2100
gaaacataag tgtgactttt tccaaacaac agcacccaaa gcaagtcaag catttggaac 2160
gcgacaaact tagattttcc tgaccgggcc caccacaccc caacctcctc acctcccaaa 2220
ctaccaacac agctcatcac catactcaca ccacccacag cggcccgccc ccactccaat 2280
cagaaaggca cccccccact ctcaagacgc gacggcgcaa tcgactgcaa ctccataacg 2340
atgccaaaac gacacaagcc aggacacggc actgtataca gcacgagggt gatctgcaac 2400
71/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
gttgtggccg aatgcagaaa atacactggg tgctggcgta aggaagatcc gcgagtaaac 2460
aacggtcttg taaacttact gcatccacca aggtacactt ccagaacgag accagacaac 2520
tacactccac acaacctgca gccacaccct atttctgcta tcataaagag cccccgcacc 2580
acataataat gccggcagac tcagtgcgcg aaacccttgt gctggacttc accacgg 2637
<210> 49
<211> 3783
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6427460CB1
<400> 49
gcactagtac cccggagccc atgggcgcgc cgagccgggc gcgggggcgc tgaacggcgg 60
agcgggagcg gccggaggag ccatggactg cagcctcgtg cggacgctcg tgcacagata 120
ctgtgcagga gaagagaatt gggtggacag caggaccatc tacgtgggac acagggagcc 180
acctccgggc gcagaggcct acatcccaca gagataccca gacaacagga tcgtctcgtc 240
caagtacaca ttttggaact ttatacccaa gaatttattt gaacaattca gaagagtagc 300
caacttttat ttccttatca tatttctggt gcagttgatt attgatacac ccacaagtcc 360
agtgacaagc ggacttccac tcttctttgt cattactgtg acggctatca aacagggtta 420
tgaagactgg cttcgacata aagcagacaa tgccatgaac eagtgtcctg ttcatttcat 480
tcagcacggc aagctcgttc ggaaacaaag tcgaaagctg cgagttgggg acattgtcat 540
ggttaaggag gacgagacct ttccctgcga cttgatcttc ctttccagca accggggaga 600
tgggacgtgc cacgtcacca ccgccagctt ggatggagaa tccagccata aaacgcatta 660
cgcggtccag gacaccaaag gcttccacac agaggaggat atcggcggac ttcacgccac 720
catcgagtgt gagcagcccc agcccgacct ctacaagttc gtgggtcgca tcaacgttta 780
cagtgacctg aatgaccccg tggtgaggcc cttaggatcg gaaaacctgc tgcttagagg 840
agctacactg aagaacactg agaaaatctt tggtgtggct atttacacgg gaatggaaac 900
caagatggca ttaaattatc aatcaaaatc tcagaagcga tctgccgtgg aaaaatcgat 960
gaatgcgttc ctcattgtgt atctctgcat tctgatcagc aaagccctga taaacactgt 1020
gctgaaatac gtgtggcaga gtgagccctt tcgggatgag ccgtggtata atcagaaaac 1080
ggagtcggaa aggcagagga atctgttcct caaggcattc acggacttcc tggccttcat 1140
ggtcctcttt aactacatca tccctgtgtc catgtacgtc acggtcgaga tgcagaagtt 1200
cctcggctct tacttcatca cctgggacga agacatgttt gacgaggaga ctggcgaggg 1260
gcctctggtg aacacgtcgg acctcaatga agagctggga caggtggagt acatcttcac 1320
agacaagacc ggcaccctca cggaaaacaa catggagttc aaggagtgct gcatcgaagg 1380
ccatgtctac gtgccccacg tcatctgcaa cgggcaggtc ctcccagagt cgtcaggaat 1440
cgacatgatt gactcgtccc ccagcgtcaa cgggagggag cgcgaggagc tgtttttccg 1500
ggccctctgt ctctgccaca ccgtccaggt gaaagacgat gacagcgtag acggccccag '1560
gaaatcgccg gacgggggga aatcctgtgt gtacatctca tcctcgcccg acgaggtggc 1620
gctggtcgaa ggtgtccaga gacttggctt tacctaccta aggctgaagg acaattacat 1680
ggagatatta aacagggaga accacatcga aaggtttgaa ttgctggaaa ttttgagttt 1740
tgactcagtc agaaggagaa tgagtgtaat tgtaaaatct gctacaggag aaatttatct 1800
gttttgcaaa ggagcagatt cttcgatatt cccccgagtg atagaaggca aagttgacca 1860
gatccgagcc agagtggagc gtaacgcagt ggaggggctc cgaactttgt gtgttgctta 1920
taaaaggctg atccaagaag aatatgaagg catttgtaag ctgctgcagg ctgccaaagt 1980
ggcccttcaa gatcgagaga aaaagttagc agaagcctat gagcaaatag agaaagatct 2040
tactctgctt ggtgctacag ctgttgagga ccggctgcag gagaaagctg cagacaccat 2100
cgaggccctg cagaaggccg ggatcaaagt ctgggttctc acgggagaca agatggagac 2160
ggccgcggcc acgtgctacg cctgcaagct cttccgcagg aacacgcagc tgctggagct 2220
gaccaccaag aggatcgagg agcagagcct gcacgacgtc ctgttcgagc tgagcaagac 2280
ggtcctgcgc cacagcggga gcctgaccag agacaacctc tccggacttt cagcagatat 2340
gcaggactac ggtttaatta tcgacggagc tgcactgtct ctgataatga agcctcgaga 2400
agacgggagt tccggcaact acagggagct cttcctggaa atctgccgga gctgcagcgc 2460
ggtgctctgc tgccgcatgg cgcccttgca gaaggctcag attgttaaat taatcaaatt 2520
ttcaaaagag cacccaatca cgttagcaat tggcgatggt gcaaatgatg tcagcatgat 2580
tctggaagcg cacgtgggca taggtgtcat cggcaaggaa ggccgccagg ctgccaggaa 2640
cagcgactat gcaatcccaa agtttaagca tttgaagaag atgctgcttg ttcacgggca 2700
tttttattac attaggatct ctgagctcgt gcagtacttc ttctataaga acgtctgctt 2760
catcttccct cagtttttat accagttctt ctgtgggttt tcacaacaga ctttgtacga 2820
caccgcgtat ctgaccctct acaacatcag cttcacctcc ctccccatcc tcctgtacag 2880
cctcatggag cagcatgttg gcattgacgt gctcaagaga gacccgaccc tgtacaggga 2940
cgtcgccaag aatgccctgc tgcgctggcg cgtgttcatc tactggacgc tcctgggact 3000
gtttgacgca ctggtgttct tctttggtgc ttatttcgtg tttgaaaata caactgtgac 3060
aagcaacggg cagatatttg gaaactggac gtttggaacg ctggtattca ccgtgatggt 3120
72/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
gttcacagtt acactaaagc ttgcattgga cacacactac tggacttgga tcaaccattt 3180
tgtcatctgg gggtcgctgc tgttctacgt tgtcttttca cttctctggg gaggagtgat 3240
ctggccgttc ctcaactacc agaggatgta ctacgtgttc atccagatgc tgtccagcgg 3300
gcccgcctgg ctggccatcg tgctgctggt gaccatcagc ctccttcccg acgtcctcaa 3360
gaaagtcctg tgccggcagc tgtggccaac agcaacagag agagtccagc agaatgggtg 3420
cgcacagcct cgggaccgcg actcagaatt cacccctctt gcctctctgc agagcccagg 3480
ctaccagagc acctgtccct cggccgcctg gtacagctcc cactctcagc aggtgacact 3540
cgcggcctgg aaggagaagg tgtccacgga gcccccaccc atcctcggcg gttcccatca 3600
ccactgcagt tccatcccaa gtcacagctg ccctaggtcc cgtgtgggaa tgctcgtgtg 3660
atggatggtc ctaagcctgt ggagactgtg cacgtgcctc ttcctggccc ccagcaggca 3720
aggagggggg tcacaggcct tgccctcgaa catggcaccc tggccgcctg gacccagcac 3780
tgt 3783
<210> 50
<211> 2105
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7474127CB1
<400> 50
ccagcgccca gggaagcggc tcaaccacct gaatccggaa aacgccaaca agtagtttct 60
cgtcggagaa gggcggctca cctgggcgcc aagactcagt cccgctgccc agagaacctc 120
gtccactcgg aaaccaaagc agaaccactt ttctctcggt ctcgttaagt catgtctgag 180
tcacagagat gggcaagatc gagaacaacg agagggtgat cctcaatgtc gggggcaccc 240
ggcacgaaac ctaccgcagc accctcaaga ccctgcctgg aacacgcctg gcccttcttg 300
cctcctccga gcccccaggc gactgcttga ccacggcggg cgacaagctg cagccgtcgc 360
cgcctccact gtcgccgccg ccgagagcgc ccccgctgtc ccccgggcca ggcggctgct 420
tcgagggcgg cgcgggcaac tgcagttccc gcggcggcag ggccagcgac catcccggtg 480
gcggccgcga gttcttcttc gaccggcacc cgggcgtctt cgcctatgtg ctcaattact 540
accgcaccgg caagctgcac tgccccgcag acgtgtgcgg gccgctcttc gaggaggagc 600
tggccttctg gggcatcgac gagaccgacg tggagccctg ctgctggatg acctaccggc 660
agcaccgcga cgccgaggag gcgctggaca tcttcgagac ccccgacctc attggcggcg 720
accccggcga cgacgaggac ctggcggcca agaggctggg catcgaggac gcggcggggc 780
tcgggggccc cgacggcaaa tctggccgct ggaggaggct gcagccccgc atgtgggccc 840
tcttcgaaga cccctactcg tccagagccg ccaggtttat tgcttttgct tctttattct 900
tcatcctggt ttcaattaca actttttgcc tggaaacaca tgaagctttc aatattgtta 960
aaaacaagac agaaccagtc atcaatggca caagtgttgt tctacaatat gaaattgaaa 1020
cagatcctgc cttgacgtat gtagaaggag tgtgtgtggt gtggtttact tttgaatttt 1080
tagtccgtat tgttttttca cccaataaac ttgaattcat caaaaatctc ttgaatatca 1140
ttgactttgt ggccatccta cctttctact tagaggtggg actcagtggg ctgtcatcca 1200
aagctgctaa agatgtgctt ggcttcctca gggtggtaag gtttgtgagg atcctgagaa 1260
ttttcaagct cacccgccat tttgtaggtc tgagggtgct tggacatact cttcgagcta 1320
gtactaatga atttttgctg ctgataattt tcctggctct aggagttttg atatttgcta 1380
ccatgatcta ctatgccgag agagtgggag ctcaacctaa cgacccttca gctagtgagc 1440
acacacagtt caaaaacatt cccattgggt tctggtgggc tgtagtgacc atgactaccc 1500
tgggttatgg ggatatgtac ccccaaacat ggtcaggcat gctggtggga gccctgtgtg 1560
ctctggctgg agtgctgaca atagccatgc cagtgcctgt cattgtcaat aattttggaa 1620
tgtactactc cttggcaatg gcaaagcaga aacttccaag gaaaagaaag aagcacatcc 1680
ctcctgctcc tcaggcaagc tcacctactt tttgcaagac agaattaaat atggcctgca 1740
atagtacaca gagtgacaca tgtctgggca aagacaatcg acttctggaa cataacagat 1800
cagtgttatc aggtgacgac agtacaggaa gtgagccgcc actatcaccc ccagaaaggc 1860
tccccatcag acgctctagt accagagaca aaaacagaag aggggaaaca tgtttcctac 1920
tgacgacagg tgattacacg tgtgcttctg atggagggat caggaaaggt tatgaaaaat 1980
