Note: Descriptions are shown in the official language in which they were submitted.
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TRANSPORTERS AND ION CHANNELS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of
transporters and ion
channels and to the use of these sequences in the diagnosis, treatment, and
prevention of transport,
neurological, muscle, immunological, and cell proliferative disorders, and in
the assessment of the
effects of exogenous compounds on the expression of nucleic acid and amino
acid sequences of
transporters and ion channels.
1o BACKGROUND OF THE INVENTION
Eukaryotic cells are surrounded and subdivided into functionally distinct
organelles by
hydrophobic lipid bilayer membranes which are highly impermeable to most polar
molecules. Cells and
organelles require trausport proteins to import and export essential nutrients
and metal ions including
K+, NH4+, Pi, SO~.2-, sugars, and vitamins, as well as various metabolic waste
products. Transport
proteins also play roles in antibiotic resistatice, toxin secretion, ion
balance, synaptic neurotransmission,
kidney function, intestinal absorption, tumor growth, and other diverse cell
functions (Griffith, J. and C.
Sansom (1998) The Transporter Facts Book, Academic Press, San Diego CA, pp. 3-
29). Transport
can occur by a passive concentration-dependent mechanism, or can be linked to
an energy source
such as ATP hydrolysis or an ion gradient. Proteins that function in transport
include carrier proteins,
2o which bind to a specific solute and undergo a conformational change that
trauslocates 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 trausporters 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 trausporters include the mammalian
glucose transporter
(SGLT1), iodide transporter (NIS), and multivitamin transporter (SMVT). All
three transporters have
twelve putative trausmembrane segments, extracellular glycosylation sites, and
cytoplasmically-
oriented N- and C-termini. 1~IS plays a crucial role in the evaluation,
diagnosis, and treatment of
various thyroid pathologies because it is the molecular basis for radioiodide
thyroid-imaging techniques
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and for specific targeting of radioisotopes to the thyroid gland (Levy, O. et
al. (1997) Proc. Natl.
Acad. Sci. USA 94:5568-5573). SMVT is expressed in the intestinal mucosa,
kidney, and placenta,
and is implicated in the transport of the water-soluble vitamins, e.g., biotin
and pantothenate (Prasad,
P.D. et al. (1998) J. Biol. Chem. 273:7501-7506).
One of the largest families of txansporters is the major facilitator
superfamily (MFS), also
called the uniporter-symporter-antiporter family. MFS transporters are single
polypeptide carriers that
transport small solutes in response to ion gradients. Members of the MFS are
found in all classes of
living organisms, and include transporters for sugars, oligosaccharides,
phosphates, nitrates,
nucleosides, monocarboxylates, and drugs. MFS transporters found in eukaryotes
all have a structure
comprising 12 transmembrane segments (Pao, S.S. et al. (1998) Microbiol.
Molec. Biol. Rev. 62:1-34).
The largest family of MFS transporters is the sugar transporter family, which
includes the seven
glucose transporters (GLUTl-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 GLUT5 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. anal 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+-licked
monocarboxylate
transporters with differing substrate and inhibitor selectivities. In
particular, cardiac muscle and tumor
cells have transporters that differ in their Km values for certain substrates,
including stereoselectivity
for L- over D-lactate, and in their sensitivity to inhibitors. There are Na+-
monocarboxylate
cotransporters on the luminal surface of intestinal and kidney epithelia,
which allow the uptake of
lactate, pyruvate, and ketone bodies in these tissues. In addition, there are
specific and selective
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transporters for organic cations and organic anions in organs including the
kidney, intestine and liver.
Organic anion taransporters 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 trausporters, such as the following diseases and their
corresponding proteins: cystic
fibrosis (CFTR, an ion channel), adrenoleukodystrophy (adrenoleukodystrophy
protein, ALDP),
Zellweger syndrome (peroxisomal membrane protein-70, PMP70), anal
hyperinsulinemic hypoglycemia
(sulfonylurea receptor, SUR). Overexpression of the multidrug resistance (MDR)
protein, another
ABC transporter, in human cancer cells makes the cells resistant to a variety
of cytotoxic drugs used
in chemotherapy (Taglicht, D. and S. Michaelis (1998) Meth. Enzymol. 292:130-
162).
A number of metal ions such as iron, zinc, copper, cobalt, manganese,
molybdenum, selenium,
nickel, and chromium are important as cofactors for a number of enzymes. For
example, copper is
involved in hemoglobin synthesis, connective tissue metabolism, and bone
development, by acting as a
cofactor in oxidoreductases such as superoxide dismutase, ferroxidase
(ceruloplasmin), and lysyl
oxidase. Copper and other metal ions must be provided in the diet, and are
absorbed by transporters in
the gastrointestinal tract. Plasma proteins transport the metal ions to the
liver and other target organs,
where specific transporters move the ions into cells and cellular organelles
as needed. Imbalances in
metal ion metabolism have been associated with a number of disease states
(Darks, D.M. (1986) J.
Med. Genet. 23:99-106).
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).
Mitochondrial carrier proteins are trausmembrane-spanning proteins which
transport ions and
charged metabolites between the cytosol and the mitochondrial matrix. Examples
include the ADP,
ATP carrier protein; the 2-oxoglutarate/malate carrier; the phosphate carrier
protein; the pyruvate
earner; the dicarboxylate carrier which transports malate, succinate,
fumarate, and phosphate; the
tricarboxylate carrier which transports citrate and malate; and the Grave's
disease carrier protein, a
protein recognized by IgG in patients with active Grave's disease, an
autoimmune disorder resulting in
hyperthyroidism. Proteins in this family consist of three tandem repeats of an
approximately 100
amino acid domain, each of which contains two transmembrane regions (Stryer,
L. (1995)
Biochemistry, W.H. Freeman and Company, New York NY, p. 551; PROSTTE PDOC00189
Mitochondrial energy transfer proteins signature; Online Mendelian Inheritance
in Man (OMIM)
*275000 Graves Disease).
This class of transporters also includes the mitochondrial uncoupling
proteins, which create
proton leaks across the inner mitochondrial membrane, thus uncoupling
oxidative phosphorylation from
ATP synthesis. The result is energy dissipation in the form of heat.
Mitochondrial uncoupling proteins
have been implicated as modulators of thermoregulation and metabolic rate, and
have been proposed
as potential targets for drugs against metabolic diseases such as obesity
(Ricquier, D. et al. (1999) J.
Int. Med. 245:637-642).
Ion Channels
The electrical potential of a cell is generated and maintained by controlling
the movement of
ions across the plasma membrane. The movement of ions requires ion channels,
which form ion-
selective pores within the membrane. There are two basic types of ion
channels, ion transporters and
gated ion channels. Ion transporters utilize the energy obtained from ATP
hydrolysis to actively
transport an ion against the ion's concentration gradient. Gated ion channels
allow passive flow of an
ion down the ion's electrochemical gradient under restricted conditions.
Together, these types of ion
channels generate, maintain, and utilize an electrochemical gradient that is
used in 1) electrical impulse
conduction down the axon of a nerve cell, 2) transport of molecules into cells
against concentration
gradients, 3) initiation of muscle contraction, and 4) endocrine cell
secretion.
Ion Transporters
Ion transporters generate and maintain the resting electrical potential of a
cell. Utilizing the
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energy derived from ATP hydrolysis, they transport ions against the ion's
concentration gradient.
These transmembrane ATPases are divided into three families. The
phosphorylated (P) class ion
transporters, including Na+-K'" ATPase, Ca2+-ATPase, and H+-ATPase, are
activated by a
phosphorylation event. P-class ion transporters are responsible for
maintaining resting potential
distributions such that cytosolic concentrations of Na+ and Caz-'' are low and
cytosolic concentration of
K~ is high. The vacuolar (V) class of ion 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 (Pi).
The P-ATPases are hexamers of a 100 kD subunit with ten transmembrane domains
and
several Iarge 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 V1
domain, a peripheral complex responsible for ATP hydrolysis; and the Vo
domain, an integral complex
responsible for proton translocation across the membrane. The F-ATPases are
structurally and
evolutionarily related to the V-ATPases. The F-ATPase Fo domain contains 12
copies of the c
subunit, a highly hydrophobic protein composed of two transmembrane domains
and containing a single
buried carboxyl group in TM2 that is essential for proton transport. The V-
ATPase Vo domain
contains three types of homologous c subunits with four or five transmembrane
domains and the
essential carboxyl group in TM4 or TM3. Both types of complex also contain a
single a subunit that
may be involved in regulating the pH dependence of activity (Forgac, M. (1999)
J. Biol. Chem.
274:12951-12954).
The resting potential of the cell is utilized in many processes involving
carrier proteins and
gated ion channels. Carrier proteins utilize the resting potential to
transport molecules into and out of
the cell. Amino acid and glucose transport into many cells is linked to sodium
ion co-transport
(symport) so that the movement of Na+ down an electrochemical gradient drives
transport of the other
molecule up a concentration gradient. Similarly, cardiac muscle links transfer
of Caz+ 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
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response to mechanical stress; voltage-gated channels (e.g., Nay, K+, Ca2+,
and Cl channels) open
their pores in response to changes in membrane potential; and ligand-gated
channels (e.g.,
acetylcholine-, serotonin-, and glutamate-gated canon channels, and GABA- and
glycine-gated
chloride channels) open their pores in the presence of a specific ion,
nucleotide, or neurotransmitter.
The gating properties of a particular ion channel (i.e., its threshold for and
duration of opening and
closing) are sometimes modulated by association with auxiliary channel
proteins and/or post
translational modifications, such as phosphorylation.
Mechanically-gated or mechanosensitive ion channels act as transducers for the
senses of
touch, hearing, and balance, and also play important roles in cell volume
regulation, smooth muscle
contraction, and cardiac rhythm generation. A stretch-inactivated channel
(SIC) was recently cloned
from rat kidney. The SIC channel belongs to a group of channels which are
activated by pressure or
stress on the cell membrane and conduct both Ca2+ and Na+ (Suzuki, M. et al.
(1999) J. Biol. Chem.
274:6330-6335).
The pore-forming subunits of the voltage-gated cation channels form a
superfamily of ion
channel proteins. The characteristic domain of these channel proteins
comprises six transmembrane
domains (S1-S6), a pore-forming region (P) located between S5 and S6, and
intracellular amino and
carboxy termini. In the Na+ and Ca2+ subfamilies, this domain is repeated four
times, while in the K+
channel subfamily, each channel is formed from a tetramer of either identical
or dissimilar subunits.
The P region contains information specifying the ion selectivity for the
channel. In the case of K+
channels, a GYG tripeptide is involved in this selectivity (Ishii, T.M. et al.
(1997) Proc. Natl. Acad.
Sci. USA 94:11651-11656).
Voltage-gated Na+ and K+ channels are necessary for the function of
electrically excitable
cells, such as nerve and muscle cells. Action potentials, which lead to
neurotransmitter release and
muscle contraction, arise from large, transient changes in the permeability of
the membrane to Na+
and K+ ions. Depolarization of the membrane beyond the threshold level opens
voltage-gated Na+
channels. Sodium ions flow into the cell, further depolarizing the membrane
and opening more
voltage-gated Na ~ channels, which propagates the depolarization down the
length of the cell.
Depolarization also opens voltage-gated potassium channels. Consequently,
potassium ions flow
outward, which leads to repolarization of the membrane. Voltage-gated channels
utilize charged
residues in the fourth transmembrane segment (S4) to sense voltage change. The
open state lasts
only about 1 millisecond, at which time the channel spontaneously converts
into an inactive state that
cannot be opened irrespective of the membrane potential. Iuactivation 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.
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Voltage-gated Na+ channels are heterotrimeric complexes composed of a 260 kDa
pore-
forming a subunit that associates with two smaller auxiliary subunits, (31 and
(32. The (32 subunit is a
integral membrane glycoprotein that contains an extracellular Ig domain, and
its association with a and
(31 subunits correlates with increased functional expression of the channel, a
change in its gating
properties, as well as an increase in whole cell capacitance due to an
increase in membrane surface
area (Isom, L.L. et al. (1995) Cell 83:433-442).
Non voltage-gated Na+ channels include the members of the amiloride-sensitive
Na+
channel/degenerin (NaC/DEG) family. Channel subunits of this family are
thought to consist of two
transmembrane domains flanking a long extracellular loop, with the amino and
carboxyl termini located
within the cell. The NaC/DEG family includes the epithelial Na+ channel (ENaC)
involved in Na+
reabsorption in epithelia including the airway, distal colon, cortical
collecting duct of the kidney, and
exocrine duct glands. Mutations in ENaC result in pseudohypoaldosteronism type
1 and Liddle's
syndrome (pseudohyperaldosteronism). The NaC/DEG family also includes the
recently characterized
H+-gated ration channels or acid-sensing ion channels (ASIC). ASIC subunits
are expressed in the
brain and form heteromultimeric Na+-permeable channels. These channels require
acid pH
fluctuations for activation. ASIC subunits show homology to the degenerins, a
family of mechanically-
gated channels originally isolated from C. elegans. Mutations in the
degenerins cause
neurodegeneration. ASIC subunits may also have a role in neuronal function, or
in pain perception,
since tissue acidosis causes pain (Waldmann, R. and M. Lazdunski (1998) Curr.
Opin. Neurobiol.
8:418-424; Eglen, R.M. et al. (1999) Trends Pharmacol. Sri. 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 acxoss
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
transmembrane/1 pore domain structure. Four subunits combine as homo- or
heterotetramers to form
7
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functional K channels. These pore-forming subunits also associate with various
cytoplasmic (3
subunits that alter channel inactivation kinetics. The Shaker-like channel
family includes the voltage-
gated K+ channels as well as the delayed rectifier type channels such as the
human ether-a-go-go
related gene (HERG) associated with long QT, a cardiac dysrythmia syndrome
(Curran, M.E. (1998)
Curr. Opin. Bioteclmol. 9:565-572; Kaczorowski, G.J. and M.L. Garcia (1999)
Curr. Opin. Chem.
Biol. 3:448-458).
A second superfamily of K+ channels is composed of the inward rectifying
channels (Kir).
Kir channels have the property of preferentially conducting K+ currents in the
inward direction. These
proteins consist of a single potassium selective pore domain and two
transmembrane domains, which
correspond to the fifth and sixth transmembrane domains of voltage-gated K+
channels. Kir subunits
also associate as tetramers. The Kir family includes ROMK1, mutations in which
lead to Banter
syndrome, a renal tubular disorder. Kir channels are also involved in
regulation of cardiac pacemaker
activity, seizures and epilepsy, and insulin regulation (Doupnik, C.A. et al.
(1995) Curr. Opin.
Neurobiol. 5:268-277; Curran, su ra).
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 transmernbrane
domains and two P domains. These proteins are probably involved in controlling
the resting pbtential
in a large set of cell types (Duprat, F. et al. (1997) EMBO J 16:5464-5471).
The voltage-gated Ca 2+ channels have been classified into several subtypes
based upon their
electrophysiological and pharmacological characteristics. L-type Ca 2+
channels are predominantly
expressed in heart and skeletal muscle where they play an essential role in
excitation-contraction
coupling. T-type channels are important for cardiac pacemaker activity, while
N-type and P/Q-type
channels are involved in the control of neurotransmitter release in the
central and peripheral nervous
system. The L-type and N-type voltage-gated Ca Z~ channels have been purified
and, though their
functions differ dramatically, they have similar subunit compositions. The
channels are composed of
three subunits. The a1 subunit forms the membrane pore and voltage sensor,
while the aZb and (3
subunits modulate the voltage-dependence, gating properties, and the current
amplitude of the channel.
These subunits are encoded by at least six al, one a28, and four (3 genes. A
fourth subunit, Y, has
been identified in skeletal muscle (Walker, D. et al. (1998) J. Biol. Chem.
273:2361-2367; McCleskey,
3o 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
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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) Cell 85:661-671;
Boulay, G. et al. (1997)
J. Biol. Chem. 272:29672-29680). Melastatin is a gene isolated in both the
mouse and human, and
whose expression in melanoma cells is inversely correlated with melanoma
aggressiveness in vivo.
The human cDNA transcript corresponds to a 1533-amino acid protein having
homology to members
of the Trp family. It has been proposed that the combined use of malastatin
mRNA expression status
and tumor thickness might allow for the determination of subgroups of patients
at both low and high
risk for developing metastatic disease (Duncan, L.M. et al (2001) J. Clip.
Oncol. 19:568-576).
Chloride channels are necessary in endocrine secretion and in regulation of
cytosolic and
organelle pH. In secretory epithelial cells, Cl- enters the cell across a
basolateral membrane through
an Na+, K+/Cl- cotransporter, accumulating in the cell above its
electrochemical equilibrium
concentration. Secretion of Cl- from the apical surface, in response to
hormonal stimulation, leads to
flow of Na+ and water into the secretory lumen. The cystic fibrosis
trausmembrane 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 trausepithelial 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) Curx. 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 neuxotrausmitters, 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,
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such as y-axninobutyric acid (GABA) and glycine, leading to hyperpolarization
of the membrane and
the subsequent generation of an action potential. Neurotransmitter-gated ion
channels have four
transmembrane domains and probably function as pentamers (Jentsch, su ra).
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 Iarge conductance (BK) channel has been purified from brain and
its subunit composition
determined. The a subunit of the BK channel has seven rather than six
transmembrane domains in
contrast to voltage-gated K+ channels. The extra transmembrane domain is
located at the subunit N-
terminus. A 28-amino-acid stretch in the C-terminal region of the subunit (the
"calcium bowl" region)
contains many negatively charged residues and is thought to be the region
responsible for calcium
binding. The (3 subunit consists of two transmembrane domains connected by a
glycosylated
extracellular loop, with intracellular N- and C-termini (Kaczorowski, su ra;
Vergara, C. et al. (1998)
Curr. Opin. Neurobiol. 8:321-329).
Cyclic nucleotide-gated (CNG) channels are gated by cytosolic cyclic
nucleotides. The best
examples of these are the cAMP-gated Na + channels involved in olfaction and
the cGMP-gated
cation channels involved in vision. Both systems involve ligand-mediated
activation of a G-protein
coupled receptor which then alters the level of cyclic nucleotide within the
cell. CNG chanuels also
represent a major pathway for Caa+ entry into neurons, and play roles in
neuronal development and
plasticity. CNG channels are tetramers containing at least two types of
subunits, au 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 trausmembrane 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 othex types of ion channel proteins may also be modulated by a
variety of
intracellular signalling proteins. Many channels have sites for
phosphorylation by one or more protein
kinases including protein kinase A, protein kinase C, tyrosine kinase, and
casein kinase II, all of which
regulate ion channel activity in cells. Kir channels are activated by the
binding of the G(3y subunits of
heterotrimeric G-proteins (Reimann, F. and F.M. Ashcroft (1999) Curr. Opin.
Cell. Biol. 11:503-508).
Other proteins are involved in the localization of ion channels to specific
sites in the cell membrane.
Such proteins include the PDZ domain proteins known as MAGUKs (membrane-
associated guanylate
CA 02422497 2003-03-14
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kinases) which regulate the clustering of ion channels at neuronal synapses
(Craven, S.E. and D.S.
Bredt (1998) CeII 93:495-498).
Disease Correlation
The etiology of numerous human diseases and disorders can be attributed to
defects in the
transport of molecules across membranes. Defects in the trafficking of
membrane-bound transporters
and ion channels are associated with several disorders, e.g., cystic fibrosis,
glucose-galactose
malabsorption syndrome, hypercholesterolemia, von Gierke disease, and certain
forms of diabetes
mellitus. Single-gene defect diseases resulting in an inability to transport
small molecules across
membranes include, e.g., cystinuria, iminoglycinuria, Harrup 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,
2o 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) C~rr.
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. (I999) Curr. Opin.
Neurobiol. 9:274-280; Cooper, supra).
Ion channels have been the target for many drug therapies. Neurotransmitter-
gated channels
have been targeted in therapies for treatment of insomnia, anxiety,
depression, and schizophrenia.
Voltage-gated channels have been targeted in therapies for arrhythmia,
ischemic stroke, head trauma,
and neurodegenerative disease (Taylor, C.P. and L.S. Narasimhan (1997) Adv.
Pharmacol. 39:47-98).
Various classes of ion channels also play an important role in the perception
of pain, and thus are
potential targets for new analgesics. These include the vanilloid-gated ion
channels, which are
activated by the vanilloid capsaicin, as well as by noxious heat. Local
anesthetics such as lidocaine
and mexiletine which blockade voltage-gated Na+ channels have been useful in
the treatment of
neuropathic pain (Eglen, su ra).
Ion channels in the immune system have recently been suggested as targets for
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immunomodulation. T-cell activation depends upon calcium signaling, and a
diverse set of T-cell
specific ion channels has been characterized that affect this signaling
process. Channel blocking
agents can inhibit secretion of lymphokines, cell proliferation, and killing
of target cells. A peptide
antagonist of the T-cell potassium channel Kvl.3 was found to suppress delayed-
type hypersensitivity
and allogenic responses in pigs, validating the idea of channel blockers as
safe and e~cacious
immunosuppressants (Cahalan, M.D. and K.G. Chandy (1997) Curr. Opin.
Biotechnol. 8:749-756).
The discovery of new transporters and ion channels, and the polynucleotides
encoding them,
satisfies a need in the art by providing new compositions which are useful in
the diagnosis, prevention,
and treatment of transport, neurological, muscle, immunological, and cell
proliferative disorders, and in
the assessment of the effects of exogenous compounds on the expression of
nucleic acid and amino
acid sequences of transporters and ion channels.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, transporters and ion channels,
referred to
collectively as "TRICIT' and individually as "TRICH-1," "TRICH-2,""TRICH-3,"
"TRICH-4,"
"TRICH-5," "TRICH-6," "TRICH-7," "TRICH-8," "TRICH-9," "TRICH-10," "TRICH-11,"
"TRICH-12," "TRICH-13," "TRICH-14," "TRICH-15," "TRICH-16," "TRICH-17," "TRTCH-
18,"
"TRICH-19," "TRICH-20," "TRICH-21," "TRICH-22," "TRICH-23," "TRICH-24," "TRTCH-
25,"
and "TRICH-26." In one aspect, the invention provides an isolated polypeptide
selected from the
group consisting of a) a polypeptide comprising an amino acid sequence
selected from the group
consisting of SEQ )D N0:1-26, 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 >D N0:1-
26, c) a biologically active fragment of a polypeptide having an amino acid
sequence selected from the
group consisting of SEQ )D NO:1-26, and d) an immunogenic fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ )D N0:1-26. In
one alternative, the
invention provides an isolated polypeptide comprising the amino acid sequence
of SEQ ID NO:1-26.
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 m N0:1-26, 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 LD N0:1-
26, c) a biologically active fragment of a polypeptide having an amino acid
sequence selected from the
group consisting of SEQ ID NO:1-26, and d) an immunogenic fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ DJ N0:1-26. In
one alternative, the
polynucleotide encodes a polypeptide selected from the group consisting of SEQ
ID N0:1-26. In
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another alternative, the polynucleotide is selected from the group consisting
of SEQ ID N0:27-52.
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 N0:1-26, 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 N0:1-26, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ m NO:1-26, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ )D NO:l-26. Iu 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 >D N0:1-26, 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:1-26, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ )~ N0:1-26, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ >D N0:1-26. The method
comprises a)
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 m NO:1-26, 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 )D N0:1-26, c) a biologically active fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ m N0:1-26, and
d) an immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
m NO:1-26.
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
)D N0:27-52, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ 1D N0:27-52,
c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide complementary to
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the polynucleotide of b), and e) an RNA equivalent of a)-d). In one
alternative, the polynucleotide
comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide selected from
the group consisting of
a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of SEQ
ID N0:27-52, 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 NO:27-52,
c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide complementary to
the polynucleotide of b), and e) an RNA equivalent of a)-d). 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
N0:27-52, b) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ m N0:27-52, c) a
polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
complementary to the
polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises
a) amplifying said
target polynucleotide or fragment thereof using polymerise 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 N0:1-26, 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:1-26, c) a biologically active fragment of a
polypeptide having au amino acid
sequence selected from the group consisting of SEQ ID N0:1-26, and d) an
immunogenic fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ lD N0:1-26,
and a pharmaceutically acceptable excipient. In one embodiment, the
composition comprises an amino
acid sequence selected from the group consisting of SEQ ~ N0:1-26. The
invention additionally
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provides a method of treating a disease or condition associated with decreased
expression of
functional TRICH, comprising administering to a patient in need of such
treatment the composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ )D N0:1-26, 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 N0:1-26, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ )Z7
N0:1-26, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ m N0:1-26. 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 )D N0:1-26, 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 )D N0:1-26, c) a
biologically active fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ 1D NO:1-26,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ >D N0:1-26. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting 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 )D NO:1-26, 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 N0:1-26, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ )D
NO:1-26, and d) an
CA 02422497 2003-03-14
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i_m_m__unoge111C fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ m N0:1-26. The method comprises a) combining the polypeptide
with at least one
test compound under suitable conditions, and b) detecting binding of the
polypeptide to the test
compound, thereby identifying a compound that specifically binds to the
polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ m NO:1-26, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ 1D N0:1-26, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected frorri the group consisting of SEQ m
N0:1-26, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ m N0:1-26. The method comprises a) combining the polypeptide
with at least one
test compound under conditions permissive for the activity of the polypeptide,
b) assessing the activity
of the polypeptide in the presence of the test compound, and c) comparing the
activity of the
polypeptide in the presence of the test compound with the activity of the
polypeptide in the absence of
the test compound, wherein a change in the activity of the polypeptide in the
presence of the test
compound is indicative of a compound that modulates the activity of the
polypeptide.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
polynucleotide sequence selected from the group consisting of SEQ ~ N0:27-52,
the method
comprising a) exposing a sample comprising the target polynucleotide to a
compound, and b) detecting
altered expression of the taxget 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 N0:27-52, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ m N0:27-52,
iii) a polynucleotide
having a sequence complementary to i), iv) a polynucleotide complementary to
the polynucleotide of
ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions
whereby a specific
hybridization complex is formed between said probe and a target polynucleotide
in the biological
sample, said target polynucleotide selected from the group consisting of i) a
polynucleotide comprising
a polynucleotide sequence selected from the group consisting of SEQ ff~ N0:27-
52, ii) a
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polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ )D N0:27-52,
iii) a polynucleotide
complementary to the polynucleotide of i), iv) a polynucleotide complementary
to the polynucleotide of
ii), and v) an RNA equivalent of i)-iv). Alternatively, the target
polynucleotide comprises a fragment
of a polynucleotide sequence selected from the group consisting of i)-v)
above; c) quantifying tha
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 Iists the cDNA andlor 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.
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It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods are now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing the
cell lines, protocols, reagents and vectors which are reported in the
publications and which might be
used in connection with the invention. Nothing herein is to be construed as an
admission that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
DEFINITIONS
"TRICH" refers to the amino acid sequences of substantially purified TRICH
obtained from
any species, particularly a mammalian species, including bovine, ovine,
porcine, 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.
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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 TR1CH.
Deliberate amino acid substitutions may be made on the basis of similarity in
polarity, charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues,
as long as the biological
or immunological activity of TRICH is retained. For example, negatively
charged amino acids may
include aspartic acid and glutamic acid, and positively charged amino acids
may include lysine and
arginine. Amino acids with uncharged polar side chains having similar
hydrophilicity values may
include: asparagine and glutamine; and serine and threonine. Amino acids with
uncharged side chains
having similar hydrophilicity values may include: leucine, isoleucine, and
valine; glycine and alanine;
and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally 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 polymerise chain reaction (PCR)
technologies well known
in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of TRICH. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of TRICH either by
directly interacting with TRICH or by acting on components of the biological
pathway in which
TRICH participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')Z, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind TRICH polypeptides can be prepared using intact
polypeptides or using fragments
containing small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide used
to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from
the translation of RNA,
or synthesized chemically, and can be conjugated to a carrier protein if
desired. Commonly used
carriers that are chemically coupled to peptides include bovine serum albumin,
thyroglobuliu, and
keyhole limpet hemocyanin (KT .Tal. 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
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which bind specifically to antigenic determinants (particular regions or three-
dimensional structures on
the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen used
to elicit the immune response) for binding to an antibody.
The term "aptamer" refers to a nucleic acid or oligonucleotide molecule that
binds to a
specific molecular target. Aptamers are derived from an in vitro evolutionary
process (e.g., SELEX
(Systematic Evolution of Ligands by EXponential Enrichment), described in U.S.
Patent No.
5,270,163), which selects for target-specific aptamer sequences from large
combinatorial libraries.
Aptamer compositions may be double-stranded or single-stranded, and may
include
deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other
nucleotide-like molecules. The
nucleotide components of an aptamer may have modified sugar groups (e.g., the
2'-OH group of a
ribonucleotide may be replaced by 2'-F or 2'-NHZ), which may improve a desired
property, e.g.,
resistance to nucleases or longer lifetime in blood. Aptamers may be
conjugated to other molecules,
e.g., a high molecular weight carrier to slow clearance of the aptamer from
the circulatory system.
Aptamers may be specifically cross-linked to their cognate ligands, e.g., by
photo-activation of a
cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-
13.)
The term "intramer" refers to an aptamer which is expressed in vivo. For
example, a vaccinia
virus-based RNA expression system has been used to express specific RNA
aptamers at high levels
in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl Acad. Sci.
USA 96:3606-3610).
The term "spiegelmer" refers to an aptamer which includes L-DNA, L-RNA, or
other left
handed nucleotide derivatives or nucleotide-like molecules. Aptamers
containing left handed
nucleotides are resistant to degradation by naturally occurring enzymes, which
act on right-handed
nucleotides.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA; RNA;
peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages
such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
designation "positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
CA 02422497 2003-03-14
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functions of a naturally occurring molecule. Likewise, "immunologically
active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic TRICH, or
of any oligopeptide thereof,
to induce a specific immune response in appropriate animals or cells and to
bind with specific
antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising a
given amino acid sequence" refer broadly to any composition containing the
given polynucleotide or
amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding TRICH or fragments
of TRICH may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., 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 5' and/or the 3' direction, and
resequenced, or which has been
assembled from one or more overlapping cDNA, EST, or genomic DNA fragments
using a computer
program for fragment assembly, such as the GELV~W fragment assembly system
(GCG, Madison
WI) or Phrap (University of Washington, Seattle WA). Some sequences have been
both extended
and assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of the
protein is conserved and not significantly changed by such substitutions. The
table below shows amino
acids which may be substituted for an original amino acid in a protein and
which are regarded as
conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
3o Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
~s Asn, Arg, Gln, Glu
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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
Tyx 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" refexs to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide can include, for example,
replacement of hydrogen by an
alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a
polypeptide which retains
at least one biological or immunological function of the natural molecule. A
derivative polypeptide is
one modified by glycosylation, pegylation, or any similar process that retains
at least one biological or
immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
"Differential expression" refers to increased or upregulated; or decreased,
downregulated, or
absent gene or protein expression, determined by comparing at least two
different samples. Such
comparisons may be carried out between, for example, a treated and an
untreated sample, or a
diseased and a normal sample.
"Exon shuffling" refers to the recombination of different coding regions
(exons). Since an
exon may represent a structural or functional domain of the encoded protein,
new proteins may be
assembled through the novel reassortment of stable substructures, thus
allowing acceleration of the
evolution of new protein functions.
A "fragment" is a unique portion of TRICH or the polynucleotide encoding TRICH
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example, a
fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
22
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used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10, 15,
16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fxagments may be preferentially selected from certain
regions.of a molecule. For
example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ II7 N0:27-52 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:27-52, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:27-52 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish SEQ
ID N0:27-52 from related polynucleotide sequences. The precise length of a
fragment of SEQ )D
N0:27-52 and the region of SEQ ID N0:27-52 to which the fragment corresponds
are routinely
determinable by one of ordinary skill in the art based on the intended purpose
for the fragment.
A fragment of SEQ ID NO:1-26 is encoded by a fragment of SEQ ID N0:27-52. A
fragment of SEQ ID N0:1-26 comprises a region of unique amino acid sequence
that specifically
identifies SEQ ID N0:1-26. For example, a fragment of SEQ ID N0:1-26 is useful
as an
immunogenic peptide for the development of antibodies that specifically
recognize SEQ )D N0:1-26.
. The precise length of a fragment of SEQ ID NO:1-26 and the region of SEQ ID
NO:1-26 to which
the fragment corresponds are routinely determinable by one of ordinary skill
in the art based on the
intended purpose for the fragment.
A "full length" polynucleotide sequence is one containing at least a
translation initiation codon
(e.g., methionine) followed by an open reading frame and a translation
termination codon. A "full
length" polynucleotide sequence encodes a "full length" polypeptide sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between two
or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide
sequences, refer to
the percentage of residue matches between at least two polynucleotide
sequences aligned using a
3o 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
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WO 02/22684 PCT/USO1/28938
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The
"weighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
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.html. The
"BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the 'BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty fof- mismatch: -2
Open Gz~p: S arad Exterasion Gap: 2 penalties
Gap x df-op-off. 50
Expect: 10
Word Size: 11
Filter-: oh
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ )D number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined 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
24
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WO 02/22684 PCT/USO1/28938
length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some alignment
methods take into account conservative amino acid substitutions. Such
conservative substitutions,
explained in more detail above, generally preserve the charge and-
hydrophobicity at the site of
substitution, thus preserving the structure (and therefore function) of the
polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version
2Ø12 (April-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matfzx: BLOS UM62
Open Gap: 11 afad Exte~asiofa Gap: 1 peyaalties
Gap x drop-off. 50
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ JD 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 1S, 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
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
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
r
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
IO 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 oxdinary skill in
the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
1% (w/v) SDS, and about 100 ~Cg/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.
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 ~Cg/ml. Organic
solvent, such as
formamide at a concentration of about 35-50% v/v, may also be used under
particular circumstances,
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WO 02/22684 PCT/USO1/28938
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., cytokiues, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide ~or oligopeptide fragment of TRICH
which is
capable of eliciting an immune response when introduced into a living
organism, for example, a
mammal. The term "immunogenic fragment" also includes any polypeptide or
oligopeptide fragment of
TRICH which is useful in any of the antibody production methods disclosed
herein or known in the art.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides,
polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of TRICH. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other biological,
functional, or immunological properties of TRICH.
. The phrases "nucleic acid" and "nucleic acid sequence" refer to a
nucleotide, oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably licked" 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
27
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WO 02/22684 PCT/USO1/28938
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 auti-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, Iigands, chemiluminescent agents,
and enzymes. "Primers"
are short nucleic acids, usually DNA oligonucleotides, wluch 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, 2"d
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Iunis, M. et
al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1992, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
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WO 02/22684 PCT/USO1/28938
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primex pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to 5,000
nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection
programs have incorporated additional features for expanded capabilities. For
example, the PrimOU
primer selection program (available to the public from the Genome Center at
University of Texas
South West Medical Center, Dallas TX) is capable of choosing specific primers
from megabase
sequences and is thus useful for designing primers on a genome-wide scope. The
Primer3 primer
selection program (available to the public from the Whitehead Institute/MTT
Center for Genome
Research, Cambridge MA) allows the user to input a "mispriming libxary," in
which sequences to
avoid as primer binding sites are user-specified. Primer3 is useful, in
particular, for the selection of
oligonucleotides for microarrays. (The source code for the latter two primer
selection programs may
also be obtained from their respective sources and modified to meet the user's
specific needs.) The
PrimeGen program (available to the public from the UK Human Genome Mapping
Project Resource
Centre, Cambridge UK) designs primers based on multiple sequence alignments,
thereby allowing
selection of primers that hybridize to either the most conserved or least
consexved regions of aligned
nucleic acid sequences. Hence, this program is useful for identification of
both unique and conserved
oligonucleotides and polynucleotide fragments. The oligonucleotides and
polynucleotide fragments
identified by any of the above selection methods are useful in hybridization
technologies, for example,
as PCR or sequencing primers, microarray elements, or specific probes to
identify fully or partially
complementary polynucleotides in a sample of nucleic acids. Methods of
oligonucleotide selection are
not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, 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.
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
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CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
(UTRs). Regulatory elements interact with host or viral proteins which contxol
transcription,
translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of
the nitrogenous base thymine are replaced with uracil, and the sugar backbone
is composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing TRICH,
nucleic acids encoding TRICH, or fragments thereof may comprise a bodily
fluid; an extract from a
cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic
DNA, RNA, or cDNA,
in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular structure
of the protein, e.g., the antigenic determinant or epitope, recogxtized by the
binding molecule. For
example, if an antibody is specific for epitope "A," the presence of a
polypeptide comprising the
epitope A, or the presence of free unlabeled A, in a reaction containing free
labeled A and the
antibody will reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 75% free, and most preferably at least 90% free from other
components with
which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides
by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" 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
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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
2o and provided in references such as Sambrook et al. (1989), su ra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99% or greater
sequence identity over a certain defined length. A variant may be described
as, for example, an
"allelic" (as defined above), "splice," "species," or "polymorphic" variant. A
splice variant may have
significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternate splicing of exons during mRNA processing. The
corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotide sequences that vary
from one species to
another. The resulting polypeptides will generally have significant amino acid
identity relative to each
other. A polymorphic variant is a variation in the polynucleotide sequence of
a particular gene
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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 SO%, 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 identih.cation number (Incyte Project ID). Each
polypeptide sequence is denoted
by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:)
and au Incyte
polypeptide sequence number (Incyte Polypeptide H~) as shown. Each
polynucleotide sequence is
denoted by both a polynucleotide sequence identification number
(Polynucleotide SEQ 117 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 aualysis 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 D7 NO:) of the nearest
GenBank homolog.
Column 4 shows the probability score for the match between each polypeptide
and its GenBank
homolog. Column 5 shows the annotation of the GenBank homolog along with
relevant citations
where applicable, all of which are expressly incorporated by reference hexein.