cccgaagctt aaacaacata gcgggcttgg caggcaatgc tctgaggctc tctccagtaa 2040
catcacccta caactctcct tgtcctctga ggcgctctcg atctcccatc ccatctatct 2100
tgtaa 2105
<210> 51
<211> 2069
<212> DNA
<213> Homo sapiens
<220>
<221> misc feature
73/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
<223> Incyte ID No: 7476949CB1
<400> 51
atgagcaagg acctggcagc aatggggcct ggagcttcag gggacggggt caggactgag 60
acagctccac acatagcact ggactccaga gttggtctgc acgcctacga catcagcgtg 120
gtggtcatct actttgtctt cgtcattgct gtggggatct ggtcgtccat ccgtgcaagt 180
cgagggacca ttggcggcta tttcctggcc gggaggtcca tgagctggtg gccaattgga 240
gcatctctga tgtccagcaa tgtgggcagt ggcttgttca tcggcctggc tgggacaggg 300
gctgccggag gccttgccgt aggtggcttc gagtggaacg caacctggct gctcctggcc 360
cttggctggg tcttcgtccc tgtgtacatc gcagcaggtg tggtcacaat gccgcagtat 420
ctgaagaagc gatttggggg ccagaggatc caggtgtaca tgtctgtcct gtctctcatc 480
ctctacatct tcaccaagat ctcgactgac atcttctctg gagccctctt catccagatg 540
gcattgggct ggaacctgta cctctccaca gggatcctgc tggtggtgac tgccgtctac 600
accattgcag gtggcctcat ggccgtgatc tacacagatg ctctgcagac ggtgatcatg 660
gtagggggag ccctggtcct catgtttctg ggctttcagg acgtgggctg gtacccaggc 720
ctggagcagc ggtacaggca ggccatccct aatgtcacag tccccaacac cacctgtcac 780
ctcccacggc ccgatgcttt ccacattctt cgggaccctg tgagcgggga catcccttgg 840
ccaggtctca ttttcgggct cacagtgctg gccacctggt gttggtgcac agaccaggtc 900
attgtgcagc ggtctctctc ggccaagagt ctgtctcatg ccaagggagg ctccgtgctg 960
gggggctacc tgaagatcct ccccatgttc ttcatcgtca tgcctggcat gatcagccgg 1020
gccctgttcc cagacgaggt gggctgcgtg gaccctgatg tctgccaaag aatctgtggg 1080
gcccgagtgg gatgttccaa cattgcctac cctaagttgg tcatggccct catgcctgtt 1140
ggtctgcggg ggctgatgat tgccgtgatc atggccgctc tcatgagctc actcacctcc 1200
atcttcaaca gcagcagcac cctgttcacc attgatgtgt ggcagcgctt ccgcaggaag 1260
tcaacagagc aggagctgat ggtggtgggc agagtgtttg tggtgttcct ggttgtcatc 1320
agcatcctct ggatccccat catccaaagc tccaacagtg ggcagctctt cgactacatc 1380
caggctgtca ccagttacct ggccccaccc atcaccgctc tcttcctgct ggccatcttc 1440
tgcaagaggg tcacagagcc cggagctttc tggggcctcg tgtttggcct gggagtgggg 1500
cttctgcgta tgatcctgga gttctcatac ccagcgccag cctgtgggga ggtggaccgg 1560
aggccagcag tgctgaagga cttccactac ctgtactttg caatcctcct ctgcgggctc 1620
actgccatcg tcattgtcat tgtcagcctc tgtacaactc ccatccctga ggaacagctc 1680
acacgcctca catggtggac tcggaactgc cccctctctg agctggagaa ggaggcccac 1740
gagagcacac cggagatatc cgagaggcca gccggggagt gccctgcagg aggtggagcg 1800
gcagagaact cgagcctggg ccaggagcag cctgaagccc caagcaggtc ctggggaaag 1860
ttgctctgga gctggttctg tgggctctct ggaacaccgg agcaggccct gagcccagca 1920
gagaaggctg cgctagaaca gaagctgaca agcattgagg aggagccact ctggagacat 1980
gtctgcaaca tcaatgctgt ccttttgctg gccatcaaca tcttcctctg gggctatttt 2040
gcgtgattca aacctggctt cactgtaga 2069
<210> 52
<211> 4245
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7477249CB1
<400> 52
gcggcggcag gctcagctgc gccgggcggg ggcggcgctg gggccgcgcc tgtaggactc 60
ggggccgacg ccgcgggatg gggacgcggc gcggggagtg aggcagtggc ggcggcggcg 120
gtaagcggaa cttcggcccg aggggctcgc ccgctcccgc ctctgtcttg tcggcctcca 180
cctgcagccc cgcggccccc gcgccccgcg ggacccggac ggcgacgacg ggggaatgtg 240
gcgctggatc cggcagcagc tgggttttga cccaccacat cagagtgaca caagaaccat 300
ctacgtagcc aacaggtttc ctcagaatgg cctttacaca cctcagaaat ttatagataa 360
caggatcatt tcatctaagt acactgtgtg gaattttgtt ccaaaaaatt tatttgaaca 420
gttcagaaga gtggcaaact tttattttct tattatattt ttggttcagc ttatgattga 480
tacacctacc agtccagtta ccagtggact tccattattc tttgtgataa cagtaactgc 540
cataaagcag ggatatgaag attggttacg gcataactca gataatgaag taaatggagc 600
tcctgtttat gttgttcgaa gtggtggcct tgtaaaaact agatcaaaaa acattcgggt 660
gggtgatatt gttcgaatag ccaaagatga aatttttcct gcagacttgg tgcttctgtc 720
ctcagatcga ctggatggtt cctgtcacgt tacaactgct agtttggacg gagaaactaa 780
cctgaagaca catgtggcag ttccagaaac agcattatta caaacagttg ccaatttgga 840
cactctagta gctgtaatag aatgccagca accagaagca gacttataca gattcatggg 900
acgaatgatc ataacccaac aaatggaaga aattgtaaga cctctggggc cggagagtct 960
cctgcttcgt ggagccagat taaaaaacac aaaagaaatt tttggtgttg cggtatacac 1020
tggaatggaa actaagatgg cattaaatta caagagcaaa tcacagaaac gatctgcagt 1080
74/7
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
agaaaagtca atgaatacat ttttgataat ttatctagta attcttatat ctgaagctgt 1140
catcagcact atcttgaagt atacatggca agctgaagaa aaatgggatg aaccttggta 1200
taaccaaaaa acagaacatc aaagaaatag cagtaaggta gagtacgtgt ttacagataa 1260
aactggtaca ctgacagaaa atgagatgca gtttcgggaa tgttcaatta atggcatgaa 1320
ataccaagaa attaatggta gacttgtacc cgaaggacca acaccagact cttcagaagg 1380
aaacttatct tatcttagta gtttatccca tcttaacaac ttatcccatc ttacaaccag 1440
ttcctctttc agaaccagtc ctgaaaatga aactgaacta attaaagaac atgatctctt 1500
ctttaaagca gtcagtctct gtcacactgt acagattagc aatgttcaaa ctgactgcac 1560
tggtgatggt ccctggcaat ccaacctggc accatcgcag ttggagtact atgcatcttc 1620
accagatgaa aaggctctag tagaagctgc tgcaaggatt ggtattgtgt ttattggcaa 1680
ttctgaagaa actatggagg ttaaaactct tggaaaactg gaacggtaca aactgcttca 1740
tattctggaa tttgattcag atcgtaggag aatgagtgta attgttcagg caccttcagg 1800
tgagaagtta ttatttgcta aaggagctga gtcatcaatt ctccctaaat gtataggtgg 1860
agaaatagaa aaaaccagaa ttcatgtaga tgaatttgct ttgaaagggc taagaactct 1920
gtgtatagca tatagaaaat ttacatcaaa agagtatgag gaaatagata aacgcatatt 1980
tgaagccagg actgccttgc agcagcggga agagaaattg gcagctgttt tccagttcat 2040
agagaaagac ctgatattac ttggagccac agcagtagaa gacagactac aagataaagt 2100
tcgagaaact attgaagcat tgagaatggc tggtatcaaa gtatgggtac ttactgggga 2160
taaacatgaa acagctgtta gtgtgagttt atcatgtggc cattttcata gaaccatgaa 2220
catccttgaa cttataaacc agaaatcaga cagcgagtgt gctgaacaat tgaggcagct 2280
tgccagaaga attacagagg atcatgtgat tcagcatggg ctggtagtgg atgggaccag 2340
cctatctctt gcactcaggg agcatgaaaa actatttatg gaagtttgca gaaattgttc 2400
agctgtatta tgctgtcgta tggctccact gcagaaagca aaagtaataa gactaataaa 2460
aatatcacct gagaaaccta taacattggc tgttggtgat ggtgctaatg acgtaagcat 2520
gatacaagaa gcccatgttg gcataggaat catgggtaaa gaaggaagac aggctgcaag 2580
aaacagtgac tatgcaatag ccagatttaa gttcctctcc aaattgcttt ttgttcatgg 2640
tcatttttat tatattagaa tagctaccct tgtacagtat tttttttata agaatgtgtg 2700
ctttatcaca ccccagtttt tatatcagtt ctactgtttg ttttctcagc aaacattgta 2760
tgacagcgtg tacctgactt tatacaatat ttgttttact tccctaccta ttctgatata 2820
tagtcttttg gaacagcatg tagaccctca tgtgttacaa aataagccca ccctttatcg 2880
agacattagt aaaaaccgcc tcttaagtat taaaacattt ctttattgga ccatcctggg 2940
cttcagtcat gcctttattt tcttttttgg atcctattta ctaataggga aagatacatc 3000
tctgcttgga aatggccaga tgtttggaaa ctggacattt ggcactttgg tcttcacagt 3060
catggttatt acagtcacag taaagatggc tctggaaact catttttgga cttggatcaa 3120
ccatctcgtt acctggggat ctattatatt ttattttgta ttttccttgt tttatggagg 3180
gattctctgg ccatttttgg gctcccagaa tatgtatttt gtgtttattc agctcctgtc 3240
aagtggttct gcttggtttg ccataatcct catggttgtt acatgtctat ttcttgatat 3300
cataaagaag gtctttgacc gacacctcca ccctacaagt actgaaaagg cacagcttac 3360
tgaaacaaat gcaggtatca agtgcttgga ctccatgtgc tgtttcccgg aaggagaagc 3420
agcgtgtgca tctgttggaa gaatgctgga acgagttata ggaagatgta gtccaaccca 3480
catcagcagg tgtgaaatct ctctaagtag cctttgctgc agatgagtat cctatctgga 3540
acaggatgaa cctgccgctc tagataccta ataaatcagc agctggtttt accaactgaa 3600
gcaggaagtc tgctatttat tagcactctt tggtggtaga tttcactttg tggctttggg 3660
gtaagggctt tttcactcac aaaggaagag aaagcacctt tgaagagact tcatctaatg 3720
aacaaaaaat tttgtttcat aatctttcta aaatgtgctc agtaggagtg tgtttatggt 3780
actcttttat ggtttgtata actttctttt ttaaattata catatactat ttccttttta 3840
tttttttaaa atttttttgc tttttgtctt tacaaaataa tctcaacata acagtgaagt 3900
caaaggcttt ccttttctta ctctgtatgt atattttcca gttggttatt tgaggctttg 3960
aggtatttat aaacacaaaa ggctgtattt ctgctcccct acctcttctt atgtctgtaa 4020
tgaagttttg aaatgagtca tgatttttaa gtttcttttg cttggtattt attgcctaat 4080
taaaagtgta tgagttagaa caggcttttt aaattatgga gtaaaagaat cttagcattt 4140
ttgtcccctc ctaaatctgt ttcttgaatg agatttatca ccatgcctgc tgttgtgcac 4200
cataacgaaa aaaaacacct tttggtaaac accatttaaa attca 4245
<210> 53
<211> 2124
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7477720CB1
<400> 53
atggctctgc agatgttcgt gacttacagt ccttggaatt gtttgctact gctagtggct 60
cttgagtgtt ctgaagcatc ttctgatttg aatgaatctg caaattccac tgctcagtat 120
gcatctaacg cttggtttgc tgctgccagc tcagagccag aggaagggat atctgttttt 180
75/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
gaactggatt atgactatgt gcaaattcct tatgaggtca ctctctggat acttctagca 240
tcccttgcaa aaataggctt ccacctctac cacaggctgc caggcctcat gccagaaagc 300
tgcctcctca tcctggtggg ggcgctggtg ggcggcatca tcttcggcac cgaccacaaa 360
tcgcctccgg tcatggactc cagcatctac ttcctgtatc tcctgccacc catcgttctg 420
gagggcggct acttcatgcc cacccggccc ttctttgaga acatcggctc catcctgtgg 480
tgggcagtat tgggggccct gatcaacgcc ttgggcattg gcctctccct ctacctcatc 540
tgccaggtga aggcctttgg cctgggcgac gtcaacctgc tgcagaacct gctgttcggc 600
agcctgatct ccgccgtgga cccagtggcc gtgctagccg tgtttgagga agcgcgcgtg 660
aacgagcagc tctacatgat gatctttggg gaggccctgc tcaatgatgg cattactgtg 720
gtcttataca atatgttaat tgcctttaca aagatgcata aatttgaaga catagaaact 780
gtcgacattt tggctggatg tgcccgattc atcgttgtgg ggcttggagg ggtattgttt 840
ggcatcgttt ttggatttat ttctgcattt atcacacgtt tcactcagaa tatctctgca 900
attgagccac tcatcgtctt catgttcagc tatttgtctt acttagctgc tgaaaccctc 960
tatctctccg gcatcctggc aatcacagcc tgcgcagtaa caatgaaaaa gtacgtggaa 1020
gaaaacgtgt cccagacatc atacacgacc atcaagtact tcatgaagat gctgagcagc 1080
gtcagcgaga ccttgatctt catcttcatg ggtgtgtcca ctgtgggcaa gaatcacgag 1140
tggaactggg ccttcatctg cttcaccctg gccttctgcc aaatctggag agccatcagc 1200
gtatttgctc tcttctatat cagtaaccag tttcggactt tccccttctc catcaaggac 1260
cagtgcatca ttttctacag tggtgttcga ggagctggaa gtttttcact tgcatttttg 1320
cttcctctgt ctctttttcc taggaagaaa atgtttgtca ctgctactct agtagttata 1380
tactttactg tatttattca gggaatcaca gttggccctc tggtcaggta cctggatgtt 1440
aaaaaaacca ataaaaaaga atccatcaat gaagagcttc atattcgtct gatggatcac 1500
ttaaaggctg gaatcgaaga tgtgtgtggg cactggagtc actaccaagt gagagacaag 1560
tttaagaagt ttgatcatag atacttacgg aaaatcctca tcagaaagaa cctacccaaa 1620
tcaagcattg tttctttgta caagaagctg gaaatgaagc aagccatcga gatggtggag 1680
actgggatac tgagctctac agctttctcc ataccccatc aggcccagag gatacaagga 7.740
atcaaaagac tttcccctga agatgtggag tccataaggg acattctgac atccaacatg 1800
taccaagttc ggcaaaggac cctgtcctac aacaaataca acctcaaacc ccaaacaagt 1860
gagaagcagg ctaaagagat tctgatccgc cgccagaaca ccttaaggga gagcatgagg 1920
aaaggtcaca gcctgccctg gggaaagccg gctggcacca agaatatccg ctacctctcc 1980
tacccctacg ggaatcctca gtctgcagga agagacacaa gggctgctgg gttctcaggt 2040
aagctgccca cctggctgct ctgctgcttt tctgtagagt caggtggtaa atatctgggg 2100
gtgtgggcca agaggcaaca ttaa 2124
<210> 54
<211> 2195
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7477852CB1
<400> 54
atggggggtt ttctacctaa ggcagaaggg cccgggagcc aactccagaa acttctgccc 60
tcctttctgg tcagagaaca agactgggac cagcacctgg acaagcttca tatgctgcag 120
cagaagagga ttctagagtc tccactgctt cgagcatcca aggaaaatga cctgtctgtt 180
cttaggcaac ttctactgga ctgcacctgt gacgttcgac aaagaggagc cctgggggag 240
acggcgctgc acatagcagc cctctatgac aacttggagg cggccttggt gctgatggag 300
gctgccccag agctggtctt tgagcccacc acatgtgagg cttttgcagg tcagactgca 360
ctgcacatcg ctgttgtgaa ccagaatgtg aacctggtgc gtgccctgct cacccgcagg 420
gccagtgtct ctgccagagc cacaggcact gccttccgcc gtagtccccg caacctcatc 480
tactttggtg agcacccttt gtcctttgct gcctgtgtga acagcgagga gatcgtgcgg 540
ctgctcattg agcatggagc tgacatcagg gcccaggact ccctgggtaa cacagtatta 600
cacatcctca tcctccagcc caacaaaacc tttgcctgcc agatgtacaa cctgctgctg 660
tcctacgatg gacatgggga ccacctgcag cccctggacc ttgtgcccaa tcaccagggt 720
ctcaccccct tcaagctggc tggagtggag ggtaacactg tgatgttcca gcacctgatg 780
cagaagcgga ggcacatcca gtggacgtat ggacccctga cctccattct ctacgacctc 840
acagagatcg actcctgggg agaggagctg tccttcctgg agcttgtggt ctcctctgat 900
aaacgagagg ctcgccaaat tctggaacag accccagtga aggagctggt gagcttcaag 960
tggaacaagt atggccggcc gtacttctgc atcctggctg ccttgtacct gctctacatg 1020
atctgcttta ccacgtgctg cgtctaccgc ccccttaagt ttcgtggtgg caaccgcact 1080
cattctcgag acatcaccat cctccagcaa aaactactac aggaggccta tgagacacgt 1140
gaagatatca tcaggctggt gggggagctg gtgagcatcg ttggggctgt gatcatcctg 1200
ctcctagaga ttccagacat cttcagggtt ggtgcctctc gctattttgg aaagacgatt 1260
cttggggggc cattccatgt catcatgatc acctatgcct ccctggtgct ggtgaccatg 1320
gtgatgcggc tcaccaacac caatggggag gtggtgccca tgtcctttgc cctggtgctg 1380
76/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
ggctggtgca gtgtcatgta tttcactcga ggattccaga tgctgggtcc cttcaccatc 1440
atgatccaga agatgatttt tggagaccta atgcgtttct gctggctgat ggctgtggtc 1500
atcttgggat ttgcctccgc gttctatatc attttccaga cagaggaccc aaccagtctg 1560
gggcaattct atgactaccc catggcactg ttcaccacct ttgagctttt tctcactgtt 1620
attgatgcac ctgccaacta cgacgtggac ttgcccttca tgttcagcat tgtcaacttc 1680
gccttcgcca tcattgccac actgctcatg ctcaacttgt tcatcgccat gatgggcgac 1740
acccactgga gggtggccca ggagagggat gagctctgga gggcccaggt cgtggccacc 1800
acagtgatgc tggagcggaa gctgcctcgc tgcctgtggc ctcgctccgg gatctgtggg 1860
tgcgaattcg ggctggggga ccgctggttc ctgcgggttg agaaccacaa tgatcagaat 1920
cctctgcgag tgcttcgcta tgtggaagtg ttcaagaact cagacaagga ggatgaccag 1980
gagcatccat ctgagaaaca gccctctggg gctgagagtg ggactctagc cagagcctct 2040
ttggctcttc caacttcctc cctgtcccgg accgcgtccc agagcagcag tcaccgaggc 2100
tgggagatcc ttcgtcaaaa caccctgggg cacttgaatc ttggactgaa ccttagtgag 2160
ggggatggag aggaggtcta ccatttttga ttaac 2195
<210> 55
<211> 2055
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1471717CB1
<400> 55
cggctctggg ccctcagcct ggctcatgca caactgtctg aagtgctctg gactatggtg 60
atgaacagcg gccttcagac gcgaggctgg ggaggaatcg tcggggtttt tattattttt 120
gccgtatttg ctgtcctgac agtagccatc cttctgatca tggagggcct ctctgctttc 180
ctgcacgccc tgcgactgca ctgggaagct gtttggggaa cttgagctat ttagaagatg 240
gcaaccaagc caacagagcc tgtcacgatc ctcagccttc ggaaattgag cctggggacc 300
gcagagccac aggttaaaga gccaaagacg ttcaccgtgg aagatgcagt ggagactatc 360
ggcttcgggc gtttccacat tgccctcttt ctgatcatgg gcagtactgg ggtggttgag 420
gccatggaga tcatgttgat agctgttgtg tctcctgtca tccgctgtga atggcaactg 480
gagaattggc aggtggcatt agtaaccacg atggtgtttt ttggctacat ggttttcagt 540
atcctctttg gcctcctggc tgacagatat ggccgctgga agattctgct catctcgttc 600
ctgtggggag cctatttctc cttgctgacc tcgtttgctc cttcgtacat ctggtttgtc 660
ttcctgcgga cgatggtggg ctgtggtgtg tccggccact cgcaagggtt aatcataaag 720
actgaatttt tgcccacgaa ataccgaggc tatatgttac ccttgtctca ggtgttctgg 780
cttgcgggct ccctgctcat cattggcttg gcctctgtga tcatccccac catcgggtgg 840
cgctggctca ttcgcgtcgc ctccatcccg ggcatcatcc tcatcgtggc cttcaagttt 900
attcctgaat ctgcccggtt caatgtctcc actgggaaca ctcgggctgc cctggccact 960
ctggagcgcg ttgccaagat gaaccgctcg gtcatgccgg aggggaagct ggtggagccc 1020
gtcctggaaa aaagaggaag atttgcagac ctattggatg ctaaatattt acggaccaca 1080
ttacagatct gggtcatatg gcttggaatc tcttttgcct actatggggt tatcctggcc 1140
agtgctgagc tgctggagcg ggacttggtc tgtggttcaa agtcagactc tgcggtggtg 1200
gtgactgggg gggactcagg ggagagccag agcccctgct actgccacat gtttgcaccc 1260
tctgactatc ggaccatgat catcagcacc atcggtgaaa ttgctttgaa tcctttaaat 1320
atactgggca tcaatttcct gggaagacgg ctgagccttt ctattaccat gggatgcacg 1380
gctttattct gccttctcct caacatttgc acttcaagtg ccggcctgat tggcttcctc 1440
ttcatgctga gggctctggt agctgcaaac ttcaacaccg tctacattta cacagctgag 1500
gtctacccca ccacgatgcg cgctttgggg atgggaacca gcggctccct gtgtcgcatt 1560
ggtgcaatgg tggcgccatt tatatcccag gttcttatga gtgcatcaat actgggggcc 1620
ctgtgtctct tctcatctgt ctgtgttgta tgcgccattt ctgcattcac tctccccatc 1680
gaaaccaaag gacgggccct ccagcaaatt aaatgaagac ctgcaaagct atgtctacca 1740
gatgagaaaa atgaattcta tcttcagaac tgcggtgcat ttttttaaaa cttggtttta 1800