Table 3 shows various structural features of the polypeptides of the
invention. Columns 1 and
2 show the polypeptide sequence identification number (SEQ 117 NO:) and the
corresponding Incyte
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polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of
the invention. Column
3 shows the number of amino acid residues in each polypeptide. Column 4 shows
potential
phosphorylation sites, and column 5 shows potential glycosylation sites, as
determined by the MOTIFS
program of the GCG sequence analysis software package (Genetics Computer
Group, Madison WI).
Column 6 shows amino acid residues comprising signature sequences, domains,
and motifs. Column 7
shows analytical methods for protein structure/function analysis and in some
cases, searchable
databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the
invention, and these
properties establish that the claimed polypeptides are transporters and ion
channels. For example,
SEQ ID N0:2 is 94% identical from amino acids 965 through 2436 to mouse abc2
transporter
(GenBank ID 8495259) as determined by the Basic Local Alignment Search Tool
(BLAST). (See
Table 2.) The BLAST probability score is 0.0, which indicates the probability
of obtaining the
observed polypeptide sequence alignment by chance. SEQ )I? N0:2 also contains
two ABC
transporter domains as determined by searching for statistically significant
matches in the hidden
Markov model (IBVVIM) based PFAM database of conserved protein family domains.
(See Table 3.)
Data from MOTIFS, and PROFILESCAN analyses provide further corroborative
evidence that SEQ
ID N0:3 is an ABC transporter. In an alternate example, SEQ 177 N0:13 is 97%
identical to human
gamma subunit precursor of muscle acetylcholine receptor (GenBank ID 8825618)
as determined by
the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST
probability score is
3.0e-273, which indicates the probability of obtaining the observed
polypeptide sequence alignment by
chance. SEQ lD N0:13 also contains a neurotransmitter-gated ion-channel domain
as determined by
searching for statistically significant matches in the hidden Markov model
(I1MM)-based PFAM
database of conserved protein family domains. (See Table 3.) Data from BLIMPS,
MOTIFS, and
PROFILESCAN analyses provide further corroborative evidence that SEQ lD N0:13
is a
neurotransmitter-gated ion-channel protein. In an alternate example, SEQ ID
N0:19 is 62% identical
to human vacuolar proton-ATPase (GenBank ID 837643) as determined by the Basic
Local
Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is
3.2e-129, which
indicates the probability of obtainixtg the observed polypeptide sequence
alignment by chance. Data
from BLAST analyses provide further corroborative evidence that SEQ ID NO:19
is a vacuolar ATP
synthase. In an alternate exampple, SEQ ID N0:22 is 94% identical to rat
GABA(A) receptor
gamma-1 subunit (GenBank 1D 856176) as determined by the Basic Local Alignment
Search Tool
(BLAST). (See Table 2.) The BLAST probability score is 4.4e-244, which
indicates the probability
of obtaining the observed polypeptide sequence alignment by chance. SEQ ID
N0:22 also contains a
neurotransmitter-gated ion channel domain as determined by searching for
statistically significant
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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 N0:22 is a neurotransmitter-gated
ion channel. In an
alternate example, SEQ m N0:26 is 61 % identical to rabbit peroxisomal Ca-
dependent solute carrier
(GenBank ID g2352427) as determined by the Basic Local Alignment Search Tool
(BLAST). (See
Table 2.) The BLAST probability score is 6.4e-156, which indicates the
probability of obtaining the
observed polypeptide sequence alignment by chance. SEQ ID N0:26 also contains
three
mitochondria) carrier protein domains, as well as three EF hand domains, as
determined by searching
for statistically significant matches in the hidden Markov model (HMM)-based
PFAM database of
conserved protein family domains. (See Table 3.) Data from BLIIVVIPS, MOTIFS,
and
PROFILESCAN analyses provide further corroborative evidence that SEQ ID N0:26
is a calcium
dependent carrier protein. In an alternate examplpe, SEQ LD N0:17 is 69%
identical to Ambystorna
tigr~inum electrogenic NaHC03 cotransporter (GenBank )D g2198815) 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 N0:17 also contains an HC03 transporter family domain as determined by
searching for
statistically significant matches in the hidden Markov model (HLVnVI)-based
PFAM database of
conserved protein family domains. (See Table 3.) Data from BLIZVVIPS, MOTIFS,
and
PROFILESCAN analyses provide further corroborative evidence that SEQ ID N0:17
is an anion
transporter. SEQ LD N0:1, SEQ ID N0:3-12, SEQ ID N0:14-16, SEQ ID N0:18, and
SEQ ID
N0:20-25 were analyzed and annotated in a similar manner. The algorithms and
parameters for the
analysis of SEQ LD N0:1-26 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 ~ 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 LD N0:27-52 or that distinguish between SEQ ID
N0:27-52 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')
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and stop (3') positions of the cDNA and/or genomic sequences in column 5
relative to their respective
full length sequences.
The identification numbers in Column 5 of Table 4 may refer specifically, for
example, to
Incyte cDNAs along with their corresponding cDNA libraries. For example,
7251266F7 is the
identification number of an Incyte cDNA sequence, and PROSTMY01 is the cDNA
library from
which it is derived. Incyte cDNAs for which cDNA libraries are not indicated
were derived from
pooled cDNA libraries (e.g., 70564238V1). Alternatively, the identification
numbers in column 5 may
refer to GenBank cDNAs or ESTs (e.g., g4689801) 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, ITK)
database (i.e., those
sequences including the designation "ENST"). Alternatively, the identification
numbers in column 5
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 5 may refer to assemblages of both cDNA and Genscan-predicted exons
brought together by
au "exon stitching" algorithm. For example, FL_~'~~XXXX Nl Nz_YYYYY_N3 Nø
represents a
"stitched" sequence in which XXXXXX is the identification number of the
cluster of sequences to
which the algorithm was applied, and YYYYY is the number of the prediction
generated by the
algorithm, and N1,2,3..., if present, represent specific exons that may have
been manually edited during
2o analysis (See Example V). Alternatively, the identification numbers in
column 5 may refer to
assemblages of exons brought together by au "exon-stretching" algorithm. For
example,
FT-X_XXXXX_gAAAAA~BBBBB_1 IV 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 NCBI RefSeq identification
number of the
nearest GenBank protein homolog, and N referring to specific exons (See
Example V). Iu 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).
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Prefix Type of analysis andlor examples of programs
GNN, GFG, Exon prediction from genomic sequences using,
for example,
ENST GENSCAN (Stanford University, CA, USA) or
FGENES
(Computer Genomics Group, The Sanger Centre,
Cambridge, UK)
GBI Hand-edited analysis of genomic sequences.
FL Stitched or stretched genomic sequences
(see Example V).
INCY Full length transcript and axon prediction
from mapping of EST
sequences to the genome. Genomic location
and EST composition
data are combined to predict the axons and
resulting transcript.
In some cases, Incyte cDNA coverage redundant with the sequence coverage shown
in
column 5 was obtained to confirm the final consensus polynucleotide sequence,
but the relevant Incyte
cDNA identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length
polynucleotide
sequences which were assembled using Incyte cDNA sequences. The representative
cDNA library
is the Incyte cDNA library which is most frequently represented by the Incyte
cDNA sequences
which were used to assemble and confirm the above polynucleotide sequences.
The tissues and
vectors which were used to construct the cDNA libraries shown in Table 5 are
described in Table 6.
The invention also encompasses TRICH variants. A preferred TRICH variant is
one which
has at least about 80%, or alternatively at least about 90%, or even at least
about 95% amino acid
sequence identity to the TRICH amino acid sequence, and which contains at
least one functional or
structural characteristic of TRICH.
The invention also encompasses polynucleotides which encode TRICH. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:27-52, which encodes TRICH. The
polynucleotide
sequences of SEQ 1D N0:27-52, as presented in the Sequence Listings 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 N0:27-
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52 which has at least about 70%, or alternatively at least about 85%, or even
at least about 95%
polynucleotide sequence identity to a nucleic acid sequence selected from the
group consisting of SEQ
ID N0:27-52. 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 axe 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 rata at which
expression of the peptide
occurs in a particular prokaryotic or eukaryotic host in accordance with the
frequency with which
particular codons are utilized by the host. Other reasons for substantially
altering the nucleotide
sequence encoding TRICH and its derivatives without altering the encoded amino
acid sequences
include the production of RNA transcripts having more desirable properties,
such as a greater half life,
than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode TRICH
and
TRICH derivatives, or fragments thereof, entirely by synthetic chemistry.
After production, the
synthetic sequence may be inserted into any of the many available expression
vectors and cell systems
using reagents well known in the art. Moreover, synthetic chemistry may be
used to introduce
mutations into a sequence encoding TRICH or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
N0:27-52 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; IKimmel, A.R. (1987) Methods
Enzymol. 152:507
511.) Hybridization conditions, including annealing and wash conditions, are
described in "Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
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of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(Applied
Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerases and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
(Applied Biosystems). Sequencing is then carried out using either the ABI 373
or 377 DNA
sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing
system
(Molecular Dynamics, SunnyVale CA), or other systems known in the art. The
resulting sequences
are analyzed using a variety of algorithms which are well known in the art.
(See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY,
unit 7.7; Meyexs,
R.A. (1995) Molecular Biolo~y and Biotechnolo~y, Wiley VCH, New York NY, pp.
856-853.)
The nucleic acid sequences encoding TRICH may be extended utilizing a partial
nucleotide
sequence and employing various PCR based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unknown
sequence from a circularized template. The template is derived from
restriction fragments comprising
a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et
al. (1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments adjacent
to known sequences in human and yeast artificial chromosome DNA. (See, e.g.,
Lagerstrom, M. et
al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme digestions and
ligations may be used to insert an engineered double-stxanded sequence into a
region of unknown
sequence before performing PCR. Other methods wluch 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 PROMOTERFTNDER libraries
(Clontech, Palo
Alto CA) to walk genomic DNA. 'This procedure avoids the need to screen
libraries and is useful in
finding intron/exon junctions. For all PCR-based methods, primers may be
designed using
commercially available software, such as OLIGO 4.06 primer analysis software
(National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30
nucleotides in length,
to have a GC content of about 50% or more, and to anneal to the template at
temperatures of about
68°C to 72°C.
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When screening for full length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5'regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence
into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
I5 In another embodiment of the invention, polynucleotide sequences or
fragments thereof which
encode TRICH may be cloned in recombinant DNA molecules that direct expression
of TRICH, or
fragments or functional equivalents thereof, in appropriate host cells. Due to
the inherent degeneracy
of the genetic code, other DNA sequences which encode substantially the same
or a functionally
equivalent amino acid sequence may be produced and used to express TRICH.
The nucleotide sequences of the present invention can be engineered using
methods generally
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 anal
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction
sites, alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
No.
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
3o 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
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WO 02/22684 PCT/USO1/28938
preferred variants may then be pooled and further subjected to recursive
rounds of DNA shuffling and
selection/screening. Thus, genetic diversity is created through "artificial"
breeding and rapid molecular
evolution. For example, fragments of a single gene containing random point
mutations may be
recombined, screened, and then reshuffled until the desired properties are
optimized. Alternatively,
fragments of a given gene may be recombined with fragments of homologous genes
in the same gene
family, either from the same or different species, thereby maximizing the
genetic diversity of multiple
naturally occurring genes in a directed and controllable manner.
In another embodiment, sequences encoding TRICH may be synthesized, in whole
or in part,
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
al. (1980) Nucleic Acids
Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.) Alternatively,
TRICH itself or a fragment thereof may be synthesized using chemical methods.
For example,
peptide synthesis can be performed using various solution-phase or solid-phase
techniques. (See, e.g.,
Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH
Freeman, New York NY, pp.
55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated
synthesis may be achieved
using the ABI 431A peptide synthesizer (Applied Biosystems). Additionally, the
amino acid sequence
of TRICH, or any part thereof, may be altered during direct synthesis and/or
combined with
sequences from other proteins, or any part thereof, to produce a variant
polypeptide or a polypeptide
having a sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by sequencing.
(See, e.g., Creighton, supra, pp. 28-53.)
In order to express a biologically active TRICH, the nucleotide sequences
encoding TRICH or
derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which contains
the necessary elements for transcriptional and translational control of the
inserted coding sequence in
a suitable host. These elements include regulatory sequences, such as
enhancers, constitutive and
inducible promoters, and 5' and 3' untranslated regions in the vector and in
polynucleotide sequences
encoding TRICH. Sueh 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
CA 02422497 2003-03-14
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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 Biology, John Wiley & Sons, New York NY, ch. 9,
13, and 16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding TRICH. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, ox cosmid DNA expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV, or
tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or
animal cell systems. (See, e.g., Sambrook, supra; Ausubel, supra; Van Heeke,
G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl.
Acad. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu,
N. (1987) EMBO
J. 6:307-311; The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw
Hill, New
York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA
81:3655-3659; and
Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors
derived from retroviruses,
adenoviruses, or herpes or vaccinia viruses, or from various bacterial
plasmids, may be used for
delivery of nucleotide sequences to the targeted organ, tissue, or cell
population. (See, e.g., Di Nicola,
M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc.
Natl. Acad. Sci. USA
90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al. (1994)
Mol. Tmmunol. 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 PBLUESCR1PT (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
41
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WO 02/22684 PCT/USO1/28938
transformed bacteria containing recombinant molecules. In addition, these
vectors may be useful for
iu 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 Saccharomyces cereyisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable integration
of foreign sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, 1995, supra;
Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et
al. (1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of TRICH. Transcription of
sequences
encoding TRICH may be driven by viral promoters, e.g., the 355 and 195
promoters of CaMV used
alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-
1680; Brogue, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105.) These
constructs can be introduced into plant cells by direct DNA transformation or
pathogen-mediated
transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technolo~y
(1992) McGraw Hill,
New York NY, pp. 191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding TRICH
may be ligated into
an adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses TRICH in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
Natl. Acad. Sei. 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,
42
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J. et al.
(1997) Nat. Genet. 15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression of
TRICH in cell lines is preferred. For example, sequences encoding TRICH can be
transformed into
cell lines using expression vectors which may contain viral origins of
replication andlor endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media before
being switched to selective media. The purpose of the selectable marker is to
confer resistance to a
selective agent, and its presence allows growth and recovery of cells which
successfully express the
introduced sequences. Resistant clones of stably transformed cells may be
propagated using tissue
cultuxe techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk and apr' cells, respectively.
(See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic, or
herbicide resistance can be used as the basis for selection. For example,
dltfr confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. (1981)
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., ttpB and hisD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. anal
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 l3-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
trausformants, but also to
quautify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest
is also present, the presence and expression of the gene may need to be
confirmed. For example, if
the sequence encoding TRICH is inserted within a marker gene sequence,
trausformed cells
containing sequences encoding TRICH can be identified by the absence of marker
gene function.
Alternatively, a marker gene can be placed iu 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
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WO 02/22684 PCT/USO1/28938
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 pxotein sequences.
T__m_munological 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 (FRCS). 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,
to 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) Tm_m__unochemical
Protocols, Humana
Press, Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled hybridization
or PCR probes for detecting sequences related to polynucleotides encoding
TRICH include
oligolabeling, nick translation, end-labeling, or PCR amplification using a
labeled nucleotide.
Alternatively, the sequences encoding TRICH, or any fragments thereof, may be
cloned into a vector
for the production of an mRNA probe. Such vectors are known in the art, are
commercially available,
and may be used to synthesize RNA probes in vitro by addition of an
appropriate RNA polymerase
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
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CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
lipidation, and acylation. Post-translational processing which cleaves a
"prepro" or "pro" form of the
protein may also be used to specify protein targeting, folding, and/or
activity. Different host cells
which have specific cellular machinery and characteristic mechanisms for post-
translational activities
(e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type
Culture
Collection (ATCC, Manassas VA) and may be chosen to ensure the correct
modification and
processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding TRICH may be Iigated to a heterologous sequence resulting
in translation of a
fusion protein in any of the aforementioned host systems. For example, a
chimeric TRICH protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of TRICH
activity. Heterologous protein and
peptide moieties may also facilitate purification of fusion proteins using
commercially available affinity
matrices. Such moieties include, but are not limited to, glutathione S-
transferase (GST), maltose
binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-
His, FLAG, c-myc, and
hemagglutinin. (HA). GST, MBP, Trx, CBP, and 6-His enable purification of
their cognate fusion
proteins on immobilized glutathione, maltose, phenylarsine oxide, calinodulin,
and metal-chelate resins,
respectively. FLAG, c-rnyc, and hemagglutinin (HA) enable immunoaffinity
purification of fusion
proteins using commercially available monoclonal and polyclonal antibodies
that specifically recognize
these epitope tags. A fusion protein may also be engineered to contain a
proteolytic cleavage site
located between the TRICH encoding sequence and the heterologous protein
sequence, so that
TRICH may be cleaved away from the heterologous moiety following purification.
Methods for
fusion protein expression and purification are discussed in Ausubel (1995, su
ra, 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
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current
Protocols in Itmnunolo~y 1(2):
Chapter S.) Similarly, the compound can be closely related to the natural
receptor to which TRICH
binds, or to at least a fragment of the receptor, e.g., the ligand binding
site. In either case, the
compound can be rationally designed using known techniques. In one embodiment,
screening for
these compounds involves producing appropriate cells which express TRICH,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or E.
coli. Cells expressing TRICH or cell membrane fractions which contain TRICH
are then contacted
with a test compound and binding, stimulation, or inhibition of activity of
either TRICH or the
compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide,
wherein binding is
detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable
label. For example, the
assay may comprise the steps of combining at least one test compound with
TRICH, either in solution
or affixed to a solid support, and detecting the binding of TRICH to the
compound. Alternatively, the
assay may detect or measure binding of a test compound in the presence of a
labeled competitor.
Additionally, the assay may be carried out using cell-free preparations,
chemical libraries, or natural
product mixtures, and the test compounds) may be free in solution or affixed
to a solid support.
TRICH of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of TRICH. Such compounds may include agonists,
antagonists, or partial or
inverse agonists. In one embodiment, an assay is performed under conditions
permissive for TRICH
activity, wherein TRICH is combined with at least one test compound, and the
activity of TRICH in
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 eithex of these assays, a test compound which modulates
the activity of
TRICH may do so indirectly and need not come in direct contact with the test
compound. At least
one and up to a plurality of test compounds may be screened.
In another embodiment, polynucleotides encoding TRICH or their mammalian
homologs may
be "knocked out" in an animal model system using homologous recombination in
embryonic stem (ES)
cells. Such techniques are well known in the art and are useful for the
generation of anumal models of
human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No.
5,767,337.) For example,
mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the
early mouse embryo and
grown in culture. The ES cells are transformed with a vector containing the
gene of interest disrupted
by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi,
M.R. (1989) Science
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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 (March, J.D.
(1996) Clip. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids
Res. 25:4323-4330).
Transformed ES cells are identified and microinjected into mouse cell
blastocysts such as those from
the C57BL/6 mouse strain. The blastocysts are surgically transferred to
pseudopregnant dams, and
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
IO human blastocysts. Human ES cells have the potential to differentiate into
at Least eight separate cell
lineages including endoderm, mesoderm, and ectodermal cell types. These cell
lineages differentiate
into, for example, neural cells, hematopoietic lineages, and cardiomyocytes
(Thomson, J.A. et aI.