cttctgtatg ctactcggta attagtaaag tgattttttt ttaaaaggca tatatgggaa 1860
tggggtaggt aactgtatat tgatctcttc cttgaggaac aatatataaa gtacttttat 1920
aaaatataat ttaagctttc aaaggggtgt gagagggaga tggtgggggg gaagatggct 1980
tttcttcgtt gaaatcaagt ctgtaaacct ttatatgaat aaatactaaa ttttaaactt 2040
acaaaaaaaa aaaaa 2055
<210> 56
<211> 4727
<212> DNA
<213> Homo sapiens
<220>
77/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
<221> misc_feature
<223> Incyte ID No: 3874406CB1
<400> 56
aagagctgct ggagtaggca cccatttaaa gaaaaaatga agaagcagca ataaagaagt 60
tgtaatcgtt acctagacaa acagagaact ggttttgaca gtgtttctag agtgcttttt 120
attattttcc tgacagttgt gttccaccat gattactttc tccttcagcg aataggctaa 180
atgaatatga aacagaaaag cgtgtatcag caaaccaaag cacttctgtg caagaatttt 240
cttaagaaat ggaggatgaa aagagagagc ttattggaat ggggcctctc aatacttcta 300
ggactgtgta ttgctctgtt ttccagttcc atgagaaatg tccagtttcc tggaatggct 360
cctcagaatc tgggaagggt agataaattt aatagctctt ctttaatggt tgtgtataca 420
ccaatatcta atttaaccca gcagataatg aataaaacag cacttgctcc tcttttgaaa 480
ggaacaagtg tcattggggc accaaataaa acacacatgg acgaaatact tctggaaaat 540
ttaccatatg ctatgggaat catctttaat gaaactttct cttataagtt aatatttttc 600
cagggatata acagtccact ttggaaagaa gatttctcag ctcattgctg ggatggatat 660
ggtgagtttt catgtacatt gaccaaatac tggaatagag gatttgtggc tttacaaaca 720
gctattaata ctgccattat agaagtagct ttggtgttcc tgatgagtgt gctgttaaag 780
aaagctgtcc tcaccaattt ggttgtgttt ctccttaccc tcttttgggg atgtctggga 840
ttcactgtat tttatgaaca acttccttca tctctggagt ggattttgaa tatttgtagc 900
ccttttgcct ttactactgg aatgattcag attatcaaac tggattataa cttgaatggt 960
gtaatttttc ctgacccttc aggagactca tatacaatga tagcaacttt ttctatgttg 1020
cttttggatg gtctcatcta cttgctattg gcattatact ttgacaaaat tttaccctat 1080
ggagatgagc gccattattc tcctttattt ttcttgaatt catcatcttg tttccaacac 1140
caaaggacta atgctaaggt tattgagaaa gaaatcgatg ctgagcatcc ctctgatgat 2200
tattttgaac cagtagctcc tgaattccaa ggaaaagaag ccatcagaat cagaaatgtt 1260
aagaaggaat ataaaggaaa atctggaaaa gtggaagcat tgaaaggctt gctctttgac 1320
atatatgaag gtcaaatcac ggcaatcctg ggtcacagtg gagctggcaa atcttcactg 1380
ctaaatattc ttaatggatt gtctgttcca acagaaggat cagttaccat ctataataaa 1440
aatctctctg aaatgcaaga cttggaggaa atcagaaaga taactggcgt ctgtcctcaa 1500
ttcaatgttc aatttgacat actcaccgtg aaggaaaacc tcagcctgtt tgctaaaata 1560
aaagggattc atctaaagga agtggaacaa gaggtacaac gaatattatt ggaattggac 1620
atgcaaaaca ttcaagataa ccttgctaaa catttaagtg aaggacagaa aagaaagctg 1680
acttttggga ttaccatttt aggagatcct caaattttgc tcttagatga accaactact 1740
ggattggatc ccttttccag agatcaagtg tggagcctcc tgagagagcg tagagcagat 1800
catgtgatcc ttttcagtac ccagtccatg gatgaggctg acatcctggc tgatagaaaa 1860
gtgatcatgt ccaatgggag actgaagtgt gcaggttctt ctatcttttt gaaaagaagg 1920
tggggtcttg gatatcacct aagtttacat aggaatgaaa tatgtaaccc agaacaaata 1980
acatccttca ttactcatca catccccgat gctaaattaa aaacagaaaa caaagaaaag 2040
cttgtatata ctttgccact ggaaaggaca aatacatttc cagatctttt cagtgatctg 2100
gataagtgtt ctgaccaggg agtgacaggt tatgacattt ccatgtcaac tctaaatgaa 2160
gtctttatga aactggaagg acagtcaact atcgaacaag atttcgaaca agtggagatg 2220
ataagagact cagaaagcct caatgaaatg gagctggctc actcttcctt ctctgaaatg 2280
cagacagctg tgagtgacat gggcctctgg agaatgcaag tctttgccat ggcacggctc 2340
cgtttcttaa agttaaaacg tcaaactaaa gtgttattga ccctattatt ggtatttgga 2400
atcgcaatat tccctttgat tgttgaaaat ataatatatg ctatgttaaa tgaaaagatc 2460
gattgggaat ttaaaaacga attgtatttt ctctctcctg gacaacttcc ccaggaaccc 2520
cgtaccagcc tgttgatcat caataacaca gaatcaaata ttgaagattt tataaaatca 2580
ctgaagcatc aaaatatact tttggaagta gatgactttg aaaacagaaa tggtactgat 2640
ggcctctcat acaatggagc tatcatagtt tctggtaaac aaaaggatta tagattttca 2700
gttgtgtgta ataccaagag attgcactgt tttccaattc ttatgaatat tatcagcaat 2760
gggctacttc aaatgtttaa tcacacacaa catattcgaa ttgagtcaag cccatttcct 2820
cttagccaca taggactctg gactgggttg ccggatggtt cctttttctt atttttggtt 2880
ctatgtagca tttctcctta tatcaccatg ggcagcatca gtgattacaa gaaaaatgct 2940
aagtcccagc tatggatttc aggcctctac acttctgctt actggtgtgg gcaggcacta 3000
gtggacgtca gcttcttcat tttaattctc cttttaatgt atttaatttt ctacatagaa 3060
aacatgcagt accttcttat tacaagccaa attgtgtttg ctttggttat agttactcct 3120
ggttatgcag cttctcttgt cttcttcata tatatgatat catttatttt tcgcaaaagg 3180
agaaaaaaca gtggcctttg gtcattttac ttcttttttg cctccaccat catgttttcc 3240
atcactttaa tcaatcattt tgacctaagt atattgatta ccaccatggt attggttcct 3300
tcatatacct tgcttggatt taaaactttt ttggaagtga gagaccagga gcactacaga 3360
gaatttccag aggcaaattt tgaattgagt gccactgatt ttctagtctg cttcataccc 3420
tactttcaga ctttgctatt cgtttttgtt ctaagatgca tggaactaaa atgtggaaag 3480
aaaagaatgc gaaaagatcc tgttttcaga atttcccccc aaagtagaga tgctaagcca 3540
aatccagaag aacccataga tgaagatgaa gatattcaaa cagaaagaat aagaacagtc 3600
actgctctga ccacttcaat cttagatgag aaacctgtta taattgccag ctgtctacac 3660
aaagaatatg caggccagaa gaaaagttgc ttttcaaaga ggaagaagaa aatagcagca 3720
agaaatatct ctttctgtgt tcaagaaggt gagattttgg gattgctagg acccagtggt 3780
78/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
gctggaaaaa gttcatctat tagaatgata tctgggatca caaagccaac tgctggagag 3840
gtggaactga aaggctgcag ttcagttttg ggccacctgg ggtactgccc tcaagagaac 3900
gtgctgtggc ccatgctgac gttgagggaa cacctggagg tgtatgctgc cgtcaagggg 3960
ctcagggaag cggacgcgag gctcgccatc gcaagattag tgagtgcttt caaactgcat 4020
gagcagctga atgttcctgt gcagaaatta acagcaggaa tcacgagaaa gttgtgtttt 4080
gtgctgagcc tcctgggaaa ctcacctgtc ttgctcctgg atgaaccatc tacgggcata 4140
gaccccacag ggcagcagca aatgtggcag gcaatccagg cagtcgttaa aaacacagag 4200
agaggtgtcc tcctgaccac ccataacctg gctgaggcgg aagccttgtg tgaccgtgtg 4260
gccatcatgg tgtctggaag gcttagatgc attggctcca tccaacacct gaaaaacaaa 4320
cttggcaagg attacattct agagctaaaa gtgaaggaaa cgtctcaagt gactttggtc 4380
cacactgaga ttctgaagct tttcccacag gctgcagggc agcaaaggta ttcctctttg 4440
ttaacctata agctgcccgt ggcagacgtt taccctctat cacagacctt tcacaaatta 4500
gaagcagtga agcataactt taacctggaa gaatacagcc tttctcagtg cacactggag 4560
aaggtattct tagagctttc taaagaacag gaagtaggaa attttgatga agaaattgat 4620
acaacaatga gatggaaact cctccctcat tcagatgaac cttaaaacct caaacctagt 4680
aattttcttg cttgatctcc tataaactta tgttttatgt aataatt 4727
<210> 57
<211> 3852
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4599654CB1
<400> 57
cgccggcgat tccgagccta cgacgcctcc gctagagccc gcggggctgc gccgactcct 60
gctctggagg ggttgcgggt acctgatggc cacagagggc tctaggaggc cgagcgtgta 120
agcggggtgg gcgccatgga ggcagagcag cggccggcgg cgggggccag cgaaggggcg 180
acccctggac tggaggcggt gcctcccgtt gctcccccgc ctgcgaccgc ggcctcaggt 240
ccgatcccca aatctgggcc tgagcctaag aggaggcacc ttgggacgct gctccagcct 300
acggtcaaca agttctccct tcgggtgttc ggcagccaca aagcagtgga aatcgagcag 360
gagcgggtga agtcagcggg ggcctggatc atccacccct acagcgactt ccggttttac 420
tgggacctga tcatgctgct gctgatggtg gggaacctca tcgtcctgcc tgtgggcatc 480
accttcttca aggaggagaa ctccccgcct tggatcgtct tcaacgtatt gtctgatact 540
ttcttcctac tggatctggt gctcaacttc cgaacgggca tcgtggtgga ggagggtgct 600
gagatcctgc tggcaccgcg ggccatccgc acgcgctacc tgcgcacctg gttcctggtt 660
gacctcatct cttctatccc tgtggattac atcttcctag tggtggagct ggagccacgg 720
ttggacgctg aggtctacaa aacggcacgg gccctacgca tcgttcgctt caccaagatc 780
ctaagcctgc tgaggctgct ccgcctctcc cgcctcatcc gctacataca ccagtgggag 840
gagatcttcc acatgaccta tgacctggcc agtgctgtgg ttcgcatctt caacctcatt 900
gggatgatgc tgctgctatg tcactgggat ggctgtctgc agttcctggt gcccatgctg 960
caggacttcc ctcccgactg ctgggtctcc atcaaccaca tggtgaacca ctcgtggggc 1020
cgccagtatt cccatgccct gttcaaggcc atgagccaca tgctgtgcat tggctatggg 1080
cagcaggcac ctgtaggcat gcccgacgtc tggctcacca tgctcagcat gatcgtaggt 1140
gccacatgct acgccatgtt catcggccat gccacggcac tcatccagtc cctggactct 1200
tcccggcgtc agtaccagga gaagtacaag caggtggagc agtacatgtc cttccacaag 1260
ctgccagcag acacgcggca gcgcatccac gagtactatg agcaccgcta ccagggcaag 1320