(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 TRZCH is injected into animal ES cells, and the
injected sequence
integrates into the animal cell genome. Transformed cells are injected into
blastulae, and the blastulae
are implanted as described above. Transgenic progeny or inbred lines are
studied and treated with
potential pharmaceutical agents to obtain information on treatment of a human
disease. Alternatively,
a mammal inbred to overexpress TRICH, e.g., by secreting TRICH in its milk,
may also serve as a
convenient source of that protein (Janne, J. et aI. (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 brain, lung, prostate, bladder, bone, hypothalamus, breast,
ileum, stomach, pancreas,
and gastrointestinal tissues and tumors of the brain and prostrate.
'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. Iu 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
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such as akinesia, amyotxophic 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, bradyarrythinia,
tachyarrythmia, hypertension, Long QT syndrome, myocarditis, cardiomyopathy,
nemaline myopathy,
centxonuclear myopathy, lipid myopathy, mitochondria) myopathy, thyrotoxic
myopathy, ethanol
myopathy, dermatomyositis, inclusion body myositis, infectious myositis,
polymyositis, neurological
disorders associated with transport, e.g., Alzheimer's disease, amnesia,
bipolar disorder, dementia,
depression, epilepsy, Tourette's disorder, paranoid psychoses, and
schizophrenia, and other disorders
associated with transport, e.g., neurofibromatosis, postherpetic neuralgia,
trigeminal neuropathy,
sarcoidosis, sickle cell anemia, Wilson's disease, cataracts, infertility,
pulmonary artery stenosis,
sensorineural autosomal deafness, hyperglycemia, hypoglycemia, Grave's
disease, goiter, Cushing's
disease, Addison's disease, glucose-galactose malabsorption syndrome,
hypercholesterolemia,
adrenoleukodystrophy, Zellweger syndrome, Menkes disease, occipital horn
syndrome, von Gierke
disease, cystinuria, iminoglycinuria, Hartup disease, and Fanconi disease; a
neurological disorder such
as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms,
Alzheimer's disease, Pick's
disease, Huntington's disease, dementia, Parkinson's disease and other
extrapyramidal disorders,
amyotrophic lateral sclerosis and other motor neuron disorders, progressive
neural muscular atrophy,
retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other
demyelinating diseases, bacterial
and viral meningitis, brain abscess, subduxal 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 herxiangioblastomatosis, 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
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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, ophthahnoplegia, 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
l0 spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune
hemolytic anemia, autoimmune
thyroiditis, autoinunune polyendocrinopathy-candidiasis-ectodermal dystrophy
(APECED), bronchitis,
cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis,
dermatomyositis, diabetes mellitus,
emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis
fetalis, erythema nodosum,
atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'
disease, Hashimoto's
thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis,
myasthenia gravis,
myocardial or pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis, polymyositis, psoriasis,
Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome,
systemic anaphylaxis,
systemic lupus erythematosus; systemic sclerosis, thrombocytopenic purpura,
ulcerative colitis, uveitis,
Werner syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral,
bacterial, fungal, parasitic, protozoal, and heltninthic infections, and
trauma; and a cell proliferative
disorder such as actinic keratosis, arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis, mixed
connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal
hemoglobinuria, polycythemia
vera, psoriasis, primary thrombocythemia, and cancers including
adenocarcinoma, leukemia,
lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular,
cancers of the adrenal
gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,
ganglia, gastrointestinal tract,
heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,
prostate, salivary glands, skin,
spleen, testis, thymus, thyroid, and uterus.
In another embodiment, a vector capable of expressing TRICH or a fragment or
derivative
thereof may beadministered to a subject to treat or prevent a disorder
associated with decreased
expression or activity of TRICH including, but not limited to, those described
above.
In a further embodiment, a composition comprising a substantially purified
TRICH in
conjunction with a suitable pharmaceutical carrier may be administered 'to a
subject to treat or prevent
a disorder associated with decreased expression or activity of TRICH
including, but not limited to,
those provided above.
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In still another embodiment, an agonist which modulates the activity of T'RICH
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 T'RICH may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of T'RICH.
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 T'RICH
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 T'RICH.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding T'RICH may be administered to a subject to treat or prevent a
disorder associated with
increased expression or activity of T'RICH including, but not limited to,
those described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be administered in combination with
other appropriate
therapeutic agents. Selection of the appropriate agents for use in combination
therapy may be made
by one of ordinary skill in the art, according to conventional pharmaceutical
principles. The
combination of therapeutic agents may act synergistically to effect the
treatment or prevention of the
various disorders described above. Using this approach, one may be able to
achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the potential for
adverse side effects.
An antagonist of TRICH may be produced using methods which are generally known
in the
art. In particular, purified TRICH may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind TRICH.
Antibodies to TRICH may
also be generated using methods that are well known in the art. Such
antibodies may include, but are
not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies,
Fab fragments, and
fragments produced by a Fab expression library. Neutralizing antibodies (i.e.,
those which inhibit
dimer formation) are generally preferred for therapeutic use..
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, humans, and
others may be immunized by injection with T'RICH or with any fragment or
oligopeptide thereof which
has immunogenic properties. Depending on the host species, various adjuvants
may be used to
increase immunological response. Such adjuvants include, but are not limited
to, Freund's, mineral gels
such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants
used in humans, BCG
(bacilli Calmette-Guerin) and Corynebacterium parvum are especially
preferable.
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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.
T_mmunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci.
USA 80:2026-2030; and
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and Takeda,
S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the art, to produce
TRICH-specific single
chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be
generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g., Burton,
D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte population
or by screening i_m_m__unoglobulin libraries or panels of highly specific
binding reagents as disclosed in
the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.
USA 86:3833-3837; Winter,
G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for TRICH may also be
generated.
For example, such fragments include, but are not limited to, F(ab')Z fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab')2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(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
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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 Scatchard analysis in conjunction with
radioimmunoassay techniques
may be used to assess the affinity of antibodies for TRICH. Affinity is
expressed as an association
constant, Ka, which is defined as the molar concentration of TRICH-antibody
complex divided by the
molar concentrations of free antigen and free antibody under equilibrium
conditions. The Ka
determined for a preparation of polyclonal antibodies, which are heterogeneous
in their affinities for
multiple TRICH epitopes, represents the average affinity, or avidity, of the
antibodies for TRICH.
The Ka determined for a preparation of monoclonal antibodies, which are
monospecific for a particular
TRICH epitope, represents a true measure of affinity. High-affinity antibody
preparations with Ka
ranging from about 109 to 1012 L/mole are preferred for use in immunoassays in
which the TRICH-
antibody complex must withstand rigorous manipulations. Low-affinity antibody
preparations with Ka
ranging from about 106 to 10' L/mole are preferred for use in
immunopurification and similar
procedures which ultimately require dissociation of TRICH, preferably in
active form, from the
antibody (Catty, D. (1988) Antibodies Volume I: A Practical Approach, IRL
Press, Washington DC;
Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies,
John Wiley & Sons,
New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to determine
the quality and suitability of such preparations for certain downstreann
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, su ra, and
Coligan et al. su ra.)
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.)
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In therapeutic use, any gene delivery system suitable for introduction of the
autisense
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 Clip. Tmmunol. 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
l0 systems known in the are. (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 deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
2o Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-
703), thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-
242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites (e.g.,
against human retroviruses, such as human immunodeficiency virus (H1V)
(Baltimore, D. (1988)
Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA.
93:11395-11399),
hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans
and Paracoccidioides
brasiliensis; and protozoan parasites such as Plasmodium falciparum and
Trypanosoma cruzi). In the
case where a genetic deficiency in TRICH expression or regulation causes
disease, the expression of
TRICH from an appropriate population of transduced cells may alleviate the
clinical manifestations
caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by
deficiencies in
TRICH are treated by constructing mammalian expression vectors encoding TRICH
and introducing
these vectors by mechanical means into TRICH-deficient cells. Mechanical
transfer technologies for
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CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
use with cells in vivo or ex vitro include (i) direct DNA microinjection into
individual cells, (ii) ballistic
gold particle delivery, (iii) liposome-mediated transfection; (iv) receptor-
mediated gene transfer, and
(v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (2993) Annu.
Rev. Biochem.
62:19'1-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H. Recipon
(1998) Curr. Opin.
Biotechnol.9:445-450).
Expression vectors that may be effective for the expression of TRICH include,
but are not
limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors
(Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La
Jolla CA),
and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
TRICH
may be expressed using (i) a constitutively active promoter, (e.g., from
cytomegalovirus (CMV), Rous
sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or (3-actin genes),
(ii) an inducible promoter
(e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992)
Proc. Natl. Acad. Sci.
USA 89:5547-5551; Gossen, M. et aI. (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 P1ND;
Invitrogen); the
FK506lrapamycin inducible promoter; or the RU486lmifepristone inducible
promoter (Rossi, F.M.V.
and Blau, H.M. supra)), or (iii) a tissue-specific promoter or the native
promoter of the endogenous
gene encoding TRICH from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KTT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of these
standardized mammalian transfection protocols. ,
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to TRICH expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding TRICH under the control of an independent promoter or
the retrovirus long
terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and
(iii) a Rev-responsive
element (RRE) along with additional retrovitus cis-acting RNA sequences and
coding sequences
required for efficient vector propagation. Retrovirus vectors (e.g., PFB and
PFBNEO) are
commercially available (Stratagene) and are based on published data (IZiviere,
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
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WO 02/22684 PCT/USO1/28938
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. e1
al. (1998) J. Virol. 72:9873-9880). U.5. Patent No. 5,910,434 to Rigg ("Method
fox obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant") discloses
a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by reference.
Propagation of retrovirus vectors, transduction of a population of cells
(e.g., CD4+ T-cells), and the
return of transduced cells to a patient are procedures well known to persons
skilled in the art of gene
therapy and have been well documented (Ranga, U. et al. (1997) J. Virol.
71:7020-7029; Bauer, G. et
l0 al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-
4716; Ranga, U. et al. (1998)
Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
In the alternative, an adenavirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding TRICH to cells which have one or moxe 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 No. 5,707,618 to Armentano ("Adenovirus vectors for
gene therapy"), hereby
incorporated by reference. For adenoviral vectors, see also Antinozzi, P.A. et
al. (1999) Annu. Rev.
2o Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature 18:389:239-242,
both incorporated by
reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding TRICH to target cells which have one or more genetic
abnormalities with
respect to the expression of TRICH. The use of herpes simplex virus (HSV)-
based vectors may be
especially valuable for introducing TRICH to cells of the central nervous
system, for which HSV has
a tropism. The construction and packaging of herpes-based vectors are well
known to those with
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
type 1-based vector has
been used to deliver a reporter gene to the eyes of primates (Liu, X. et al.
(1999) Exp. Eye Res.
169:385-395). The construction of a HSV-1 virus vector has also been disclosed
in detail in U.S.
Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is hereby
incorporated by reference. U.S. Patent No. 5,804,413 teaches the use of
recombinant HSV d92
which consists of a genome containing at least one exogenous gene to be
transferred to a cell under
the control of the appropriate promoter for purposes including human gene
therapy. Also taught by
this patent are the construction and use of recombinant HSV strains deleted
for ICP4, ICP27 and
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
ICP22. For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-
532 and Xu, H. et al.
(1994) Dev. Biol. 163:152-161, hereby incorporated by reference. The
manipulation of cloned
herpesvirus sequences, the generation of recombinant virus following the
transfection of multiple
plasmids containing different segments of the large herpesvirus genomes, the
growth and propagation
of herpesvirus, and the infection of cells with herpesvirus are techniques
well known to those of
ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding TRICH to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based
on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During
alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full length
genomic RNA,
resulting in the overproduction of capsid proteins relative to the viral
proteins with enzymatic activity
(e.g., protease and polymerase). Similarly, inserting the coding sequence for
TRICH into the
alphavirus genome in place of the capsid-coding region results in the
production of a large number of
TRICH-coding RNAs and the synthesis of high levels of TRICH in vector
transduced cells. While
alphavirus infection is typically associated with cell lysis within a few
days, the ability to establish a
persistent infection in hamster normal kidney cells (BHK-21) with a variant of
Sindbis virus (SIN)
indicates that the lytic replication of alphaviruses can be altered to suit
the needs of the gene therapy
application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host
range of alphaviruses will
allow the introduction of TRICH into a variety of cell types. The specific
transduction of a subset of
cells in a population may require the sorting of cells prior to transduction.
The methods of
manipulating infectious cDNA clones of alphaviruses, performing alphavirus
cDNA and RNA
transfections, and performing alphavirus infections, are well known to those
with ordinary skill in the
art.
Oligonucleotides derived from the transcription initiation site, e.g., between
about positions -10
and +10 from the start site, may also be employed to inhibit gene expression.
Similarly, inhibition can
be achieved using triple helix base-pairing methodology. Triple helix pairing
is useful because it causes
inhibition of the ability of the double helix to open sufficiently for the
binding of polymerases,
transcription factors, or regulatory molecules. Recent therapeutic advances
using triplex DNA have
been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in
Huber, B.E. and B.I. Carr,
Molecular and hnmunolo~ic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-
177.) A
complementary sequence or antisense molecule may also be designed to block
translation of mRNA
by preventing the transcript from binding to rib~osomes.
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Ribozyrnes, 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 riboz5nne 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 polymerise promoters such as T7 or SP6.
Alternatively, these cDNA
constructs that synthesize complementary RNA, constitutively or inducibly, can
be introduced into cell
lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3' ends
of the molecule, or the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages
within the backbone of the molecule. This concept is inherent in the
production of PNAs and can be
extended in all of these molecules by the inclusion of nontraditional bases
such as inosine, queosine,
and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified
forms of adenine, cytidine,
guanine, thymine, and uridine which are not as easily recognized by endogenous
endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding TRICH. Compounds
which may be effective in altering expression of a specific polynucleotide may
include, but are not
limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming
oligonucleotides,
transcription factors and other polypeptide transcriptional regulators, and
non-macromolecular
chemical entities which are capable of interacting with specific
polynucleotide sequences. Effective
compounds may alter polynucleotide expression by acting as either inhibitors
or promoters of
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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 the
polynucleotide. A screen for a compound effective in altering expression of a
specific polynucleotide
can be carried out, for example, using a Schizosaccharom ces 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 txansfection, by liposome injections, or by polycationic amino
polymers may be achieved
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using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
Biotechnol. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
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 Remiugton's
Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may
consist of TRICH,
antibodies to TRICH, and mimetics, agonists, antagonists, or inhibitors of
TRICH.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, infra-
arterial, intramedullary, intrathecal,
intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal,
intranasal, enteral, topical,
sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
case of small molecules (e.g. traditional low molecular weight organic chugs),
aerosol delivery of fast-
acting formulations is well-known in the art. In the case of macromolecules
(e.g. larger peptides and
proteins), recent developments in the field of pulmonary delivery via the
alveolar region of the lung
have enabled the practical delivery of drugs such as insulin. to blood
circulation (see, e.g., Patton, J.S.
et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage of
administration without
needle injection, and obviates the need for potentially toxic penetration
enhancers.
Compositions suitable for use in the invention include compositions wherein
the active
ingredients are contained in au effective amount to achieve the intended
purpose. The determination
of an effective dose is well within the capability of those skilled in the
art.
Specialized forms of compositions may be prepared for direct intracellular
delivery of
macromolecules comprising TRICH or fragments thereof. For example, Iiposome
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 fxom 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).
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For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs, monkeys,
or pigs. An animal model may also be used to determine the appropriate
concentration range and
route of administration. Such information can then be used to determine useful
doses and routes for
administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example
TRICH or fragments thereof, antibodies of TRICH, and agonists, antagonists or
inhibitors of TRICH,
which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity
may be determined
by standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDso (the dose therapeutically effective in 50°l0 of
the population) or LDso (the dose
lethal to 50°Io 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. 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 ~.g to 100,000 ~cg, up to a
total dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind 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
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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 axe 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 from a highly specific region, e.g., the 5'regulatory region, or from a
less specific region, e.g., a
conserved motif, and the stringency of the hybridization or amplification will
determine whether the
probe identifies only naturally occurring sequences encoding TRICH, allelic
variants, or related
sequences.
Probes may also be used for the detection of related sequences, and may have
at least 50%
sequence identity to any of the TRICH encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ )D
N0:27-52 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 yitro by means of the addition of the
appropriate RNA
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polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as 32P or 355,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding TRICH may be used for the diagnosis of
disorders
associated with expression of TRICH. Examples of such disorders include, but
are not limited to, a
transport disorder such as akinesia, amyotrophic lateral sclerosis, ataxia
telangiectasia, cystic fibrosis,
Becker's muscular dystrophy, Bell's palsy, Charcot-Marie Tooth disease,
diabetes mellitus, diabetes
insipidus, diabetic neuropathy, Duchenne muscular dystrophy, hyperkalemic
periodic paralysis,
normokalemic periodic paralysis, Parkinson's disease, malignant hyperthermia,
multidrug resistance,
myasthenia gravis, myotonic dystrophy, catatonia, tardive dyskinesia,
dystonias, peripheral neuropathy,
cerebral neoplasms, prostate cancer, cardiac disorders associated with
transport, e.g., angina,
bradyarrythmia, tachyarrythmia, hypertension, Long QT syndrome, myocarditis,
cardiomyopathy,
nemaline myopathy, centronuclear myopathy, lipid myopathy, mitochondrial
myopathy, thyrotoxic
myopathy, ethanol myopathy, dermatomyositis, inclusion body myositis,
infectious myositis,
polymyositis, neurological disorders associated with transport, e.g.,
Alzheimer's disease, amnesia,
bipolar disorder, dementia, depression, epilepsy, Tourette's disorder,
paranoid psychoses, and
schizophrenia, and other disorders associated with transport, e.g.,
neurofibromatosis, postherpetic
neuralgia, trigeminal neuropathy, sarcoidosis, sickle cell anemia, Wilson's
disease, cataracts, infertility,
pulmonary artery stenosis, sensorineural autosomal deafness, hyperglycemia,
hypoglycemia, Grave's
disease, goiter, Cushing's disease, Addison's disease, glucose-galactose
malabsorption syndrome,
hypercholesterolemia, adrenoleukodystrophy, Zellweger syndrome, Menkes
disease, occipital horn
syndrome, von Gierke disease, cystinuria, iminoglycinuria, Hartup disease, and
Fanconi disease; a
neurological disorder such as epilepsy, ischemic cerebrovascular disease,
stroke, cerebral neoplasms,
Alzheimer's disease, Pick's disease, Huntington's disease, dementia,
Parkinson's disease and other
extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron
disorders, progressive
neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple
sclerosis and other
demyelinating diseases, bacterial and viral meningitis, brain abscess,
subdural empyema, epidural
abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis,
viral central nervous system
disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and
Gerstmann-Straussler-Scheinker
syndrome, fatal familial insomnia, nutritional and metabolic diseases of the
nervous system,
neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis,
encephalotrigeminal
syndrome, mental retardation and other developmental disorders of the central
nervous system
including Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic
nervous system
disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy
and other neuromuscular
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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 at~thritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial,
fungal, parasitic, protozoal, and hehninthic infections, and trauma; and a
cell proliferative disorder such
as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis,
hepatitis, mixed connective
tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria,
polycythemia 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,
gastarointestinal 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
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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 dia~osis 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
2o polynucleotide is used. Standard values obtained in this manner may be
compared with values
obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard
values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the development
of the disease, or may provide a means for detecting the disease prior to the
appearance of actual
clinical symptoms. A more definitive diagnosis of this type may allow health
professionals to employ
preventative measures or aggressive treatment earlier thereby preventing the
development or further
progression of the cancer.
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Additional diagnostic uses for oligonucleotides designed from the sequences
encoding TRICH
may involve the use of PCR. These oligomers may be chemically synthesized,
generated
enzymatically, or produced in vitro. Oligomers will preferably contain a
fragment of a polynucleotide
encoding TRICH, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
TRICH, and will be employed under optimized conditions for identification of a
specific gene or
condition. Oligomers may also be employed under less stringent conditions for
detection or
quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding TRICH may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are
substitutions, insertions and deletions that are a frequent cause of inherited
or acquired genetic disease
in humans. Methods of SNP detection include, but are not limited to, single-
stranded conformation
polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP,
oligonucleotide primers
derived from the polynucleotide sequences encoding TRICH are used to amplify
DNA using the
polymerase chain reaction (PCR). The DNA may be derived, for example, from
diseased or normal
tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause
differences in the
secondary and tertiary structures of PCR products in single-stranded form, and
these differences are
detectable using gel electrophoresis in non-denaturing gels. In fSCCP, the
oligonucleotide primers are
fluorescently labeled, which allows detection of the amplimers in high-
throughput equipment such as
DNA sequencing machines. Additionally, sequence database analysis methods,
termed in silico SNP
(isSNP), are capable of identifying polymorphisms by comparing the sequence of
individual
overlapping DNA fragments which assemble into a common consensus sequence.
These computer-
based methods filter out sequence variations due to laboratory preparation of
DNA and sequencing
errors using statistical models and automated analyses of DNA sequence
chromatograms. In the
alternative, SNPs may be detected and characterized by mass spectrometry
using, for example, the
high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
Methods which may also be used to quantify the expression of TRICH include
radiolabeling or
biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Tmmunol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples maybe
3o accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives rapid
quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
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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
TRICI~may
be used as elements on a microarray. The microarray may be used to monitor or
measure protein-
protein interactions, drug-target interactions, and gene expression profiles,
as described above.
A particular embodiment relates to the use of the polynucleotides of the
present invention to
generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern of
gene expression by a particular tissue or cell type. Global gene expression
patterns are analyzed by
quantifying the number of expressed genes and their relative abundance under
given conditions and at
a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent No.
5,840,484, expressly incorporated by reference herein.) Thus a transcript
image may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines, biopsies,
or other biological samples. The transcript image may thus reflect gene
expression in vivo, as in the
case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the
present invention
may also be used in conjunction with in vitro model systems and preclinical
evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and toxicity
(Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L.
Anderson (2000)
Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein).
If a test compound has a
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signature similar to that of a compound with known toxicity, it is likely to
share those toxic properties.
These fingerprints or signatures are most useful and refined when they contain
expression information
from a large number of genes and gene families. Ideally, a genome-wide
measurement of expression
pxovides 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:/lwww.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 au
untreated biological sample. Differences in the transcript levels between the
two samples are
indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a proteome
can be subjected individually to further analysis. Proteome expression
patterns, or profiles, are
analyzed by quantifying the number of expressed proteins and their relative
abundance under given
conditions and at a given time. A profile of a cell's proteome may thus be
generated by separating
and analyzing the polypeptides of a particular tissue or cell type. In one
embodiment, the separation is
achieved using two-dimensional gel electrophoresis, in which proteins from a
sample are separated by
isoelectric focusing in the first dimension, and then according to molecular
weight by sodium dodecyl
sulfate slab gel electrophoresis in the second dimension (Steiner and
Anderson, supra). The proteins
are visualized in the gel as discrete and uniquely positioned spots, typically
by staining the gel with an
agent such as Coomassie Blue or silver or fluorescent stains. The optical
density of each protein spot
is generally proportional to the level of the protein in the sample. The
optical densities of equivalently
positioned protein spots from different samples, for example, from biological
samples either treated or
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untreated with a test compound or therapeutic agent, are compared to identify
any changes in protein
spot density related to the treatment. The proteins in the spots are partially
sequenced using, for
example, standard methods employing chemical or enzymatic cleavage followed by
mass
spectrometry. The identity of the protein in a spot may be determined by
comparing its partial
sequence, preferably of at least 5 contiguous amino acid residues, to the
polypeptide sequences of the
present invention. In some cases, further sequence data may be obtained for
definitive protein
identification.
A proteomic profile may also be generated using antibodies specific for TRICH
to quantify
the levels of TRICH expression. In one embodiment, the antibodies are used as
elements on a
microarray, and protein expression levels are quantified by exposing the
microarray to the sample and
detecting the levels of protein bound to each array element (Lueking, A. et
al. (1999) Anal. Biochem.
270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection
may be performed by
a variety of methods known in the art, for example, by reacting the proteins
in the sample with a thiol-
or amino-reactive fluorescent compound and detecting the amount of
fluorescence bound at each
1 S array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
correlation between transcript and protein abundances for some proteins in
some tissues (Anderson,
N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures maybe
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to rapid
degradation of mRNA, so proteomic profiling may be more reliable and
informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
each protein is compared to the amount of the corresponding protein in an
untreated biological sample.
A difference in the amount of protein between the two samples is indicative of
a toxic response to the
test compound in the treated sample. Individual proteins are identified by
sequencing the amino acid
residues of the individual proteins and comparing these partial sequences to
the polypeptides of the
present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are incubated
with antibodies specific to the polypeptides of the present invention. The
amount of protein recognized
by the antibodies is quantified. The amount of protein in the treated
biological sample is compared
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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.,
Brennau, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad.
Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application
W095/251116; Shalon, D. et
al. (1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA
94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.)
Various types of
microarrays are well known and thoroughly described in DNA Microarrays: A
Practical Approach,
M. Schena, ed. (1999) Oxford University Press, London, hereby expressly
incorporated by reference.
1o In another embodiment of the invention, nucleic acid sequences encoding
TRICH may be
used to generate hybridization probes useful in mapping the naturally
occurring genomic sequence.
Either coding or noncoding sequences may be used, and in some instances,
noncoding sequences may
be preferable over coding sequences. For example, conservation of a coding
sequence among
members of a multi-gene family may potentially cause undesired cross
hybridization during
chromosomal mapping. The sequences may be mapped to a particular chromosome,
to a specific
region of a chromosome, or to artificial chromosome constructions, e.g., human
artificial chromosomes
(HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes
(BACs), bacterial P1
constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop
genetic liukage 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, supra, pp. 965-
968.) Examples of genetic
map data can be found in various scientific journals or at the Online
Mendelian Inheritauce in Man
(OMIM) World Wide Web site. Correlation between the location of the gene
encoding TRICH on a
physical map and a specific disorder, or a predisposition to a specific
disorder, may help define the
region of DNA associated with that disorder and thus may further positional
cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
may reveal associated markers even if the exact chromosomal locus is not
known. This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
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techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely ,
localized by genetic linkage to a particular genomic xegion, 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.
Tn another embodiment of the invention, TRICH, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used fox 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 intracellulaxly. The
formation of binding complexes
between T12ICH 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 WO84/03564.) Iu 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.
2o 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 axe currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following embodiments are, therefore,
to be construed as merely illustrative, and not limitative of the remainder of
the disclosure in any way
whatsoever.
The disclosures of all patents, applications and publications, mentioned above
and below and
including U.S. Ser. No. 60/232,685, U.S. Ser. No. 60/234,842, U.S. Ser. No.
60/236,882, U.S. Ser.
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No. 60/239,057, U.S. Ser. No. 60/240,54b, and U.S. Ser. No. 60!241,700 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 lysates were
centrifuged over CsCl
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 txeated 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 mR.NA purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the
UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life
Technologies), using
the recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra,
units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
1000 bp) using SEPHACRYL 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.,
PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies),
PCDNA2.1 plasmid
(3nvitrogen, Carlsbad CA), PBK-CMV plasnud (Stratagene), PCR2-TOPOTA
(Invitrogen), PCMV-
ICIS (Stratagene), or pINCY (Incyte Genomics, Palo Alto CA), or derivatives
thereof Recombinant
plasmids were transformed into competent E. coli cells including XL1-Blue, XL1-
BlueMRF, or SOLR
from Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones
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Plasmids obtained as described in Example I were recovered from host cells by
in viyo
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, Gaithexsburg 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 1I 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 (Iiamilton) 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, supra, unit 7.7). Some of the cDNA sequences were selected for extension
using the
techniques disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by
removing
vector, linker, and poly(A) sequences and by masking ambiguous bases, using
algorithms and
programs based on BLAST, dynamic programming, and dinucleotide nearest
neighbor analysis. The
Incyte cDNA sequences or translations thereof were then queried against a
selection of public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases, and
BLOCKS, PRINTS, DOMO, PRODOM, and hidden Markov model (I-hVIM)-based protein
family
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databases such as PFAM. (HIYhVI 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
I~IVIMER.
The Incyte cDNA sequences were assembled to produce full length polynucleotide
sequences.
Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched
sequences, or
Genscan-predicted coding sequences (see Examples IV and V) were used to extend
Incyte cDNA
assemblages.to full length. Assembly was performed using programs based on
Phred, Phrap, and
Consed, and cDNA assemblages were screened for open reading frames using
programs based on
GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were
translated to derive
the corresponding full length polypeptide sequences. Alternatively, a
polypeptide of the invention may
begin at any of the metluonine 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.
2o Table 7 summarizes the tools, programs, and algorithms used for the
analysis and assembly of
Incyte cDNA and full length sequences and provides applicable descriptions,
references, and threshold
parameters. The first column of Table 7 shows the tools, programs, and
algorithms used, the second
column provides brief descriptions thereof, the third column presents
appropriate references, all of
which are incorporated by reference herein in their entirety, and the fourth
column presents, where
applicable, the scores, probability values, and other parameters used to
evaluate the strength of a
match between two sequences (the higher the score or the lower the probability
value, the greater the
identity between two sequences).
The programs described above for the assembly and analysis of full length
polynucleotide and
polypeptide sequences were also used to identify polynucleotide sequence
fragments from SEQ m
N0:27-52. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
amplification technologies are described in Table 4, column 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
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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 Iucyte 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 Jncyte 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
Iucyte 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" Sequences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification program described in Example IV. Partial cDNAs assembled as
described in Example
I(I 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 progranuning to integrate cDNA and genomic
information,
generating possible splice variants that were subsequently confirmed, edited,
or extended to create a
full length sequence. Sequence intervals in which the entire length of the
interval was present on
more than one sequence in the cluster were identified, and intervals thus
identified were considered to
be equivalent by transitivity. For example, if an interval was present on a
cDNA and two genomic
sequences, then all three intervals were considered to be equivalent. This
process allows unrelated
but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals
thus identified were then "stitched" together by the stitching algorithm in
the order that they appear
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along their parent sequences to generate the longest possible sequence, as
well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence
(cDNA to cDNA or
genomic sequence to genomic sequence) were given preference over linkages
which change parent
type (cDNA to genomic sequence). The resultant stitched sequences were
translated and compared
by BLAST analysis to the genpept and gbpri public databases. Incorrect exons
predicted by Genscan
were corrected by comparison to the top BLAST hit from genpept. Sequences were
further extended
with additional cDNA sequences, or by inspection of genomic DNA, when
necessary.
"Stretched" Seguences
Partial DNA sequences ware 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 resultaut 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 pxobes 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 N0:27-52 were compared with
sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ m N0:27-52 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for
Genome Research (WIGR), and Genethon were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ ID NO:, to that
map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map
position of an interval, in centiMorgans, is measured relative to the terminus
of the chromosome's p-
arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb)
of DNA in
CA 02422497 2003-03-14
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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.nhn.nih.gov/genernapn, can be employed to determine if
previously identified disease
genes map within or in proximity to the intervals indicated above.
In this manner, SEQ ID N0:31 was mapped to chromosome 1 within the interval
from 133.00
to 137.30 centiMorgans. SEQ lD N0:33 was mapped to chromosome 12 within the
interval from
120.50 to the q terminal, or more specifically, within the interval from
126.10 to 145.70 centiMorgans.
1o 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,
su ra, 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 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
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other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
Alternatively, polynucleotide sequences encoding TRICH are analyzed with
respect to the
tissue sources from which they were derived. For example, some full length
sequences are
assembled, at least in part, with overlapping Incyte cDNA sequences (see
Example DI). Each cDNA
sequence is derived from a cDNA library constructed from a human tissue. Each
human tissue is
classified into one of the following organ/tissue categories: cardiovascular
system; connective tissue;
digestive system; embryonic structures; endocrine system; exocrine glands;
genitalia, female; genitalia,
male; germ cells; heroic and immune system; liver; musculoskeletal system;
nervous system;
pancreas; respiratory system; sense organs; skin; stomatognathic system;
unclassified/mixed; or
urinary tract. 'The number of libraries in each category is counted and
divided by the total number of
libraries across all categories. Similarly, each human tissue is classified
into one of the following
disease/condition categories: cancer, cell lice, 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
2o fragment of the full length molecule using oligonucleotide primers designed
from this fragment. One
primer was synthesized to initiate 5' extension of the known fragment, and the
other primer was
synthesized to initiate 3' extension of the known fragment. The initial
primers were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68°C to about 72°C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer; reaction buffer
containing Mg2+, (NH4)ZSp4,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE
enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the
following parameters
for primer pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 60°C, 1 min;
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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 SI~+
were as follows: Step 1: 94°C,
3 min; Step 2: 94°C, 15 sec; Step 3: 57°C, 1 min; Step 4:
68°C, 2 min; Step 5: Steps 2, 3, and 4
repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 p1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 p,1 of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 /.c1 to 10 ,u1 aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose gel to determine which reactions were
successful in extending the
sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to relegation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended
clones were religated 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 trausfected into competent E. coli cells. Transformed
cells were selected on
antibiotic-containing media, and individual colonies were picked and cultured
overnight at 37 °C in 384-
well plates in LB/2x curb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the
following
parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min; Step
5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7:
storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted weth
20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy trausfer
sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGD1'E Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotede sequences are verified using the
above procedure or
are used to obtain 5' regulatory sequences using the above procedure along
with oligonucleotides
designed for such extension, and an appropriate genomic library.
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IX. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:27-52 are employed to screen
cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide
fragments. Oligonucleotides are designed using state-of the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 ~cCi of
[Y-3zP] 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 NIA. 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, arid derivatives thereof. The substrate in each of
the aforementioned
technologies should be uniform and solid with a non-porous surface (Schena
(1999), su ra).
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; Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and
J. Hodgson (1998)
Nat. Biotechnol. 16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The
array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
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biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element on
the microarray may be assessed. In one embodiment, microarray preparation and
usage is described
in detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
1o 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/ul oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 units/p.l RNase inhibitor, 500 ~,M dATP, 500 ~,M dGTP, 500
~.M dTTP, 40 ~.M
dCTP, 40 p,M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)+ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling), is treated with 2.5 ml of 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 ~tl 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 ~,g.
Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water, and
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coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are
cured in a 110°C
oven.
Array elements are applied to the coated glass substrate using a procedure
descizbed in U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 ~Cl of the array
element DNA, at an average
concentration of 100 ng/~tl, is loaded into the open capillary printing
element by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°
C followed by washes in 0.2%
SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 ~Cl of sample mixture consisting of 0.2 ~Cg
each of Cy3 and
Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65° C for 5 minutes and is aliquoted onto the
microarray surface and covered with
an 1.8 cm2 coversJip. The arrays are transferred to a waterproof chamber
having a cavity just slightly
larger than a microscope slide. The chamber is kept at 100% humidity
internally by the addition of 140
p,1 of 5X SSC in a corner of the chamber. The chamber containing the arrays is
incubated for about
6.5 hours at 60° C. The arrays are washed for 10 min at 45° C in
a first wash buffer (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 ou wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores. Appropriate
i~tlters positioned between the array and the photomultiplier tubes are used
to filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
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typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that location
to be correlated with a weight ratio of hybridizing species of 1:100,000. When
two samples from
different sources (e.g., representing test and control cells), each labeled
with a different fluorophore,
are hybridized to a single array for the purpose of identifying genes that are
differentially expressed,
the calibration is done by labeling samples of the calibrating cDNA with the
two fluorophores and
adding identical amounts of each to the hybridization mixture.
The output of the photomultiplier tube is digitized using a 12-bit RT'I-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping emission
spectra) between the fluorophores using each fluorophore's emission spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each spot
is centered in each element of the grid. The fluorescence signal within each
element is then integrated
to obtain a numerical value corresponding to the average intensity of the
signal. The software used
for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
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 compxising from about 15 to 30 base pairs is described,
essentially the same
procedure is used with smaller or with larger sequence fragments. Appropriate
oligonucleotides are
designed using OLIGO 4.06 software (National Biosciences) and the coding
sequence of TRICH. To
inhibit transcription, a complementary oligonucleotide is designed from the
most unique 5' sequence
and used to prevent promoter binding to the coding sequence. To inhibit
translation, a complementary
oligonucleotide is designed to prevent ribosomal binding to the TRICH-encoding
transcript.
XII. Expression of TRICH
Expression and purification of TRICH is achieved using bacterial or virus-
based expression
systems. For expression of TRICH in bacteria, cDNA is subcloned into an
appropriate vector
containing an antibiotic resistance gene and an inducible promoter that
directs high levels of cDNA
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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 (1PTG). Expression of TRICH in eukaryotic cells is
aclueved by infecting
insect or mammalian cell lines with recombinant Autographica californica
nuclear polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding TRICH by either homologous recombination or
bacterial-mediated
transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect Spodoptera fru~iperda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus. (See Engelhard, E.K. et
al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)
Hum. Gene Ther.
7:1937-1945.)
In most expression systems, TRICH is synthesized as a fusion protein with,
e.g., glutathione
S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His,
permitting rapid, single-step,
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-kilodalton
enzyme from Schistosoma iaponicum, 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
immunoafhnity 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, XV1I, and XVJII 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
contain the cytomegalovirus promoter. 5-10 ~cg of recombinant vector are
transiently transfected into
a human cell line, for example, an endothelial or hematopoietic cell line,
using either liposome
formulations or electroporation. 1-2 ~g of an additional plasmid containing
sequences encoding a
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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 'tn cell size and granularity as measured by forward light scatter and
90 degree side light
1o scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as
measured by reactivity with
specific antibodies; and alterations in plasma membrane composition as
measured by the binding of
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M.G. (1994) Flow C_ ometry, 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). Trausfected 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 TRICH Specific Antibodies
TRICH substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used to
immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the TRICH amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
KLH (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) to
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increase immunogenicity. (See, e.g., Ausubel, 1995, s-unra.) Rabbits are
immunized with the
oligopeptide-I~LH complex in complete Freund's adjuvant. Resulting antisera
are tested for
antipeptide and anti-TRICH activity by, for example, binding the peptide or
TRICH to a substrate,
blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting
with radio-iodinated goat
anti-rabbit IgG.