atgttcgatg aggaaagcat cctgggcgag ctgagcgagc cgcttcgcga ggagatcatt 1380
aacttcacct gtcggggcct ggtggcccac atgccgctgt ttgcccatgc cgaccccagc 1440
ttcgtcactg cagttctcac caagctgcgc tttgaggtct tccagccggg ggatctcgtg 1500
gtgcgtgagg gctccgtggg gaggaagatg tacttcatcc agcatgggct gctcagtgtg 1560
ctggcccgcg gcgcccggga cacacgcctc accgatggat cctactttgg ggagatctgc 1620
ctgctaacta ggggccggcg cacagccagt gttcgggctg acacctactg ccgcctttac 1680
tcactcagcg tggaccattt caatgctgtg cttgaggagt tccccatgat gcgccgggcc 1740
tttgagactg tggccatgga tcggctgctc cgcatcggca agaagaattc catactgcag 1800
cggaagcgct ccgagccaag tccaggcagc agtggtggca tcatggagca gcatttggtg 1860
caacatgaca gagacatggc tcggggtgtt cggggtcggg ccccgagcac aggagctcag 1920
cttagtggaa agccagtact gtgggagcca ctggtacatg cgccccttca ggcagctgct 1980
gtgacctcca atgtggccat tgccctgact catcagcggg gccctctgcc cctctcccct 2040
gactctccag ccaccctcct tgctcgctct gcttggcgct cagcaggctc tccagcttcc 2100
ccgctggtgc ccgtccgagc tggcccatgg gcatccacct cccgcctgcc cgccccacct 2160
gcccgaaccc tgcacgccag cctatcccgg gcagggcgct cccaggtctc cctgctgggt 2220
ccccctccag gaggaggtgg acggcggcta ggacctcggg gccgcccact ctcagcctcc 2280
caaccctctc tgcctcagcg ggcaacaggc gatggctctc ctgggcgtaa gggatcagga 2340
agtgagcggc tgcctccctc agggctcctg gccaaacctc caaggacagc ccagcccccc 2400
79/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
aggccaccag tgcctgagcc agccacaccc cggggtctcc agctttctgc caacatgtaa 2460
aacctttgag tacatccagc cttagttctt ggggtgcagt agtatgtacc caagggcaga 2520
tgcctcttgg ggaaggccat ggggacctga aacattgccc catggaaatg tcgaccctgt 2580
gcggacattc cgcatactgc catgaagacg gtctctgtgt cctcagctca agaatcctgt 2640
agcttgtccc atcataatcc attcacccgt tcatcatgtg tactgagcag ctaccatgtt 2700
caaggtaata tgccaggcgc tgtatgtctc cactgccaag tagaagtga.c tcaaaaccct 2760
ctgacaagga tattcccttg gctatggtcc tgccaggtgc aggcccaggc ccatgacccc 2820
acctttacta agcacaagta cttgccactg ccatcactgc caagtaacta gatgtctctg 2880
tttccctgcc aatgatcctg caggttctgc ccggtctggt tatcttcctg tttcctgtag 2940
catagccagg cactgccagt cacctgtgcc cccattgctg tcagcagatg tcttgggtcc 3000
tgagtgtggg tatccacttt tacccgctca ctgccacctg tggacactct gtgtctaccc 3060
tctgagtggg aacatacttc taagttccct gcagtctctg tcctgtggta gaccatcttt 3120
ttgtaaactg cgagcttcct cttccctgta ccctctgccc cagtcgtgac cccctaaaag 3180
ttaaggggta gttggcacct ccttattaat atgccagcct agatcccccc cggtggaggg 3240
gcaaatggct gaatccttgt gtgatatttt tttcttcgct tgtttattta ttcatttatt 3300
taattgtatt tattcattta ctaactttat gtgttaccaa ttaattttgt ttacccattc 3360
ctttatccat ccctcccctc cttttcaggt aaggagacag gaggagtagg aggaggcagg 3420
gcctctccat gccagcctct gtggtccttg cccaaaccca tcagcgcaat acttgaacct 3480
tctcccaggt aggggcagga ggagccacat gagagaggga gaaggaccgc gtttaccttt 3540
agagttttgt tttgtttttt ccttctgagt ttgctgttgg tgcaggaata agggaaaggc 3600
ccaaggtatc caagcctggg gaagggcagg ccagccagca cctctgcctt ctcagggaca 3660
agagtagtcc tttaccaccc tcactctgcc tgtcccctct cctactctac agcattaaag 3720
actgtgggac caggacccta agtctccttt ccttctgggt ggggagttct aggggttctt 3780
ggtgtgtggg agaagtttta taattgcttc caaacagctg ggtttaaata taaaatagac 3840
acactcaaaa as 3852
<210> 58
<211> 1917
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5047435CB1
<400> 58
atggcagaag gtgaaagggg agcagacgtg ccacatggcc tcggggcctg gctggccgac 60
gtggcgttgg cggcgctgcg cgcgggaggg cagggcagga gggacagagg cgggggcggg 120
ccggaaagtt tgtccggcgg cagcggcgtt ggggactccg gcgggggatg cgcgcccggc 180
ccctcagcgc ccccagcacg ccgccgagtc ccgctcgcca tgggccactc cccacctgtc 240
ctgcctttgt gtgcctctgt gtctttgctg ggtggcctga cctttggtta tgaactggca 300
gtcatatcag gtgccctgct gccactgcag cttgactttg ggctaagctg cttggagcag 360
gagttcctgg tgggcagcct gctcctgggg gctctcctcg cctccctggt tggtggcttc 420
ctcattgact gctatggcag gaagcaagcc atcctcggga gcaacttggt gctgctggca 480
ggcagcctga ccctgggcct ggctggttcc ctggcctggc tggtcctggg ccgcgctgtg 540
gttggcttcg ccatttccct ctcctccatg gcttgctgta tctacgtgtc agagctggtg 600
gggccacggc agcggggagt gctggtgtcc ctctatgagg caggcatcac cgtgggcatc 660
ctgctctcct atgccctcaa ctatgcactg gctggtaccc cctggggatg gaggcacatg 720
ttcggctggg ccactgcacc tgctgtcctg caatccctca gcctcctctt cctccctgct 780
ggtacagatg agactgcaac acacaaggac ctcatcccac tccagggagg tgaggccccc 840
aagctgggcc cggggaggcc acggtactcc tttctggacc tcttcagggc acgcgataac 900
atgcgaggcc ggaccacagt gggcctgggg ctggtgctct tccagcaact aacagggcag 960
cccaacgtgc tgtgctatgc ctccaccatc ttcagctccg ttggtttcca tgggggatcc 1020
tcagccgtgc tggcctctgt ggggcttggc gcagtgaagg tggcagctac cctgaccgcc 1080
atggggctgg tggaccgtgc aggccgcagg gctctgttgc tagctggctg tgccctcatg 1140
gccctgtccg tcagtggcat aggcctcgtc agctttgccg tgcccatgga ctcaggccca 1200
agctgtctgg ctgtgcccaa tgccaccggg cagacaggcc tccctggaga ctctggcctg 1260
ctgcaggact cctctctacc tcccattcca aggaccaatg aggaccaaag ggagccaatc 1320
ttgtccactg ctaagaaaac caagccccat cccagatctg gagacccctc agcccctcct 1380
cggctggccc tgagctctgc cctccctggg ccccctctgc ccgctcgggg gcatgcactg 1440
ctgcgctgga ccgcactgct gtgcctgatg gtctttgtca gtgccttctc ctttgggttt 1500
gggccagtga cctggcttgt cctcagcgag atctaccctg tggagatacg aggaagagcc 1560
ttcgccttct gcaacagctt caactgggcg gccaacctct tcatcagcct ctccttcctc 1620
gatctcattg gcaccatcgg cttgtcctgg accttcctgc tctacggact gaccgctgtc 1680
ctcggcctgg gcttcatcta tttatttgtt cctgaaacaa aaggccagtc gttggcagag 1740
atagaccagc agttccagaa gagacggttc accctgagct ttggccacag gcagaactcc 1800
actggcatcc cgtacagccg catcgagatc tctgcggcct cctgaggaat ccgtctgcct 1860
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
ggaaattctg gaactgtggc tttggcagac catctccagc atcctgcttc ctaggcc 1917
<210> 59
<211> 6791
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7475603CB1
<400> 59
cgcgctccct gcctgctgct gggcggaggg aaggcggcaa gagctgcgga gcccctggaa 60
gagcttccag gaaccctgcg ctgtgggata aaggaatgag gttcagaaag gggcaggagt 120
tgcccgcagc cgcaccgcac gtcttcagcc cgaccgttgt cctgacctct ctgtcccgtc 180
ccctgcccag tctcaccatg gccttctgga cacagctgat gctgctgctc tggaagaatt 240
tcatgtatcg ccggagacag ccggtccagc tcctggtcga attgctgtgg cctctcttcc 300
tcttcttcat cctggtggct gttcgccact cccacccgcc cctggagcac catgaatgcc'360
acttcccaaa caagccactg ccatcggcgg gcaccgtgcc ctggctccag ggtctcatct 420
gtaatgtgaa caacacctgc tttccgcagc tgacaccggg cgaggagccc gggcgcctga 480
gcaacttcaa cgactccctg gtctcccggc tgctagccga tgcccgcact gtgctgggag 540
gggccagtgc ccacaggacg ctggctggcc tagggaagct gatcgccacg ctgagggctg 600
cacgcagcac ggcccagcct caaccaacca agcagtctcc actggaacca cccatgctgg 660
atgtcgcgga gctgctgacg tcactgctgc gcacggaatc cctggggttg gcactgggcc 720
aagcccagga gcccttgcac agcttgttgg aggccgctga ggacctggcc caggagctcc 780
tggcgctgcg cagcctggtg gagcttcggg cactgctgca gagaccccga gggaccagcg 840
gccccctgga gttgctgtca gaggccctct gcagtgtcag gggacctagc agcacagtgg 900
gcccctccct caactggtac gaggctagtg acctgatgga gctggtgggg caggagccag 960
aatccgccct gccagacagc agcctgagcc ccgcctgctc ggagctgatt ggagccctgg 1020
acagccaccc gctgtcccgc ctgctctgga gacgcctgaa gcctctgatc ctcgggaagc 1080
tactctttgc accagataca ccttttaccc ggaagctcat ggcecaggtg aaccggacct 1140
tcgaggagct caccctgctg agggatgtcc gggaggtgtg ggagatgctg ggaccccgga 1200
tcttcacctt catgaacgac agttccaatg tggccatgct gcagcggctc ctgcagatgc 1260
aggatgaagg aagaaggcag cccagacctg gaggccggga ccacatggag gccctgcgat 1320
cctttctgga ccctgggagc ggtggctaca gctggcagga cgcacacgct gatgtggggc 1380
acctggtggg cacgctgggc cgagtgacgg agtgcctgtc cttggacaag ctggaggcgg 1440
caccctcaga ggcagccctg gtgtcgcggg ccctgcaact gctcgcggaa catcgattct 1500
gggccggcgt cgtcttcttg ggacctgagg actcttcaga ccccacagag cacccaaccc 1560
cagacctggg ccccggccac gtgcgcatca aaatccgcat ggacattgac gtggtcacga 1620
ggaccaataa gatcagggac aggttttggg accctggccc agccgcggac cccctgaccg 1680
acctgcgcta cgtgtggggc ggcttcgtgt acctgcaaga cctggtggag cgtgcagccg 1740
tccgcgtgct cagcggcgcc aacccccggg ccggcctcta cctgcagcag atgccctatc 1800
cgtgctatgt ggacgacgtg ttcctgcgtg tgctgagccg gtcgctgccg ctcttcctga 1860