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
to CNBr-activated SEPHAROSE (Arnersham 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
antibodyfTRICH binding (e.g., a buffer of pH 2 to pH 3, or a high
concentration of a chaotrope, such
as urea or thiocyanate ion), and TRICH is collected.
XVI. Identification of Molecules Which Interact with TRICH
Molecules which interact with TRICH may include transporter substrates,
agonists or
antagonists, modulatory proteins such as G(3y proteins (Reimann, supra) or
proteins involved in TRICH
localization or clustering such as MAGUI~s (Craven, su ra). TRICH, or
biologically active fragments
thereof, are labeled with lasl 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 bixtding 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 pxoteins with an activation
domain) reconstitute a
transactivation function that is observed by expression of a reporter gene.
Yeast 2-hybrid systems are
commercially available, and methods for use of the yeast 2-hybrid system with
ion channel proteins
are discussed in Niethammer, M. and M. Sheng (1998, Meth. Enzymol. 293:104-
122).
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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 alI interactions
between the proteins encoded by two Large libraries of genes (Nandabalan, K.
et al. (2000) U.S.
Patent No. 6,057,101).
Potential TRICH agonists or antagonists may be tested for activation or
inhibition of TRICH
ion channel activity using the assays described in section XVIB.
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 !3-galactosidase are stained blue when a suitable
colorimetric
substrate is added to the culture media under conditions that are well known
in the art. Stained cells
are tested for differences in membrane conductance by electrophysiological
techniques that are well
known in the art. Untransformed cells, and/or cells transformed with either
vector sequences alone or
13-galactosidase sequences alone, are used as controls and tested in parallel.
Cells expressixtg TRICH
will have higher anion or cation conductance relative to control cells. The
contribution of TRICH to
conductance can be confirmed by incubating the cells using antibodies specific
for TRICH. The
antibodies will bind to the extracellular side of TRICH, thereby blocking the
pore in the ion channel,
and the associated conductance.
Alternatively, ion channel activity of TRICH is measured as current flow
across a TRICH-
containing Xenopus laeyis oocyte membrane using the two-electrode voltage-
clamp technique (Ishi et
al., supra; Jegla, T. and L. Salkoff (1997) J. Neurosci. 17:32-44). TRICH is
subcloned into an
appropriate Xenopus oocyte expression vector, such as pBF, and 0.5-5 ng of
mRNA is injected into
mature stage 1V oocytes. Injected oocytes are incubated at 18 °C for 1-
5 days. Inside-out
macropatches are excised into an intracellular solution containing 116 mM K-
gluconate, 4 mM KCl,
and 10 mM Hepes (pH 7.2). The intracellular solution is supplemented with
varying concentrations of
the TRICH mediator, such as CAMP, cGMP, or Ca+2 (in the form of CaCl2), where
appropriate.
Electrode resistance is set at 2-S MSZ and electrodes are filled with the
intracellular solution lacking
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mediator. Experiments are performed at room temperature from a holding
potential of 0 mV. Voltage
ramps (2.5 s) from -100 to 100 mV are acquired at a sampling frequency of 500
Hz. Current
measured is proportional to the activity of TRICH in the assay. In particular,
the activity of TRICH-
25 is measured as Cl- conductance.
Transport activity of TRICH is assayed by measuring uptake of labeled
substrates into
Xenopus laevis oocytes. Oocytes at stages V and VI are injected with TRICH
mRNA (10 ng per
oocyte) and incubated for 3 days at 18°C in OR2 medium (82.5mM NaCI,
2.5 mM KCl, 1mM CaCl2,
1mM MgCl2, 1mM Na2HP04, 5 mM Hepes, 3.8 mM NaOH , 50~Cg/mI gentamycin, pH 7.8)
to allow
expression of TRICH. Oocytes are then transferred to standard uptake medium
(100mM NaCl, 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 amino
acids for TRICH-1, xanthine
and uracil for TRICH-3, melibiose for TRICH-18, monocarboxylate for TRICH-20,
neurotransmitters
such as gamma-aminobutyric acid (GABA) for TRICH-22, and nucleosides for TRICH-
23.
ATPase activity associated with TRICH can be measured by hydrolysis of
radiolabeled ATP-
[y-32P], separation of the hydrolysis products by chromatographic methods, and
quantitation of the
recovered 32P using a scintillation counter. The reaction mixture contains ATP-
['y-~ZP] and varying
amounts of TRICH in a suitable buffer incubated at 37 °C for a suitable
period of time, The reaction
is terminated by acid precipitation with trichloroacetic acid and then
neutralized with base, and an
aliquot of the reaction mixture is subjected to membrane or filter paper-based
chromatography to
separate the reaction products. The amount of 32P liberated is counted in a
scintillation counter. The
amount of radioactivity recovered is proportional to the ATPase activity of
TRICH in the assay.
XVIII. Identification of TRICH Agonists and Antagonists
TRICH is expressed in a eukaryotic cell line such as CHO (Chinese Hamster
Ovary) or HEK
(Human Embryonic Kidney) 293. Ion channel activity of the transformed cells is
measured in the
presence and absence of candidate agonists or antagonists. Ion channel
activity is assayed using
patch clamp methods well known in the art or as described in Example XVJI.
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
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Ca2+ indicator Fluo-4 AM, sodium-sensitive dyes such as SBFI and sodium green,
or the Cl- indicator
MQAE (all available from Molecular Probes) in combination with the FLIPR
fluorimetric plate reading
system (Molecular Devices). In a more generic version of this assay, changes
in membrane potential
caused by ionic flux across the plasma membrane are measured using oxonyl dyes
such as DiBAC4
(Molecular Probes). DiBAC4 equilibrates between the extracellular solution and
cellular sites
according to the cellular membrane potential. The dye's fluorescence intensity
is 20-fold greater
when bound to hydrophobic intracellular sites, allowing detection of DiBAC~
entry into the cell
(Gonzalez, J.E. and P.A. Negulescu (1998) Curr. Opin. Biotechnol. 9:624-631).
Candidate agonists or
antagonists may be selected from known ion channel agonists or antagonists,
peptide libraries, or
combinatorial chemical libraries.
Various modifications and variations of the described methods and systems of
the invention
will be apparent to those skilled in the art without departing from the scope
and spirit of the invention.
Although the invention has been described in connection with certain
embodiments, it should be
understood that the invention as claimed should not be unduly limited to such
specific embodiments.
Indeed, various modifications of the described modes for carrying out the
invention which are obvious
to those skilled in molecular biology or related fields are intended to be
within the scope of the
following claims.
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CA 02422497 2003-03-14
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A
H
N
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CA 02422497 2003-03-14
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<110> INCYTE GENOMICS, INC.
LEE, Ernestine A.
YUE, Henry
LAL, Preeti G.
WALIA, Narinder K.
BAUGHN, Mariah R.
WARREN, Bridget A.
LEE, Sally
SANJANWALA, Madhu S.
YAO, Monique G.
RAMKUMAR, Jayalaxmi
THORNTON, Michael
GANDHI, Ameena R.
POLICKY, Jennifer L.
ELLIOTT, Viclci S.
ARVIZU, Chandra
RAUMANN, Brigitte E.
BRUNS, Christopher M.
NAINA, Amir
HAFALIA, April J.A.
NGUYEN, Danniel B.
XU, Yuming
LU, Dyung Aina M.
ISON, Craig H.
GRIFFIN, Jennifer A.
REDDY, Roopa M.
BURFORD, Neil
<120> TRANSPORTERS AND ION CHANNELS
<130> PI-0217 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/232,685; 60/234,842; 60/236,882; 60/239,057; 60/240,540;
60/241,700
<151> 2000-09-15; 2000-09-22; 2000-09-29; 2000-10-05; 2000-13-10;
2000-10-18
<160> 52
<170> PERL Program
<210> 1
<211> 619
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1687189CD1
<400> 1 .
Met Pro Trp Gln Ala Phe Arg Arg Phe Gly Gln Lys Leu Val Arg
1 5 10 15
Arg Arg Thr Leu Glu Ser Gly Met Ala Glu Thr Arg Leu Ala Arg
1/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
20 25 30
Cys Leu Ser Thr Leu Asp Leu Val Ala Leu Gly Val Gly Ser Thr
35 40 45
Leu Gly Ala Gly Val Tyr Val Leu Ala Gly Glu Val Ala Lys Asp
50 55 60
Lys Ala Gly Pro Ser Ile Val Ile Cys Phe Leu Val Ala Ala Leu
65 70 75
Ser Ser Val Leu Ala Gly Leu Cys Tyr Ala Glu Phe Gly Ala Arg
80 85 90
Val Pro Arg Ser Gly Ser Ala Tyr Leu Tyr Ser Tyr Val Thr Val
95 100 105
Gly Glu Leu Trp Ala Phe Thr Thr Gly Trp Asn Leu Ile Leu Ser
110 115 120
Tyr Val Ile Gly Thr Ala Ser Val Ala Arg Ala Trp Ser Ser Ala
125 130 135
Phe Asp Asn Leu Ile Gly Asn His Ile Ser Lys Thr Leu Gln Gly
140 145 150
Ser Ile Ala Leu His Val Pro His Val Leu Ala Glu Tyr Pro Asp
155 160 165
Phe Phe Ala Leu Gly Leu Val Leu Leu Leu Thr Gly Leu Leu Ala
170 175 180
Leu Gly Ala Ser Glu Ser Ala Leu Val Thr Lys Val Phe Thr Gly
185 190 195
Val Asn Leu Leu Val Leu Gly Phe Va1 Met Ile Ser Gly Phe Val
200 205 210
Lys Gly Asp Val His Asn Trp Lys Leu Thr Glu Glu Asp Tyr Glu
215 220 225
Leu Ala Met Ala Glu Leu Asn Asp Thr Tyr Ser Leu Gly Pro Leu
230 235 240
Gly Ser Gly Gly Phe Val Pro Phe Gly Phe Glu Gly Ile Leu Arg
245 250 255
Gly Ala Ala Thr Cys Phe Tyr Ala Phe Val Gly Phe Asp Cys Ile
260 265 270
Ala Thr Thr Gly Glu Glu Ala Gln Asn Pro Gln Arg Ser Ile Pro
275 280 285
Met Gly Ile Val Ile Ser Leu Ser Val Cys Phe Leu Ala Tyr Phe
290 295 300
Ala Val Ser Ser Ala Leu Thr Leu Met Met Pro Tyr Tyr Gln Leu
305 310 315
Gln Pro Glu Ser Pro Leu Pro Glu Ala Phe Leu Tyr Ile Gly Trp
320 325 330
Ala Pro Ala Arg Tyr Val Val Ala Val Gly 5er Leu Cys Ala Leu
335 340 345
Ser Thr Ser Leu Leu Gly Ser Met Phe Pro Met Pro Arg Val Ile
350 355 360
Tyr Ala Met Ala Glu Asp Gly Leu Leu Phe Arg Val Leu Ala Arg
365 370 375
Ile His Thr Gly Thr Arg Thr Pro Tle Ile A1a Thr Val Val Ser
380 385 390
Gly Ile Ile Ala Ala Phe Met Ala Phe Leu Phe Lys Leu Thr Asp
395 400 405
Leu Val Asp Leu Met Ser I1e Gly Thr Leu Leu Ala Tyr Ser Leu
410 415 420
Val Ser Ile Cys Val Leu Ile Leu Arg Tyr Gln Pro Asp Gln Glu
425 430 435
Thr Lys Thr,Gly Glu Glu Val Glu Leu Gln Glu Glu Ala Ile Thr
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440 445 450
Thr Glu Ser Glu Lys Leu Thr Leu Trp Gly Leu Phe Phe Pro Leu
455 460 465
Asn Ser Ile Pro Thr Pro Leu Ser Gly Gln Ile Val Tyr Val Cys
470 475 480
Ser Ser Leu Leu Ala Val Leu Leu Thr Ala Leu Cys Leu Val Leu
485 490 495
Ala Gln Trp Ser Val Pro Leu Leu Ser Gly Asp Leu Leu Trp Thr
500 505 510
Ala Val Val Val Leu Leu Leu Leu Leu Ile Ile Gly Ile Ile Val
515 520 525
Val Ile Trp Arg Gln Pro Gln Ser Ser Thr Pro Leu His Phe Lys
530 535 540
Val Pro Ala Leu Pro Leu Leu Pro Leu Met Ser Ile Phe Val Asn
545 550 555
Ile Tyr Leu Met Met Gln Met Thr Ala Gly Thr Trp Ala Arg Phe
560 565 570
Gly Val Trp Met Leu Tle Gly Phe Ala Ile Tyr Phe Gly Tyr Gly
575 580 S85
Ile Gln His Ser Leu Glu Glu Ile Lys Ser Asn Gln Pro Ser Arg
590 595 600
Lys Ser Arg Ala Lys Thr Val Asp Leu Asp Pro Gly Thr Leu Tyr
605 610 615
Val His Ser Val
<210> 2
<211> 2436
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7078207CD1
<400> 2
Met Gly Phe Leu His Gln Leu Gln Leu Leu Leu Trp Lys Asn Val
1 5 10 15
Thr Leu Lys Arg Arg Ser Pro Trp Val Leu Ala Phe Glu I1e Phe
20 25 30
Ile Pro Leu Val Leu Phe Phe Ile Leu Leu Gly Leu Arg Gln Lys
35 40 45
Lys Pro Thr Ile Ser Val Lys Glu Val Ser Phe Tyr Thr Ala Ala
50 55 60
Pro Leu Thr Ser Ala Gly Ile Leu Pro Val Met Gln Ser Leu Cys
65 70 75
Pro Asp Gly Gln Arg Asp Glu Phe Gly phe Leu Gln Tyr Ala Asn
80 85 90
Ser Thr Val Thr Gln Leu Leu Glu Arg Leu Asp Arg Val Val Glu
95 l00 205
Glu Gly Asn Leu Phe Asp Pro Ala Arg pro Ser Leu Gly Ser Glu
110 115 120
Leu Glu Ala Leu Arg Gln His Leu Glu Ala Leu Ser Ala Gly Pro
125 130 135
G1y Thr Ser Gly Ser His Leu Asp Arg Ser Thr Va1 Ser Ser Phe
140 145 150
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Ser Leu Asp Ser Val Ala Arg Asn Pro Gln Glu Leu Trp Arg Phe
155 160 165
Leu Thr Gln Asn Leu Ser Leu Pro Asn Ser Thr Ala Gln Ala Leu
170 175 180
Leu Ala Ala Arg Val Asp Pro Pro Glu Val Tyr His Leu Leu Phe
185 190 195
Gly Pro Ser Ser A1a Leu Asp Ser Gln Ser Gly Leu His Lys Gly
200 ~ 205 210
Gln Glu Pro Trp Ser Arg Leu Gly Gly Asn Pro Leu Phe Arg Met
215 220 225
Glu Glu Leu Leu Leu Ala Pro Ala Leu Leu Glu Gln Leu Thr Cys
230 235 240
Thr Pro Gly Ser Gly Glu Leu Gly Arg Ile Leu Thr Val Pro Glu
245 250 255
Ser Gln Lys Gly Ala Leu Gln Gly Tyr Arg Asp Ala Val Cys Ser
260 265 270
Gly Gln Ala Ala A1a Arg Ala Arg Arg Phe Ser Gly Leu Ser Ala
275 280 285
Glu Leu Arg Asn G1n Leu Asp Val Ala Lys Val Ser Gln Gln Leu
290 295 300
Gly Leu Asp Ala Pro Asn Gly Ser Asp Ser Ser Pro Gln Ala Pro
305 310 315
Pro Pro Arg Arg Leu Gln Ala Leu Leu Gly Asp Leu Leu Asp Ala
320 325 330
Gln Lys Val Leu G1n Asp Val Asp Val Leu Ser Ala Leu Ala Leu
335 340 345
Leu Leu Pro Gln Gly Ala Cys Thr Gly Arg Thr Pro Gly Pro Pro
350 355 360
Ala Ser Gly Ala Gly Gly Ala Ala Asn Gly Thr Gly Ala Gly Ala
365 370 375
Val Met Gly Pro Asn Ala Thr Ala' Glu Glu Gly Ala Pro Ser Ala
380 385 390
Ala Ala Leu Ala Thr Pro Asp Thr Leu Gln Gly G1n Cys Ser A1a
395 400 405
Phe Val Gln Leu Trp Ala Gly Leu Gln Pro Ile Leu Cys Gly Asn
410 415 420
Asn Arg Thr Ile Glu Pro Glu Ala Leu Arg Arg Gly Asn Met Ser
425 430 435
Ser Leu Gly Phe Thr Ser Lys Glu Gln Arg Asn Leu Gly Leu Leu
440 ~ 445 450
Val His Leu Met Thr Ser Asn Pro Lys Ile Leu Tyr Ala Pro Ala
455 460 465
Gly Ser G1u Val Asp Arg Val Ile Leu Lys Ala Asn Glu Thr Phe
470 475 480
Ala Phe Val Gly Asn Val Thr His Tyr Ala Gln Val Trp Leu Asn
485 490 495
Ile Ser Ala Glu Ile Arg Ser Phe Leu Glu Gln Gly Arg Leu Gln
500 505 510
Gln His Leu Arg Trp Leu Gln Gln Tyr Val Ala Glu Leu Arg Leu
515 520 525
His Pro Glu Ala Leu Asn Leu Ser Leu Asp Glu Leu Pro Pro Ala
530 535 540
Leu Arg Gln Asp Asn Phe Ser Leu Pro Ser Gly Met Ala Leu Leu
545 550 555
Gln Gln Leu Asp Thr Ile Asp Asn Ala Ala Cys Gly Trp Ile Gln
560 565 570
4/71
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Phe Met Ser Lys Val Ser Val Asp Ile Phe Lys Gly Phe Pro Asp
575 580 585
Glu Glu Ser Ile Val Asn Tyr Thr Leu Asn Gln Ala Tyr Gln Asp
590 595 600
Asn Val Thr Val Phe Ala Ser Val Ile Phe Gln Thr Arg Lys Asp
605 610 615
Gly Ser Leu Pro Pro His Val His Tyr Lys Ile Arg Gln Asn Ser
620 625 630
Ser Phe Thr Glu Lys Thr Asn Glu Ile Arg Arg Ala Tyr Trp Arg
635 640 645
Pro Gly Pro Asn Thr Gly Gly Arg Phe Tyr Phe Leu Tyr Gly Phe
650 655 660
Val Trp Ile Gln Asp Met Met Glu Arg Ala Ile Ile Asp Thr Phe
665 670 675
Val Gly His Asp Val Val Glu Pro Gly Ser Tyr Val Gln Met Phe
680 685 690
Pro Tyr Pro Cys Tyr Thr Arg Asp Asp Phe Leu Phe Val Ile Glu
695 700 705
His Met Met Pro Leu Cys Met Val Ile Ser Trp Val Tyr Ser Val
710 715 720
Ala Met Thr Ile Gln His Ile Val Ala Glu Lys Glu His Arg Leu
725 730 735
Lys Glu Val Met Lys Thr Met Gly Leu Asn Asn Ala Val His Trp
740 745 750
Val Ala Trp Phe Ile Thr Gly Phe Val Gln Leu Ser Ile Ser Val
755 760 765
Thr Ala Leu Thr Ala Ile Leu Lys Tyr Gly Gln Val Leu Met His
770 775 780
Ser His Val Val Ile Ile Trp Leu Phe Leu Ala Val Tyr Ala Val
785 790 795
Ala Thr Ile Met Phe Cys Phe Leu Val Ser Val Leu Tyr Ser Lys
800 805 810
Ala Lys Leu Ala Ser Ala Cys Gly Gly Ile Ile Tyr Phe Leu Ser
815 820 825
Tyr Val Pro Tyr Met Tyr Val Ala Ile Arg Glu Glu Val Ala His
830 835 840
Asp Lys Ile Thr Ala Phe Glu Lys Cys Ile Ala Ser Leu Met Ser
845 850 855
Thr Thr Ala Phe Gly Leu G1y Ser Lys Tyr Phe Ala Leu Tyr Glu
860 865 870
Val Ala Gly Val Gly Ile Gln Trp His Thr Phe Ser Gln Ser Pro
875 880 885
Val Glu Gly Asp Asp Phe Asn Leu Leu Leu Ala Val Thr Met Leu
890 895 900
Met Val Asp Ala Val Val Tyr Gly Ile Leu Thr Trp Tyr Ile Glu
905 910 915
Ala Va1 His Pro Gly Met Tyr Gly Leu Pro Arg Pro Trp Tyr Phe
920 925 930
Pro Leu Gln Lys Ser Tyr Trp Leu Gly Ser Gly Arg Thr Glu Ala
935 940 945
Trp Glu Trp Ser Trp Pro Trp Ala Arg Thr Pro Arg Leu Ser Val
950 955 960
Met G1u Glu Asp Gln Ala Cys Ala Met Glu Ser Arg Arg Phe Glu
965 970 975
Glu Thr Arg Gly Met Glu Glu Glu Pro Thr His Leu Pro Leu Val
980 985 990
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Va1 Cys Val Asp Lys Leu Thr Lys Val Tyr Lys Asp Asp Lys Lys
995 1000 1005
Leu Ala Leu Asn Lys Leu Ser Leu Asn Leu Tyr Glu Asn Gln Val
1010 1015 1020
Val Ser Phe Leu Gly His Asn Gly Ala Gly Lys Thr Thr Thr Met
1025 1030 1035
Sex Ile Leu Thr Gly Leu Phe Pro Pro Thr Ser Gly Ser Ala Thr
1040 1045 1050
Ile Tyr Gly His Asp Ile Arg Thr Glu Met Asp Glu Ile Arg Lys
1055 1060 1065
Asn Leu Gly Met Cys Pro Gln His Asn Val Leu Phe Asp Arg Leu
1070 1075 1080
Thr Val Glu Glu His Leu Trp Phe Tyr Ser Arg Leu Lys Ser Met
1085 1090 1095
Ala Gln Glu Glu Ile Arg Arg Glu Met Asp Lys Met Ile Glu Asp
1100 1105 1110
Leu Glu Leu Ser Asn Lys Arg His Ser Leu Val Gln Thr Leu Ser
1115 1120 1125
Gly Gly Met Lys Arg Lys Leu Ser Val Ala Ile Ala Phe Val Gly
1130 1135 1140
Gly Ser Arg Ala Ile Ile Leu Asp Glu Pro Thr Ala Gly Val Asp
1145 1150 1155
Pro Tyr Ala Arg Arg Ala Ile Trp Asp Leu Ile Leu Lys Tyr Lys
1160 1165 1170
Pro Gly Arg Thr Ile Leu Leu Ser Thr His His Met Asp Glu Ala
1175 1180 1185
Asp Leu Leu Gly Asp Arg Ile Ala Ile Ile Ser His Gly Lys Leu
1190 1195 1200
Lys Cys Cys Gly Ser Pro Leu Phe Leu Lys Gly Thr Tyr Gly Asp
1205 1210 1215
Gly Tyr Arg Leu Thr Leu Val Lys Arg Pro A1a Glu Pro Gly Gly
1220 1225 1230
Pro Gln Glu Pro Gly Leu A1a Ser Ser Pro Pro Gly Arg Ala Pro
1235 1240 1245
Leu Ser Ser Cys Ser Glu Leu Gln Val Ser Gln Phe Ile Arg Lys
1250 1255 1260
His Val Ala Ser Cys Leu Leu Val Ser Asp Thr Ser Thr Glu Leu
1265 1270 1275
Ser Tyr Ile Leu Pro Ser Glu Ala Ala Lys Lys Gly Ala Phe Glu
1280 1285 1290
Arg Leu Phe Gln His Leu Glu Arg Ser Leu Asp Ala Leu His Leu
1295 1300 1305
Ser Ser Phe Gly Leu Met Asp Thr Thr Leu Glu Glu Val Phe Leu
1310 1315 1320
Lys Val Ser Glu Glu Asp Gln Ser Leu Glu Asn Ser Glu Ala Asp
1325 1330 1335
Val Lys Glu Ser Arg Lys Asp Val Leu Pro Gly Ala Glu Gly Pro
1340 1345 1350
Ala Ser Gly Glu Gly His Ala Gly Asn Leu Ala Arg Cys Ser Glu
1355 1360 1365
Leu Thr Gln Ser Gln Ala Ser Leu Gln Ser Ala Ser Ser Val Gly
1370 1375 1380
Ser Ala Arg Gly Asp Glu Gly Ala Gly Tyr Thr Asp Val Tyr Gly
1385 1390 1395
Asp Tyr Arg Pro Leu Phe Asp Asn Pro Gln Asp Pro Asp Asn Va1
1400 1405 1410
6/71
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Ser Leu Gln Glu Val Glu Ala Glu Ala Leu Ser Arg Val Gly Gln
1415 1420 1425
Gly Ser Arg Lys Leu Asp Gly Gly Trp Leu Lys Val Arg Gln Phe
1430 1435 ~ 1440
His Gly Leu Leu Val Lys Arg Phe His Cys Ala Arg Arg Asn Ser
1445 1450 1455
Lys Ala Leu Phe Ser.Gln Ile Leu Leu Pro Ala Phe Phe Val Cys
1460 1465 1470
Va1 Ala Met Thr Val Ala Leu Ser Val Pro Glu Ile Gly Asp Leu
1475 1480 1485
Pro Pro Leu Val Leu Ser Pro Ser Gln Tyr His Asn Tyr Thr Gln
1490 1495 1500
Pro Arg Gly Asn Phe Ile Pro Tyr Ala Asn Glu Glu Arg Arg Glu
1505 1510 1515
Tyr Arg Leu Arg Leu Ser Pro Asp Ala Ser Pro Gln Gln Leu Val
1520 1525 1530
Ser Thr Phe Arg Leu Pro Ser Gly Val Gly Ala Thr Cys Val Leu
1535 1540 1545
Lys Ser Pro Ala Asn Gly Ser Leu Gly Pro Thr Leu Asn Leu Ser
1550 1555 1560
Ser Gly Glu Ser Arg Leu Leu Ala Ala Arg Phe Phe Asp Ser Met
1565 1570 1575
Cys Leu Glu Ser Phe Thr Gln Gly Leu Pro Leu Ser Asn Phe Val
1580 1585 1590
Pro Pro Pro Pro Ser Pro Ala Pro Ser Asp Ser Pro Ala Ser Pro
1595 1600 1605
Asp Glu Asp Leu Gln Ala Trp Asn Val Ser Leu Pro Pro Thr Ala
1610 1615 1620
Gly Pro Glu Met Trp Thr Ser Ala Pro Ser Leu Pro Arg Leu Val
1625 1630 1635
Arg Glu Pro Val Arg Cys Thr Cys Ser Ala Gln Gly Thr Gly Phe
1640 1645 1650
Ser Cys Pro Ser Ser Val Gly Gly His Pro Pro Gln Met Arg Val
1655 1660 1665
Val Thr Gly Asp Ile Leu Thr Asp Ile Thr Gly His Asn Val Ser
1670 1675 1680
Glu Tyr Leu Leu Phe Thr Ser Asp Arg Phe Arg Leu His Arg Tyr
1685 1690 1695
Gly Ala Ile Thr Phe Gly Asn Val Leu Lys Ser Tle Pro Ala Ser
1700 1705 1710
Phe Gly Thr Arg Ala Pro Pro Met Val Arg Lys Ile Ala Val Arg
1715 1720 1725
Arg Ala Ala Gln Val Phe Tyr Asn Asn Lys Gly Tyr His Ser Met
1730 1735 1740
Pro Thr Tyr Leu Asn Ser Leu Asn Asn Ala Ile Leu Arg Ala Asn
1745 1750 1755
Leu Pro Lys Ser Lys Gly Asn Pro A1a Ala Tyr Gly Ile Thr Val
1760 1765 1770
Thr Asn His Pro Met Asn Lys Thr Ser Ala Ser Leu Ser Leu Asp
1775 1780 1785
Tyr Leu Leu Gln G1y Thr Asp Val Val Ile Ala Ile Phe Ile Ile
1790 1795 1800
Val Ala Met Ser Phe Val Pro Ala Ser Phe Val Val Phe Leu Val
1805 1810 1815
Ala Glu Lys Ser Thr Lys Ala Lys His Leu Gln Phe Val Ser Gly
1820 1825 1830
7/71
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Cys Asn Pro Ile Ile Tyr Trp Leu Ala Asn Tyr Va1 Trp Asp Met
1835 1840 1845
Leu Asn Tyr Leu Val Pro Ala Thr Cys Cys Val Ile Tle Leu Phe
1850 1855 1860
Val Phe Asp Leu Pro Ala Tyr Thr Ser Pro Thr Asn Phe Pro Ala
1865 1870 1875
Val Leu Ser Leu Phe Leu Leu Tyr Gly Trp Sex Ile Thr Pro I1e
1880 1885 1890
Met Tyr Pro Ala Ser Phe Trp Phe Glu Val Pro Ser Ser Ala Tyr
1895 1900 1905
Val Phe Leu Ile Val Ile Asn Leu Phe Ile Gly Ile Thr Ala Thr
1910 1915 1920
Val Ala Thr Phe Leu Leu Gln Leu Phe Glu His Asp Lys Asp Leu
1925 1930 1935
Lys Val Val Asn Ser Tyr Leu Lys Ser Cys Phe Leu Ile Phe Pro
1940 1945 1950
Asn Tyr Asn Leu Gly His Gly Leu Met Glu Met Ala Tyr Asn Glu
1955 1960 1965
Tyr Ile Asn Glu Tyr Tyr Ala Lys Ile Gly Gln Phe Asp Lys Met
1970 1975 1980
Lys Ser Pro Phe Glu Trp Asp Ile Val Thr Arg Gly Leu Val Ala
1985 1990 1995
Met Ala Val Glu Gly Val Val Gly Phe Leu Leu Thr Ile Met Cys
2000 2005 2010
Gln Tyr Asn Phe Leu Arg Arg Pro Gln Arg Met Pro Val Ser Thr
2015 2020 2025
Lys Pro Val Glu Asp Asp Val Asp Val Ala Ser Glu Arg Gln Arg
2030 2035 2040
Val Leu Arg Gly Asp A1a Asp Asn Asp Met Val Lys Ile Glu Asn
2045 2050 2055
Leu Thr Lys Val Tyr Lys Ser Arg Lys Ile Gly Arg Ile Leu Ala
2060 2065 2070
Val Asp Arg Leu Cys Leu G1y Val Arg Pro Gly Glu Cys Phe Gly
2075 2080 2085
Leu Leu Gly Val Asn Gly A1a Gly Lys Thr Ser Thr Phe Lys Met
2090 2095 2100
Leu Thr Gly Asp G1u ~Ser Thr Thr Gly Gly Glu Ala Phe Val Asn
2105 2110 2115.
Gly His Ser Val Leu Lys Glu Leu Leu Gln Val Gln Gln Ser Leu
2120 2125 2130
Gly Tyr Cys Pro Gln Cys Asp Ala Leu Phe Asp Glu Leu Thr Ala
2135 2140 2145
Arg Glu His Leu Gln Leu Tyr Thr Arg Leu Arg Gly Ile Ser Trp
2150 2155 2160
Lys Asp Glu A1a Arg Val Val Lys Trp Ala Leu Glu Lys Leu Glu
2165 2170 21?5
Leu Thr Lys Tyr Ala Asp Lys Pro Ala Gly Thr Tyr Ser Gly Gly
2180 2185 2190
Asn Lys Arg Lys Leu Ser Thr Ala Ile Ala Leu Ile Gly Tyr Pro
2195 2200 2205
A1a Phe Ile Phe Leu Asp G1u Pro Thr Thr Gly Met Asp Pro Lys
2210 2215 2220
Ala Arg Arg Phe Leu Trp Asn Leu Ile Leu Asp Leu Ile Lys Thr
2225 2230 2235
Gly Arg Ser Val Val Leu Thr Ser His Ser Met Glu Glu Cys Glu
2240 2245 2250
8/71
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Ala Leu Cys Thr Arg Leu Ala Ile Met Val Asn Gly Arg Leu Arg
2255 2260 2265
Cys Leu Gly Ser Ile Gln His Leu Lys Asn Arg Phe Gly Asp Gly
2270 2275 2280
Tyr Met Ile Thr Val Arg Thr Lys Ser Ser G1n Ser Val Lys Asp
2285 2290 2295
Val Val Arg Phe Phe Asn Arg Asn Phe Pro Glu Ala Met Leu Lys
2300 2305 2310
G1u Arg His His Thr Lys Val Gln Tyr Gln Leu Lys Ser Glu His
2315 2320 2325
I1e Ser Leu Ala Gln Val Phe Ser Lys Met Glu Gln Val Ser Gly
2330 2335 2340
Val Leu G1y Ile Glu Asp Tyr Ser Val Ser Gln Thr Thr Leu Asp
2345 2350 2355
Asn Val Phe Val Asn Phe Ala Lys Lys Gln Ser Asp Asn Leu Glu
2360 2365 2370
Gln Gln Glu Thr Glu Pro Pro Ser Ala Leu Gln Ser Pro Leu Gly
2375 2380 2385
Cys Leu Leu Ser Leu Leu Arg Pro Arg Ser Ala Pro Thr G1u Leu
2390 2395 2400
Arg Ala Leu Val Ala Asp Glu Pro G1u Asp Leu Asp Thr G1u Asp
2405 2410 2415
Glu Gly Leu Ile Ser Phe Glu Glu Glu Arg Ala Gln Leu Ser Phe
2420 2425 2430
Asn Thr Asp Thr Leu Cys
2435
<210> 3
<211> 610
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1560619CD1
<400> 3
Met Ser Arg Ser Pro Leu Asn Pro Ser Gln Leu Arg Ser Val Gly
1 5 10 15
Ser Gln Asp Ala Leu Ala Pro Leu Pro Pro Pro Ala Pro Gln Asn
20 25 30
Pro Ser Thr His Ser Trp Asp Pro Leu Cys Gly Ser Leu Pro Trp
35 40 45
Gly Leu Ser Cys Leu Leu Ala Leu Gln His Val Leu Val Met Ala
50 55 60
Ser Leu Leu Cys Val Ser His Leu Leu Leu Leu Cys Ser Leu Ser
65 70 75
Pro Gly Gly Leu Ser Tyr Ser Pro Ser Gln Leu Leu Ala Ser Ser
80 85 90
Phe Phe Ser Cys Gly Met Ser Thr Ile Leu Gln Thr Trp Met Gly
95 100 105
Ser Arg Leu Pro Leu Val Gln Ala Pro Ser Leu Glu Phe Leu Ile
110 115 120
Pro Ala Leu Val Leu Thr Ser Gln Lys Leu Pro Arg A1a Ile Gln
125 130 135
Thr Pro Gly Asn Ser Ser Leu Met Leu His Leu Cys Arg Gly Pro
9/71
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140 145 150
Ser Cys His Gly Leu Gly His Trp Asn Thr Ser Leu Gln Glu Val
155 l60 165
Ser Gly Ala Val Val Val Ser Gly Leu Leu Gln Gly Met Met Gly
170 l75 180
Leu Leu Gly Ser Pro Gly His Val Phe Pro His Cys Gly Pro Leu
185 190 195
Val Leu Ala Pro Ser Leu Val Val Ala Gly Leu Ser Ala His Arg
200 205 210
Glu Val Ala Gln Phe Cys Phe Thr His Trp Gly Leu Ala Leu Leu
215 220 225
Val Ile Leu Leu Met Val Val Cys Ser Gln His Leu Gly Ser Cys
230 235 240
Gln Phe His Val Cys Pro Trp Arg Arg Ala Ser Thr Ser Ser Thr
245 250 255
His Thr Pro Leu Pro Val Phe Arg Leu Leu Ser Val Leu Ile Pro
260 265 270
Val Ala Cys Val Trp Ile Val Ser Ala Phe Val Gly Phe Ser Val
275 280 285
Ile Pro Gln Glu Leu Ser Ala Pro Thr Lys Ala Pro Trp Ile Trp
290 295 300
Leu Pro His Pro Gly Glu Trp Asn Trp Pro Leu Leu Thr Pro Arg