cgctggcctg gatctactcc gtgacactga cagtgaaggc cgtggtgcgg gagaaggaga 1920
cgcggctgcg ggacaccatg cgcgccatgg ggctcagccg cgcggtgctc tggctaggct 1980
ggttcctcag ctgcctcggg cccttcctgc tcagcgccgc gctgctggtt ctggtgctca 2040
agctggggga catcctcccc tacagccacc cgggcgtggt cttcctgttc ttggcagcct 2100
tcgcggtggc cacggtgacc cagagcttcc tgctcagcgc cttcttctcc cgcgccaacc 2160
tggctgcggc ctgcggcggc ctggcctact tctccctcta cctgccctac gtgctgtgtg 2220
tggcttggcg ggaccggctg cccgcgggtg gccgcgtggc cgcgagcctg ctgtcgcccg 2280
tggccttcgg cttcggctgc gagagcctgg ctctgctgga ggagcagggc gagggcgcgc 2340
agtggcacaa cgtgggcacc cggcctacgg cagacgtctt cagcctggcc caggtctctg 2400
gccttctgct gctggacgcg gcgctctacg gcctcgccac ctggtacctg gaagctgtgt 2460
gcccaggcca gtacgggatc cctgaaccat ggaattttcc ttttcggagg agctactggt 2520
gcggacctcg gccccccaag agtccagccc cttgccccac cccgctggac ccaaaggtgc 2580
tggtagaaga ggcaccgccc ggcctgagtc ctggcgtctc cgttcgcagc ctggagaagc 2640
gctttcctgg aagcccgcag ccagccctgc gggggctcag cctggacttc taccagggcc 2700
acatcaccgc cttcctgggc cacaacgggg ccggcaagac caccaccctg tccatcttga 2760
gtggcctctt cccacccagt ggtggctctg ccttcatcct gggccacgac gtccgctcca 2820
gcatggccgc catccggccc cacctgggcg tctgtcctca gtacaacgtg ctgtttgaca 2880
tgctgaccgt ggacgagcac gtctggttct atgggcggct gaagggtctg agtgccgctg 2940
tagtgggccc cgagcaggac cgtctgctgc aggatgtggg gctggtctcc aagcagagtg 3000
tgcagactcg ccacctctct ggtgggatgc aacggaagct gtccgtggcc attgcctttg 3060
tgggcggctc ccaagttgtt atcctggacg agcctacggc tggcgtggat cctgcttccc 3120
gccgcggtat ttgggagctg ctgctcaaat accgagaagg tcgcacgctg atcctctcca 3180
cccaccacct ggatgaggca gagctgctgg gagaccgtgt ggccgtggtg gcaggtggcc 3240
gcttgtgctg ctgtggatcc ccactcttcc tgcgccgtca cctgggctcc ggctactacc 3300
81/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
tgacgctggt gaaggcccgc ctgcccctga ccaccaatga gaaggctgac actgacatgg 3360
agggcagtgt ggacaccagg caggaaaaga agaatggcag ccagggcagc agagtcggca 3420
ctcctcagct gctggccctg gtacagcact gggtgcccgg ggcacggctg gtggaggagc 3480
tgccacacga gctggtgctg gtgctgccct acacgggtgc ccatgacggc agcttcgcca 3540
cactcttccg agagctagac acgcggctgg cggagctgag gctcactggc tacgggatct 3600
ccgacaccag cctcgaggag atcttcctga aggtggtgga ggagtgtgct gcggacacag 3660
atatggagga tggcagctgc gggcagcacc tatgcacagg cattgctggc ctagacgtaa 3720
ccctacggct caagatgccg ccacaggaga cagcgctgga gaacggggaa ccagctgggt 3780
cagccccaga gactgaccag ggctctgggc cagacgccgt gggccgggta cagggctggg 3840
cactgacccg ccagcagctc caggccctgc ttctcaagcg ctttctgctt gcccgccgca 3900
gccgccgcgg cctgttcgcc cagatcgtgc tgcctgccct ctttgtgggc ctggccctcg 3960
tgttcagcct catcgtgcct cctttcgggc actacccggc tctgcggctc agtcccacca 4020
tgtacggtgc tcaggtgtcc ttcttcagtg aggacgcccc aggggaccct ggacgtgccc 4080
ggctgctcga ggcgctgctg caggaggcag gactggagga gcccccagtg cagcatagct 4140
cccacaggtt ctcggcacca gaagttcctg ctgaagtggc caaggtcttg gccagtggca 4200
actggacccc agagtctcca tccccagcct gccagtgtag ccggcccggt gcccggcgcc 4260
tgctgcccga ctgcccggct gcagctggtg gtccccctcc gccccaggca gtgaccggct 4320
ctggggaagt ggttcagaac cagacaggcc ggaacctgtc tgacttcctg gtcaagacct 4380
acccgcgcct ggtgcgccag ggcctgaaga ctaagaagtg ggtgaatgag gtcagatacg 4440
gaggcttctc gctggggggc cgagacccag gcctgccctc gggccaagag ttgggccgct 4500
cagtggagga gttgtgggcg ctgctgagtc ccctgcctgg cggggccctc gaccgtgtcc 4560
tgaaaaacct cacagcctgg gctcacagcc tggatgctca ggacagtctc aagatctggt 4620
tcaacaacaa aggctggcac tccatggtgg cctttgtcaa ccgagccagc aacgcaatcc 4680
tccgtgctca cctgccccca ggcccggccc gccacgccca cagcatcacc acactcaacc 4740
accccttgaa cctcaccaag gagcagctgt ctgaggctgc actgatggcc tcctcggtgg 4800
acgtcctcgt ctccatctgt gtggtctttg ccatgtcctt tgtcccggcc agcttcactc 4860
ttgtcctcat tgaggagcga gtcacccgag ccaagcacct gcagctcatg gggggcctgt 4920
cccccaccct ctactggctt ggcaactttc tctgggacat gtgtaactac ttggtgccag 4980
catgcatcgt ggtgctcatc tttctggcct tccagcagag ggcatatgtg gcccctgcca 5040
acctgcctgc tctcctgctg ttgctactac tgtatggctg gtcgatcaca ccgctcatgt 5100
acccagcctc cttcttcttc tccgtgccca gcacagccta tgtggtgctc acctgcataa 5160
acctctttat tggcatcaat ggaagcatgg ccacctttgt gcttgagctc ttctctgatc 5220
agaagctgca ggaggtgagc cggatcttga aacaggtctt ccttatcttc ccccacttct 5280
gcttgggccg ggggctcatt gacatggtgc ggaaccaggc catggctgat gcctttgagc 5340
gcttgggaga caggcagttc cagtcacccc tgcgctggga ggtggtcggc aagaacctct 5400
tggccatggt gatacagggg cccctcttcc ttctcttcac actactgctg cagcaccgaa 5460
gccaactcct gccacagccc agggtgaggt ctctgccact cctgggagag gaggacgagg 5520
atgtagcccg tgaacgggag cgggtggtcc aaggagccac ccagggggat gtgttggtgc 5580
tgaggaactt gaccaaggta taccgtgggc agaggatgcc agctgttgac cgcttgtgcc 5640
tggggattcc ccctggtgag tgttttgggc tgctgggtgt gaatggagca gggaagacgt 5700
ccacgtttcg catggtgacg ggggacacat tggccagcag gggcgaggct gtgctggcag 5760
gccacagcgt ggcccgggaa cccagtgctg cgcacctcag catgggatac tgccctcaat 5820
ccgatgccat ctttgagctg ctgacgggcc gcgagcacct ggagctgctt gcgcgcctgc 5880
gcggtgtccc ggaggcccag gttgcccaga ccgctggctc gggcctggcg cgtctgggac 5940
tctcatggta cgcagaccgg cctgcaggca cctacagcgg agggaacaaa cgcaagctgg 6000
cgacggccct ggcgctggtt ggggacccag ccgtggtgtt tctggacgag ccgaccacag 6060
gcatggaccc cagcgcgcgg cgcttccttt ggaacagcct tttggccgtg gtgcgggagg 6120
gccgttcagt gatgctcacc tcccatagca tggaggagtg tgaagcgctc tgctcgcgcc 6180
tagccatcat ggtgaatggg cggttccgct gcctgggcag cccgcaacat ctcaagggca 6240
gattcgcggc gggtcacaca ctgaccctgc gggtgcccgc cgcaaggtcc cagccggcag 6300
cggccttcgt ggcggccgag ttccctgggg cggagctgcg cgaggcacat ggaggccgcc 6360
tgcgcttcca gctgccgccg ggagggcgct gcgccctggc gcgcgtcttt ggagagctgg 6420
cggtgcacgg cgcagagcac ggcgtggagg acttttccgt gagccagacg atgctggagg 6480
aggtattctt gtacttctcc aaggaccagg ggaaggacga ggacaccgaa gagcagaagg 6540
aggcaggagt gggagtggac cccgcgccag gcctgcagca ccccaaacgc gtcagccagt 6600
tcctcgatga ccctagcact gccgagactg tgctctgagc ctccctcccc tgcggggccg 6660
cggggaggcc ctgggaatgg caagggcaag gtagagtgcc taggagccct ggactcaggc 6720
tggcagaggg gctggtgccc tggagaaaat aaagagaagg ctggagagaa gccgtggtgg 6780
tgaaaaaaaa a 6791
<210> 60
<211> 5214
<212> DNA
<213> Homo sapiens
<220>
<221> misc feature
82187
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
<223> Incyte ID No: 7477845CB1
<400> 60
atgctcaaaa ggaagcagag ttccagggtg gaagcccagc cagtcactga ctttggtcct 60
gatgagtctc tgtcggataa tgctgacatc ctctggatta acaaaccatg ggttcactct 120
ttgctgcgca tctgtgccat catcagcgtc atttctgttt gtatgaatac gccaatgacc 180
ttcgagcact atcctccact tcagtatgtg accttcactt tggatacatt attgatgttt 240
ctctacacgg cagagatgat agcaaaaatg cacatccggg gcattgtcaa gggggatagt 300
tcctatgtga aagatcgctg gtgtgttttt gatggattta tggtcttttg cctttgggtt 360
tctttggtgc tacaggtgtt tgaaattgct gatatagttg atcagatgtc accttggggc 420
atgttgcgga ttccacggcc actgattatg atccgagcat tccggattta tttccgattt 480
gaactgccaa ggaccagaat tacaaatatt ttaaagcgat cgggagaaca aatatggagt 540
gtttccattt ttctactttt ctttctactt ctttatggaa ttttaggagt tcagatgttt 600
ggaacattta cttatcactg tgttgtaaat gacacaaagc cagggaatgt aacctggaat 660
agtttagcta ttccagacac acactgctca ccagagctag aagaaggcta ccagtgccca 720
cctggattta aatgcatgga ccttgaagat ctgggactta gcaggcaaga gctgggctac 780
agtggcttta atgagatagg aactagtata ttcaccgtct atgaggccgc ctcacaggaa 840
ggctgggtgt tcctcatgta cagagcaatt gacagctttc cccgttggcg ttcctacttc 900
tatttcatca ctctcatttt cttcctcgcc tggcttgtga agaacgtgtt tattgctgtt 960
atcattgaaa catttgcaga aatcagagta cagtttcaac aaatgtgggg atcgagaagc 1020
agcactacct caacagccac cacccagatg tttcatgaag atgctgctgg aggttggcag 1080
ctggtagctg tggatgtcaa caagccccag ggacgcgccc cagcctgcct ccagaaaatg 1140
atgcggtcat ccgttttcca catgttcatc ctgagcatgg tgaccgtgga cgtgatcgtg 1200
gcggctagca actactacaa aggagaaaac ttcaggaggc agtacgacga gttctacctg 1260
gcggaggtgg cttttacagt actttttgat ttggaagcac ttctgaagat atggtgtttg 1320
ggatttactg gatatattag ctcatctctc cacaaattcg aactactact cgtaattgga 1'380
actactcttc atgtataccc agatctttat cattcacaat tcacgtactt tcaggtactc 1440
cgagtagttc ggctgattaa gatttcacct gcattagaag actttgtgta caagatattt 1500
ggtcctggaa aaaagcttgg gagtttggtt gtatttactg ccagcctctt gattgttatg 1560
tcagcaatta gtttgcagat gttctgcttt gtcgaagaac tggacagatt tactacgttt 1620
ccgagggcat ttatgtccat gttccagatc ctcacccagg aaggatgggt ggacgtaatg 1680
gaccaaactc taaatgctgt gggacatatg tgggcacccg tggttgccat ctatttcatt 1740
ctctatcatc tttttgccac tctgatcctc ctgagtttgt ttgttgctgt tattttggac 1800
aacttagaac ttgatgaaga cctaaagaag cttaaacaat taaagcaaag tgaagcaaat 1860
gcggacacca aagaaaagct ccctttacgc ctgcgaatct ttgaaaaatt tccaaacaga 1920
cctcaaatgg tgaaaatctc aaagcttcct tcagatttta cagttcctaa aatcagggag 1980
agttttatga agcagtttat tgaccgccag caacaggaca catgttgcct tctgagaagc 2040
ctcccgacca cctcttcctc ctcctgcgac cactccaaac gctcagcaat tgaggacaac 2100
aaatacatcg accaaaaact tcgcaagtct gttttcagca tcagggcaag gaaccttctg 2160
gaaaaggaga ccgcagtcac taaaatctta agggcttgca cccgacagcg catgctgagc 2220
ggatcatttg aggggcagcc cgcaaaggag aggtcaatcc tcagcgtgca gcatcatatc 2280
cgccaagagc gcaggtcact aagacatgga tcaaacagcc agaggatcag caggggaaaa 2340
tctcttgaaa ctttgactca agatcattgc aatacagtga tatatagaaa tgctcaaaga 2400
gaagtcagtg aaataaagat gattcaggaa aaaaaggagc tagcagagat gcttcaagga 2460
aagtgcaaaa aggaactcag agagagccac ccatacttcg ataagccact gttcattgtc 2520
gggcgagaac acaggttcag aaacttttgc cgggtggtgg tccgagcacg cttcaacgcg 2580
tctaaaacag accctgtcac aggagctgtg aaaaatacaa agtaccatct tctttatgat 2640
ttgctgggat tggtcactta cctggactgg gtcatgatca tcgtaacctc tgactcttgc 2700
atttccatga tgtttgagtc cccgtttcga agagtcatgc atgcacctac tttgcagatt 2760
gctgagtatg tgtttgtgat attcatgagc attgagctta atctgaagat tatggcagat 2820
ggcttatttt tcactccaac tgctgtcatc agggacttcg gtggagtaat ggacatattt 2880
atatatcttg tgagcttgat atttctttgt tggatgcctc aaaatgtacc tgctgaatcg 2940
ggagctcagc ttctaatggt ccttcggtgc ctgagacctc tgcgcatatt caaactggtg 3000
ccccagatga ggaaagttgt tcgagaactt ttcagcggct tcaaggaaat ttttttggtc 3060
tccattcttt tgctgacatt aatgctcgtt tttgcaagct ttggagttca gctttttgct 3120
ggaaaactgg ccaagtgcaa tgatcccaac attattagaa gggaagattg caatggcata 3180
ttcagaatta atgtcagtgt gtcaaagaac ttaaatttaa aattgaggcc tggagagaaa 3240
aaacctggat tttgggtgcc ccgtgtttgg gcgaatcctc ggaactttaa tttcgacaat 3300
gtgggaaacg ctatgctggc gttgtttgaa gttctctcct tgaaaggctg ggtggaagtg 3360
agagatgtta ttattcatcg tgtggggccg atccatggaa tctatattca tgtttttgta 3420
ttcctgggtt gcatgattgg actgaccctt tttgttggag tagttattgc taatttcaat 3480
gaaaacaagg ggacggcttt gctgaccgtc gatcagagaa gatgggaaga cctgaagagc 3540
cgactgaaga tcgcacagcc tcttcatctt ccgcctcgcc cggataatga tggttttaga 3600
gctaaaatgt atgacataac ccagcatcca ttttttaaga ggacaatcgc attactcgtc 3660
ctggcccagt cggtgttgct ctctgtcaag tgggacgtcg aggacccggt gaccgtacct 3720
ttggcaacaa tgtcagttgt tttcaccttc atctttgttc tggaggtaac catgaagatc 3780
atagcaatgt cgcctgctgg cttctggcaa agcagaagaa accgatacga tctcctggtg 3840
83/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
acgtcgcttg gcgttgtatg ggtggtgctt cactttgccc tcctgaatgc atatacttac 3900
atgatgggcg cttgtgtgat tgtatttagg tttttctcca tctgtggaaa acatgtaacg 3960
ctaaagatgc tcctcttgac agtggtcgtc agcatgtaca agagcttctt tatcatagta 4020
ggcatgtttc tcttgctgct gtgttacgct tttgctggag ttgttttatt tggtactgtg 4080
aaatatgggg agaatattaa caggcatgca aatttttctt cggctggaaa agctattacc 4140
gtactgttcc gaattgtcac aggtgaagac tggaacaaga ttatgcatga ctgtatggta 4200
cagcctccgt tttgtactcc agatgaattt acatactggg caacagactg tggaaattat 4260
gctggggcac ttatgtattt ctgttcattt tatgtcatca ttgcctacat catgctaaat 4320
ctgcttgtag ccataattgt ggagaatttc tccttgattt attccactga ggaggaccag 4380
cttttaagtt acaatgatct tcgccacttt caaataatat ggaacatggt ggatgataaa 4440
agagaggtat tccccacgtt ccgcgtcaag ttcctgctgc ggctactgcg tgggaggctg 4500
gaggtggacc tggacaagga caagctcctg tttaagcaca tgtgctacga aatggagagg 4560
ctccacaatg gcggcgacgt caccttccat gatgtcctga gcatgctttc ataccggtcc 4620
gtggacatcc ggaagagctt gcagctggag gaactcctgg cgagggagca gctggagtac 4680
accatagagg aggaggtggc caagcagacc atccgcatgt ggctcaagaa gtgcctgaag 4740
cgcatcagag ctaaacagca gcagtcgtgc agtatcatcc acagcctgag agagagtcag 4800
cagcaagagc tgagccggtt tctgaacccg cccagcatcg agaccaccca gcccagtgag 4860
gacacgaatg ccaacagtca ggacaacagc atgcaacctg agacaagcag ccagcagcag 4920
ctcctgagcc ccacgctgtc ggatcgagga ggaagtcggc aagatgcagc cgacgcaggg 4980
aaaccccaga ggaaatttgg gcagtggcgt ctgccctcag ccccaaaacc aataagccat 5040
tcagtgtcct cagtcaactt acggtttgga ggaaggacaa ccatgaaatc tgtcgtgtgc 5100
aaaatgaacc ccatgactga cgcggcttcc tgcggttctg aagttaagaa gtggtggacc 5160
cggcagctga ctgtggagag cgacgaaagt ggggatgacc ttctggatat ttag 5214
<210> 61
<211> 1818
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 168827CB1
<400> 61
ggaaattgct tccgtgaccc tgctgcagat gggagagagg gcccattaag aagagagtgg 60
ggtcaggatc aacacacaca cttagtgtga tttaaggaaa ggaaatattt tctctttgaa 120
cttatctgga tacagtcatt ttgtctcctc ttggggatca cttgtccagc ctcaatggcc 180
tttcaggacc tcctagatca agttggaggc ctggggagat tccagatcct tcagatggtt 240
ttccttataa tgttcaacgt catagtatac catcaaactc agctggagaa cttcgcagca 300
ttcatacttg atcatcgctg ctgggttcat atactggaca atgacactat ccctgacaat 360
gaccctggga ccctcagcca ggatgccctc ctgagaatct ccatcccatt cgactcaaat 420
ctgaggccag agaagtgtcg tcgctttgtc catccccagt ggaagctcat tcatctgaat 480
gggaccttcc ccaacacgag tgagccagat acagagccct gtgtggatgg ctgggtatat 540
gaccaaagct ccttcccttc caccattgtg actaagtggg atctggtatg cgaatctcaa 600
ccactgaatt cagtagctaa atttctattc atggctggaa tgatggtggg aggcaaccta 660
tatggccatt tgtcagacag gtttgggaga aagttcgtgc tcagatggtc ttacctccag 720
ctcgccattg taggcacctg tgcggccttt gctcccacca tcctcgtata ctgctccctg 780
cgcttcttgg ctggggctgc tacatttagc atcattgtaa atactgtttt gttaattgta 840
gagtggataa ctcaccaatt ctgtgccatg gcattgacat tgacactttg tgctgctagt 900
attggacata taaccctggg aagcctggct tttgtcattc gagaccagtg catcctccag 960
ttggtgatgt ctgcaccatg ctttgtcttc tttctgttct caaggtggct ggcagagtct 1020
gctcggtggc tcattatcaa caacaaacca gaagagggct taaaggaact tacaaaagct 1080
gcacacagga atggaatgaa gaatgctgaa gacatcctaa ccatggaggt tttgaaatcc 1140
accatgaagc aagaactgga ggcagcacag aaaaagcatt ctctttgtga attgctccgc 1200
atacccaaca tatgtaaaag aatctgtttc ctgtcctttg tgagatttgc aagtaccatc 1260
cctttttggg gccttacttt gcacctccag catctgggaa acaatgtttt cctgttgcag 1320
actctctttg gtgcagtcac cctcctggcc aattgtgttg caccttgggc actgaatcac 1380
atgagccgtc gactaagcca gatgcttctc atgttcctac tggcaacctg ccttctggcc 1440
atcatatttg tgcctcaaga aatgcagacc ctgcgtgtgg ttttggcaac cctgggtgtg 1500
ggagctgctt ctcttggcat tacctgttct actgcccaag aaaatgaact aattccttcc 1560
ataatcaggg gaagagctac tggaatcact ggaaactttg ctaatattgg gggagccctg 1620
gcttccctca tgatgatcct aagcatatat tctcgacccc tgccctggat catctatgga 1680
gtctttgcca tcctctctgg ccttgttgtc ctcctccttc ctgaaaccag gaaccagcct 1740
cttcttgaca gcatccagga tgtggaaaat gagggagtaa atagcctagc tgcccctcag 1800
aggagctctg tgctatag 1818
<210> 62
84/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
<211> 2245
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472734CB1
<400> 62
cccttggaac agtaggatgt tggtgatgca aaagtcaatg tttaaactca acattccact 60
ttcctttaac taagaatagt ttttattaac ttttagtaaa actcagtcct agtccaaaaa 120
aagccctgct ctctgatctt tgtacaagaa catcataaag caattcactt tggattttct 180
aatatcccat ttctgagaag aatggcagac tattgaacag gtgtatttta ggtcacgtgg 240
ggactgcatc cacctgaaaa tccaccgttg acttatcagg aaactcagag atcaggatct 300
ttcacagagt agtcttttaa gaagattcag ttgteaacag ctagcagtct ctttgccaaa 360
taattatatc tgtgacttct gaaactattt ggctgcctaa agttaaagga cttggggaaa 420
gtccctccac tgctcttctg cagtagtgtc acaccactca gtgcagggcc caccaagaag 480
aaagcagtgt caggatccac atggcactat ggtaactttg tgaaagggga cattttctcc 540
ctctgaactt ctcttcataa agtcattgtg cttcctcttg gggatcaoct gttcagtctc 600
aatgggcttt gatgtgctcc tggatcaagt gggtggcatg gggagattcc agatttgtct 660
gatagctttc ttttgcatca ccaacatcct actgttccct aatattgtgt tggagaactt 720
cactgcattc acccctagtc atcgctgctg ggtccccctc ctggacaatg acactgtgtc 780
tgacaatgat accgggaccc tcagcaagga tgacctcctg agaatctcca tcccactgga 840
ctcaaacctg aggccacaga agtgtcagcg ctttatccat ccccagtggc agctccttca 900
cctgaacggg accttcccca acacaaatga gccagacacg gagccctgtg tggatggctg 960
ggtgtacgac agaagctctt tcctctccac catcgtgact gagtgggacc tggtatgtga 1020
atctcagtca ctaaaatcaa tggttcaatc cctatttatg gctgggtcac ttctgggagg 1080
tctaatatat ggccatcttt cagacaggtt tgggagaaag ttcgtgctca gatggtctta 1140
cctccagctc gccattgtag gcacctgtgc ggcctttgct cccaccatcc tcgtatactg 1200
ctccctgcgc ttcttggctg gggctgctac atttagcatc attgtaaata ctgttttgtt 1260
aattgtagag tggataactc accaattctg tgccatggca ttgacattga cactttgtgc 1320
tgctagtatt ggacatataa ccctgggaag cctggctttt gtcattcgag accagtgcat 1380
cctccagttg gtgatgtctg caccatgctt tgtcttcttt ctgttctcaa ggtggctggc 1440
agagtctgct cggtggctca ttatcaacaa caaaccagaa gagggcttaa aggaacttag 1500
aaaagctgca cacaggaatg gaatgaagaa tgctgaagac atcctaacca tggaggtttt 1560
gaaatccacc atgaagcaag aactggaggc agcacagaaa aagcattctc tttgtgaatt 1620
gctccgcata cccaacatat gtaaaagaat ctgtttcctg tcctttgtga gatttgcaag 1680
taccatccct ttttggggcc ttactttgca cctccagcat ctgggaaaca atgttttcct 1740
gttgcagact ctctttggtg cagtcaccct cctggccaat tgtgttgcac cttgggcact 1800
gaatcacatg agccgtcgac taagccagat gcttctcatg ttcctactgg caacctgcct 1860
tctggccatc atatttgtgc ctcaagaaat gcagaccctg cgtgtggttt tggcaaccct 1920
gggtgtggga gctgcttctc ttggcattac ctgttctact gcccaagaaa atgaactaat 1980
tccttccata atcaggggaa gagctactgg aatcactgga aactttgcta atattggggg 2040
agccctggct tccctcatga tgatcctaag catatattct cgacccctgc cctggatcat 2100
ctatggagtc tttgccatcc tctctggcct tgttgtcctc ctccttcctg aaaccaggaa 2160
ccagcctctt cttgacagca tccaggatgt ggaaaatgag ggagtaaata gcctagctgc 2220
ccctcagagg agctctgtgc tatag 2245
<210> 63
<211> 3196
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473473CB1
<400> 63
gcggcggccg ggggagcgct actaccatga actgcctggt cctcctcccc agagctgctc 60
atccgggtcg ggctggagac acagtcaggg gaccccgtcg ccgccgccgc gccccctctt 120
ctttcggctc aatcttctct tccacctttt cctcctcttc ctccaccttc tttgcctgca 180
tccccccctc ccccgccgcg gatcctggcc gctgctctcc agacccagga tgccgggggg 240
caagagaggg ctggtggcac cgcagaacac atttttggag aacatcgtca ggcgctccag 300
tgaatcaagt ttcttactgg gaaatgccca gattgtggat tggcctgtag tttatagtaa 360
tgacggtttt tgtaaactct ctggatatca tcgagctgac gtcatgcaga aaagcagcac 420
ttgcagtttt atgtatgggg aattgactga caagaagacc attgagaaag tcaggcaaac 480
ttttgacaac tacgaatcaa actgctttga agttcttctg tacaagaaaa acagaacccc 540
85/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
tgtttggttt tatatgcaaa ttgcaccaat aagaaatgaa catgaaaagg tggtcttgtt 600
cctgtgtact ttcaaggata ttacgttgtt caaacagcca atagaggatg attcaacaaa 660
aggttggacg aaatttgccc gattgacacg ggctttgaca aatagccgaa gtgttttgca 720
gcagctcacg ccaatgaata aaacagaggt ggtccataaa cattcaagac tagctgaagt 780
tcttcagctg ggatcagata tccttcctca gtataaacaa gaagcgccaa agacgccacc 840
acacattatt ttacattatt gtgcttttaa aactacttgg gattgggtga ttttaattct 900
taccttctac accgccatta tggttcctta taatgtttcc ttcaaaacaa agcagaacaa 960
catagcctgg ctggtactgg atagtgtggt ggacgttatt tttctggttg acatcgtttt 1020
aaattttcac acgactttcg tggggcccgg tggagaggtc atttctgacc ctaagctcat 1080
aaggatgaac tatctgaaaa cttggtttgt gatcgatctg ctgtcttgtt taccttatga 1140
catcatcaat gcctttgaaa atgtggatga gggaatcagc agtctcttca gttctttaaa 1200
agtggtgcgt ctcttacgac tgggccgtgt ggctaggaaa ctggaccatt acctagaata 1260
tggagcagca gtcctcgtgc tcctggtgtg tgtgtttgga ctggtggccc actggctggc 1320
ctgcatatgg tatagcatcg gagactacga ggtcattgat gaagtcacta acaccatcca 1380
aatagacagt tggctctacc agctggcttt gagcattggg actccatatc gctacaatac 1440
cagtgctggg atatgggaag gaggacccag caaggattca ttgtacgtgt cctctctcta 1500
ctttaccatg acaagcctta caaccatagg atttggaaac atagctccta ccacagatgt 1560
ggagaagatg ttttcggtgg ctatgatgat ggttggcgct cttctttatg caactatttt 1620
tggaaatgtt acaacaattt tccagcaaat gtatgccaac accaaccgat accatgagat 1680
gctgaataat gtacgggact tcctaaaact ctatcaggtc ccaaaaggcc ttagtgagcg 1740
agtcatggat tatattgtct caacatggtc catgtcaaaa ggcattgata cagaaaaggt 1800
cctctccatc tgtcccaagg acatgagagc tgatatctgt gttcatctaa accggaaggt 1860
ttttaatgaa catcctgctt ttcgattggc cagcgatggg tgtctgcgcg ccttggcggt 1920
agagttccaa accattcact gtgctcccgg ggacctcatt taccatgctg gagaaagtgt 1980
ggatgccctc tgctttgtgg tgtcaggatc cttggaagtc atccaggatg atgaggtggt 2040
ggctatttta gggaagggtg atgtatttgg agacatcttc tggaaggaaa ccacccttgc 2100
ccatgcatgt gcgaacgtcc gggcactgac gtactgtgac ctacacatca tcaagcggga 2160
agccttgctc aaagtcctgg acttttatac agcttttgca aactccttct caaggaatct 2220
cactcttact tgcaatctga ggaaacggat catctttcgt aagatcagtg atgtgaagaa 2280
agaggaggag gagcgcctcc ggcagaagaa tgaggtgacc ctcagcattc ccgtggacca 2340
cccagtcaga aagctcttcc agaagttcaa gcagcagaag gagctgcgga atcagggctc~2400
aacacagggt gaccctgaga ggaaccaact ccaggtagag agccgctcct tacagaatgg 2460
agcctccatc accggaacca gcgtggtgac tgtgtcacag attactccca ttcagacgtc 2520
tctggcctat gtgaaaacca gtgaatccct taagcagaac aaccgtgatg ccatggaact 2580
caagcccaac ggcggtgctg accaaaaatg tctcaaagtc aacagcccaa taagaatgaa 2640
gaatggaaat ggaaaagggt ggctgcgact caagaataat atgggagccc atgaggagaa 2700
aaaggaagac tggaataatg tcactaaagc tgagtcaatg gggctattgt ctgaggaccc 2760
caagagcagt gattcagaga acagtgtgac caaaaaccca ctaagaaaaa cagattcttg 2820
tgacagtgga attacaaaaa gtgaccttcg tttggataag gctggggagg cccgaagtcc 2880
gctagagcac agtcccatcc aggctgatgc caagcacccc ttttatccca tccccgagca 2940
ggccttacag accacactgc aggaagtcaa acacgaactc aaagaggaca tccagctgct 3000
cagctgcaga atgactgccc tagaaaagca ggtggcagaa attttaaaaa tactgtcgga 3060
aaaaagcgta ccccaggcct catctcccaa atcccaaatg ccactccaag taccccccca 3120
gataccatgt caggatattt ttagtgtctc aaggcctgaa tcacctgaat ctgacaaaga 3180
tgaaatccac ttttaa 3196
<210> 64
<211> 1602
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7477725CB1
<400> 64
atggcctttg aggagctctt gagtcaagtt ggaggccttg ggagatttca gatgcttcat 60
ctggttttta ttcttccctc tctcatgtta ttaatccctc atatactgct agagaacttt 120
gctgcagcca ttcctggtca tcgttgctgg gtccacatgc tggacaataa tactggatct 180
ggtaatgaaa ctggaatcct cagtgaagat gccctcttga gaatctctat cccactagac 240
tcaaatctga ggccagagaa gtgtcgtcgc tttgtccatc cccagtggca gcttcttcac 300
ctgaatggga ctatccacag cacaagtgag gcagacacag aaccctgtgt ggatggctgg 360
gtatatgatc aaagctactt cccttcgacc attgtgacta agtgggacct ggtatgtgat 420
tatcagtcac tgaaatcagt ggttcaattc ctacttctga ctggaatgct ggtgggaggc 480
atcataggtg gccatgtctc agacaggttt gggcgaagat ttattctcag atggtgtttg 540
ctccagcttg ccattactga cacctgcgct gccttcgctc ccaccttccc tgtttactgt 600
gtactacgct tcttggcagg tttttcttcc atgatcatta tatcaaataa ttctttgccc 660
86/87
CA 02415808 2003-O1-06
WO 02/04520 PCT/USO1/21448
attactgagt ggataaggcc caactctaaa gccctggtag taatattgtc atctggtgcc 720
cttagtattg gacagataat cctgggaggc ttggcttatg tcttccgaga ctggcaaacc 780
ctgcacgtgg tggcgtctgt acctttcttt gtcttctttc ttctttcaag gtggctggtg 840
gaatctgctc ggtggttgat aatcaccaat aaactagatg agggcttaaa ggcacttaga 900
aaagttgcac gcacaaatgg aataaagaat gctgaagaaa ccctgaacat agaggttgta 960
agatccacca tgcaggagga gctggatgca gcacagacca aaactactgt gtgtgacttg 1020
ttccgcaacc ccagtatgcg taaaaggatc tgtatcctgg tatttttgag atttgcaaac 1080
acaatacctt tttatggtac catggtcaat cttcagcatg tggggagcaa cattttcctg 1140
ttgcaggtac tttatggagc tgtcgctctc atagttcgat gtcttgctct tttgacacta 1200
aatcatatgg gccgtcgaat aagccagata ttgttcatgt tcctggtggg cctttccatt 1260
ttggccaaca cgtttgtgcc caaagaaatg cagaccctgc gtgtggcttt ggcatgtctg 1320
ggaatcggct gttctgctgc tactttttcc agtgttgctg ttcacttcat tgaactcatc 1380
cccactgttc tcagggcaag agcttcagga atagatttaa cggctagtag gattggagca 1440
gcactggctc ccctcttgat gaccttaacg gtatttttta ccactttgcc atggatcatt 1500
tatggaatct tccccatcat tggtggcctt attgtcttcc tcctaccaga aaccaagaat 1560
ctgcctttgc ctgacaccat caaggatgtg gaaaatcagt ga 1602
87/87