305 310 315
Ala Leu Ala Ala Gly Ile Ser Met Ala Leu Ala Ala Ser Thr Ser
320 325 330
Ser Leu Gly Cys Tyr Ala Leu Cys Gly Arg Leu Leu His Leu Pro
335 340 345
Pro Pro Pro Pro His Ala Cys Ser Arg Gly Leu Ser Leu Glu Gly
350 355 360
Leu Gly Ser Val Leu Ala Gly Leu Leu Gly Ser Pro Met Gly Thr
365 370 375
Ala Ser Ser Phe Pro Asn Val Gly Lys Val Gly Leu Ile Gln Ala
380 385 390
Gly Ser Gln,Gln Val Ala His Leu Val Gly Leu Leu Cys Val Gly
395 400 405
Leu Gly Leu Ser Pro Arg Leu Ala Gln Leu Leu Thr Thr Tle Pro
410 415 420
Leu Pro Val Val Gly Gly Val Leu Gly Val Thr Gln Ala Val Val
425 430 435
Leu Ser Ala Gly Phe Ser Ser Phe Tyr Leu A1a Asp Ile Asp 5er
440 445 450
Gly Arg Asn Ile Phe Tle Val Gly Phe Ser Ile Phe Met Ala Leu
455 460 465
Leu Leu Pro Arg Trp Phe Arg Glu Ala Pro Val Leu Phe Ser Thr
470 475 480
Gly Trp Ser Pro Leu Asp Val Leu Leu His Ser Leu Leu Thr Gln
485 490 495
Pro Ile Phe Leu Ala Gly Leu Ser Gly Phe Leu Leu Glu Asn Thr
500 505 510
Ile Pro Gly Thr Gln Leu Glu Arg Gly Leu G1y Gln Gly Leu Pro
515 520 525
Ser Pra Phe Thr Ala Gln Glu Ala Arg Met Pro Gln Lys Pro Arg
530 535 540
Glu Lys Ala Ala Gln Val Tyr Arg Leu Pro Phe Pro Ile Gln Asn
545 550 555
Leu Cys Pro Cys Ile Pro Gln Pro Leu His Cys Leu Cys Pro Leu
10/71
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560 565 570
Pro Glu Asp Pro Gly Asp Glu Glu Gly Gly Ser Ser Glu Pro Glu
575 580 585
Glu Met Ala Asp Leu Leu Pro Gly Ser Gly Glu Pro Cys Pro Glu
590 595 600
Ser Ser Arg Glu Gly Phe Arg Ser Gln Lys
605 610
<210> 4
<211> 372
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2614283CD1
<400> 4
Met Glu Ala Lys Glu Lys Gln His Leu Leu Asp Ala Arg Pro Ala
1 5 10 15
Ile Arg Ser Tyr Thr Gly Ser Leu Trp Gln Glu Gly Ala Gly Trp
20 25 30
Ile Pro Leu Pro Arg Pro Gly Leu Asp Leu Gln Ala Ile Glu Leu
35 40 45
Ala Ala Gln Ser Asn His His Cys His Ala Gln Lys Gly Pro Asp
50 55 60
Ser His Cys Asp Pro Lys Lys Gly Lys Ala Gln Arg Gln Leu Tyr
65 70 75
Val Ala Ser Ala Ile Cys Leu Leu Phe Met Ile Gly Glu Val Val
80 85 90
Gly Gly Tyr Leu Ala His Ser Leu Ala Val Met Thr Asp Ala Ala
95 100 105
His Leu Leu Thr Asp Phe Ala Ser Met Leu Ile Ser Leu Phe Ser
110 115 l20
Leu Trp Met Ser Ser Arg Pro Ala Thr Lys Thr Met Asn Phe Gly
125 130 135
Trp Gln Arg Ala Glu Ile Leu Gly Ala Leu Val Ser Val Leu Ser
140 145 150
Ile Trp Val Val Thr Gly Val Leu Val Tyr Leu Ala Val Glu Arg
155 160 165
Leu Ile Ser Gly Asp Tyr Glu Ile Asp Gly G1y Thr Met Leu Ile
170 175 180
Thr Ser Gly Cys Ala Val Ala Val Asn Ile Ile Met Gly Leu Thr
185 190 195
Leu His Gln Ser Gly His Gly His Ser His Gly Thr Thr Asn G1n
200 205 210
Gln Glu Glu Asn Pro Ser Val Arg Ala Ala Phe Ile His Val Ile
215 220 225
Gly Asp Phe Met Gln Ser Met Gly Val Leu Val Ala Ala Tyr Ile
230 235 240
Leu Tyr Phe Lys Pro Glu Tyr Lys Tyr Val Asp Pro Ile Cys Thr
245 250 255
Phe Val Phe Ser Ile Leu Val Leu Gly Thr Thr Leu Thr I1e Leu
260 265 270
Arg Asp Val Ile Leu Val Leu Met Glu Gly Thr Pro Lys Gly Val
275 280 285
11/71
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Asp Phe Thr Ala Val Arg Asp Leu Leu Leu Ser Val Glu Gly Val
290 295 300
Glu Ala Leu His Ser Leu His Ile Trp Ala Leu Thr Val Ala Gln
305 310 315
Pro Val Leu Ser Val His Ile Ala Ile Ala Gln Asn Thr Asp Ala
320 325 330
Gln Ala Val Leu Lys Thr Ala Ser Ser Arg Leu Gln Gly Lys Phe
335 340 345
His Phe His Thr Val Thr Ile Gln Ile Glu Asp Tyr Ser Glu Asp
350 355 360
Met Lys Asp Cys Gln Ala Cys Gln Gly Pro Ser Asp
365 370
<210> 5
<211> 490
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte ID No: 2667691CD1
<400> 5
Met Thr Gln Gly Lys Lys Lys Lys Arg Ala Ala Asn Arg Ser Ile
1 5 10 15
Met Leu Ala Lys Lys Ile Ile Ile Lys Asp Gly Gly Thr Pro Gln
20 25 30
Gly Ile Gly Ser Pro Ser Val Tyr His Ala Val Ile Val Ile Phe
35 40 45
Leu Glu Phe Phe Ala Trp Gly Leu Leu Thr Ala Pro Thr Leu Val
50 55 60
Val Leu His Glu Thr Phe Pro Lys His Thr Phe Leu Met Asn G1y
65 70 75
Leu Ile Gln Gly Val Lys Gly Leu Leu Ser Phe Leu Ser Ala Pro
80 85 90
Leu Ile Gly Ala Leu Ser Asp Val Trp Gly Arg Lys Ser Phe Leu
95 100 105
Leu Leu Thr Val Phe Phe Thr Cys Ala Pro Ile Pro Leu Met Lys
110 115 120
Ile Ser Pro Trp Trp Tyr Phe Ala Val Ile Ser Va1 Ser Gly Val
125 130 135
Phe Ala Val Thr Phe Ser Val Val Phe Ala Tyr Val Ala Asp Ile
140 145 150
Thr Gln Glu His Glu Arg Ser Met Ala Tyr Gly Leu Val Ser Ala
155 160 165
Thr Phe A1a Ala Ser Leu Val Thr Ser Pro Ala Ile Gly Ala Tyr
170 175 180
Leu Gly Arg Val Tyr Gly Asp Ser Leu Val Val Val Leu Ala Thr
185 190 195
Ala I1e Ala Leu Leu Asp Ile Cys Phe Ile Leu Val Ala Val Pro
200 205 ' 210
Glu Ser Leu Pro Glu Lys Met Arg Pro Ala Ser Trp Gly Ala Pro
215 220 225
Ile Ser Trp Glu Gln Ala Asp Pro Phe Ala Ser Leu Lys Lys Val
230 235 240
Gly Gln Asp Ser Ile Val Leu Leu Ile Cys Ile Thr Val Phe Leu
12/71
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245 250 255
Ser Tyr Leu Pro Glu Ala Gly Gln Tyr Ser Ser Phe Phe Leu Tyr
260 265 270
Leu Arg Gln Ile Met Lys Phe Ser Pro Glu Ser Va1 Ala Ala Phe
275 280 285
Ile Ala Val Leu Gly Ile Leu Ser Ile Ile Ala Gln Thr Ile Val
290 295 300
Leu Ser Leu Leu Met Arg Ser Ile Gly Asn Lys Asn Thr Ile Leu
305 310 315
Leu Gly Leu Gly Phe Gln Ile Leu Gln Leu Ala Trp Tyr Gly Phe
320 325 330
Gly Ser Glu Pro Trp Met Met Trp Ala Ala Gly Ala Val Ala Ala
335 340 345
Met Ser Ser Ile Thr Phe Pro Ala Val Ser Ala Leu Val Ser Arg
350 355 360
Thr Ala Asp Ala Asp Gln Gln Gly Val Val Gln Gly Met Ile Thr
365 370 375
Gly Ile Arg Gly Leu Cys Asn Gly Leu Gly Pro Ala Leu Tyr Gly
380 385 390
Phe Ile Phe Tyr Ile Phe His Val Glu Leu Lys Glu Leu Pro Ile
395 400 405
Thr Gly Thr Asp Leu Gly Thr Asn Thr Ser Pro Gln His His Phe
410 415 420
Glu Gln Asn Ser Ile Ile Pro Gly Pro Pro Phe Leu Phe Gly Ala
425 430 435
Cys Ser Val Leu Leu Ala Leu Leu Val Ala Leu Phe Ile Pro Glu
440 445 450
His Thr Asn Leu Ser Leu Arg Ser Ser Ser Trp Arg Lys His Cys
455 460 465
Gly Ser His Ser His Pro His Asn Thr Gln Ala Pro Gly Glu Ala
470 475 480
Lys Glu Pro Leu Leu Gln Asp Thr Asn Val
485 490
<210> 6
<211> 377
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No. 3211415CD1
<400> 6
Met Leu Pro Leu Ser Ile Lys Asp Asp Glu Tyr Lys Pro Pro Lys
1 5 10 15
Phe Asn Leu Phe GIy Lys Ile Ser Gly Trp Phe Arg Ser Ile Leu
20 25 30
Ser Asp Lys Thr Ser Arg Asn Leu Phe Phe Phe Leu Cys Leu Asn
35 40 45
Leu Ser Phe Ala Phe Val Glu Leu Leu Tyr Gly Ile Trp Ser Asn
50 55 60
Cys Leu Gly Leu Ile Ser Asp Ser Phe His Met Phe Phe Asp Ser
65 70 75
Thr Ala Ile Leu Ala Gly Leu Ala Ala Ser Val Ile Ser Lys Trp
80 85 90
13/71
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Arg Asp Asn Asp Ala Phe Ser Tyr Gly Tyr Val Arg Ala Glu Val
95 100 105
Leu Ala Gly Phe Val Asn Gly Leu Phe Leu Tle Phe Thr Ala Phe
110 115 120
Phe Ile Phe. Ser Glu Gly Val Glu Arg Ala Leu Ala Pro Pro Asp
125 130 135
Val His His Glu Arg Leu Leu Leu Val Ser Ile Leu Gly Phe Val
140 145 150
Val Asn Leu Ile Gly Ile Phe Val Phe Lys His Gly Gly His Gly
155 160 165
His Ser His Gly Ser Gly Gly His Gly His Ser His Ser Leu Phe
170 175 180
Asn Gly Ala Leu Asp Gln Ala His Gly His Val Asp His Cys His
185 190 195
Ser His Glu Val Lys His Gly Ala Ala His Ser His Asp His Ala
200 205 210
His Gly His Gly His Phe His Ser His Asp Gly Pro Ser Leu Lys
215 220 225
Glu Thr Thr Gly Pro Ser Arg Gln Ile Leu Gln Gly Val Phe Leu
230 235 240
His Ile Leu Ala Asp Thr Leu Gly Ser Ile Gly Val Ile Ala Ser
245 250 255
Ala Ile Met Met Gln Asn Phe Gly Leu Met Ile Ala Asp Pro Ile
260 265 270
Cys Ser Ile Leu Ile Ala Ile Leu Tle Val Val Ser Val Ile Pro
275 280 285
Leu Leu Arg Glu Ser Val Gly Ile Leu Met Gln Arg Thr Pro Pro
290 295 300
Leu Leu Glu Asn Ser Leu Pro Gln Cys Tyr Gln Arg Val Gln Gln
305 310 315
Leu Gln Gly Val Tyr Ser Leu Gln Glu Gln His Phe Trp Thr Leu
320 325 330
Cys Ser Asp Val Tyr Val Gly Thr Leu Lys Leu Ile Val Ala Pro
335 340 345
Asp Ala Asp Ala Arg Trp Ile Leu Ser Gln Thr His.Asn Ile Phe
350 355 360
Thr Gln Ala Gly Val Arg Gln Leu Tyr Val Gln Ile Asp Phe Ala
365 370 375
Ala Met
<210> 7
<211> 340
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4739923CD2
<400> 7
Met Ala Asp Thr Ala Thr Thr Ala Ser Ala Ala Ala Ala Ser Ala
1 5 10 15
Ala Ser Ala Ser Ser Asp Ala Pro Pro Phe Gln Leu Gly Lys Pro
20 25 30
Arg Phe Gln Gln Thr Ser Phe Tyr Gly Arg Phe Arg His Phe Leu,
14/71
CA 02422497 2003-03-14
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35 40 45
Asp Ile Ile Asp Pro Arg Thr Leu Phe Val Thr Glu Arg Arg Leu
50 55 60
Arg Glu Ala Val Gln Leu Leu Glu Asp Tyr Lys His Gly Thr Leu
65 70 75
Arg Pro Gly Val Thr Asn Glu Gln Leu Trp Ser Ala Gln Lys Ile
80 85 90
Lys Gln Ala Ile Leu His Pro Asp Thr Asn Glu Lys Ile Phe Met
95 100 105
Pro Phe Arg Met Pro Gly Tyr Ile Pro Phe Gly Thr Pro Ile Val
110 115 120
Val Gly Leu Leu Leu Pro Asn Gln Thr Leu Ala Ser Thr Val Phe
125 130 135
Trp Gln Trp Leu Asn Gln Ser His Asn Ala Cys Val Asn Tyr Ala
140 145 150
Asn Arg Asn AIa Thr Lys Pro Ser Pro Ala Ser Lys Phe Ile Gln
155 160 165
Gly Tyr Leu Gly Ala Val Ile Ser Ala Va1 Ser Ile Ala Val Gly
170 175 180
Leu Asn Val Leu Val Gln Lys Ala Asn Lys Leu Thr Pro Ala Thr
185 190 195
Arg Leu Leu Ile Gln Arg Phe Val Pro Phe Pro Ala Val Ala Ser
200 205 210
Ala Asn Ile Cys Asn Va1 Val Leu Met Arg Tyr Gly Glu Leu Glu
215 220 225
Glu Gly Ile Asp Val Leu Asp Ser Asp Gly Asn Leu Val Gly Ser
230 235 240
Ser Lys Ile AIa Ala Arg His Ala Leu Leu Glu Thr Ala Leu Thr
245 250 255
Arg Val Val Leu Pro Met Pro Ile Leu Val Leu Pro Pro Ile Val
260 265 270
Met Ser Met Leu Glu Lys Thr Ala Leu Leu Gln Ala Arg Pro Arg
275 280 285
Leu Leu Leu Pro Val Gln Ser Leu Val Cys Leu Ala Ala Phe Gly
290 295 300
Leu Ala Leu Pro Leu Ala Ile Ser Leu Phe Pro Gln Met Ser Glu
305 310 315
Ile Glu Thr Ser Gln Leu Glu Pro Glu Ile Ala Gln Ala Thr Ser
320 325 330
Ser Arg Thr Val Val Tyr Asn Lys Gly Leu
335 340
<210> 8
<211> 1274
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 55030459CD1
<400>
8
Met Arg Gln Glu Glu Glu Glu Ala Val Ala Arg
A1a Pro Thr Ala
1 5 10 15
Arg Pro Pro Trp Leu Leu Cys Val Ala Cys Trp
Arg Leu Leu Leu
20 25 30
15/71
CA 02422497 2003-03-14
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Leu Gly Ala Gly Ala Glu A1a Asp Phe Ser Ile Leu Asp Glu Ala
35 40 45
Gln Val Leu Ala Ser Gln Met Arg Arg Leu Ala Ala Glu Glu Leu
50 55 60
Gly Val Val Thr Met Gln Arg Ile Phe Asn Ser Phe Val Tyr Thr
65 70 75
Glu Lys Ile Ser Asn Gly Glu Ser Glu Val Gln Gln Leu Ala Lys
80 85 90
Lys Ile Arg Glu Lys Phe Asn Arg Tyr Leu Asp Val Val Asn Arg
95 100 105
Asn Lys Gln Val Val Glu Ala Ser Tyr Thr Ala His Leu Thr Ser
110 115 120
Pro Leu Thr Ala Ile Gln Asp Cys Cys Thr Ile Pro Pro Ser Met
125 130 135
Met Glu Phe Asp Gly Asn Phe Asn Thr Asn Val Ser Arg Thr Ile
140 145 150
Ser Cys Asp Arg Leu Ser Thr Thr Val Asn Ser Arg Ala Phe Asn
155 160 165
Pro Gly Arg Asp Leu Asn Ser Val Leu Ala Asp Asn Leu Lys Ser
170 175 180
Asn Pro Gly Ile Lys Trp Gln Tyr Phe Ser Ser Glu Glu Gly Ile
185 190 195
Phe Thr Val Phe Pro Ala His Lys Phe Arg Cys Lys Gly Ser Tyr
200 205 210
Glu His Arg Ser Arg Pro Ile Tyr Val Ser Thr Val Arg Pro Gln
215 220 225
Ser Lys His Ile Va1 Val Ile Leu Asp His Gly Ala Ser Val Thr
230 235 240
Asp Thr Gln Leu Gln Ile Ala Lys Asp Ala Ala Gln Val Ile Leu
245 250 255
Ser Ala Ile Asp Glu His Asp Lys Ile Ser Val Leu Thr Val Ala
260 265 270
Asp Thr Val Arg Thr Cys Ser Leu Asp GIn Cys Tyr Lys Thr Phe
275 280 285
Leu Ser Pro Ala Thr Ser Glu Thr Lys Arg Lys Met Ser Thr Phe
290 295 300
Val Ser Ser Val Lys Ser Ser Asp Ser Pro Thr Gln His Ala Val
305 310 315
Gly Phe Gln Lys Ala Phe Gln Leu Ile Arg Ser Thr Asn Asn Asn
320 325 330
Thr Lys Phe Gln Ala Asn Thr Asp Met Val Ile Ile Tyr Leu Ser
335 340 345
Ala Gly Ile Thr Ser Lys Asp Ser Ser Glu Glu Asp Lys Lys Ala
350 355 360
Thr Leu Gln Val Tle Asn Glu Glu Asn Ser Phe Leu Asn Asn Ser
365 370 375
Val Met Ile Leu Thr Tyr A1a Leu Met Asn Asp Gly Val Thr Gly
380 385 390
Leu Lys Glu Leu A1a Phe Leu Arg Asp Leu Ala Glu Gln Asn Ser
395 400 405
Gly Lys Tyr Gly Va1 Pro Asp Arg Thr A1a Leu Pro Val I1e Lys
410 415 420
Gly Ser Met Met Val Leu Asn G1n Leu Ser Asn Leu Glu Thr Thr
425 430 435
Val Gly Arg Phe Tyr Thr Asn Leu Pro Asn Arg Met Ile Asp Glu
440 445 450
16/71
CA 02422497 2003-03-14
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Ala Val Phe Ser Leu Pro Phe Ser Asp Glu Met Gly Asp Gly Leu
455 460 465
Ile Met Thr Val Ser Lys Pro Cys Tyr Phe Gly Asn Leu Leu Leu
470 475 480
Gly Ile Val Gly Val Asp Val Asn Leu Ala Tyr Ile Leu Glu Asp
485 490 495
Val Thr Tyr Tyr Gln Asp Ser Leu Ala Ser Tyr Thr Phe Leu Ile
500 505 510
Asp Asp Lys Gly Tyr Thr Leu Met His Pro Ser Leu Thr Arg Pro
515 ° 520 525
Tyr Leu Leu Ser Glu Pro Pro Leu His Thr Asp Ile Ile His Tyr
530 535 540
Glu Asn Ile Pro Lys Phe Glu Leu Val Arg Gln Asn Ile Leu Ser
545 550 555
Leu Pro Leu Gly Ser Gln I1e Ile Ala Val Pro Val Asn Ser Ser
560 565 570
Leu Ser Trp His Ile Asn Lys Leu Arg Glu Thr Gly Lys Glu Ala
575 580 585
Tyr Asn Val Ser Tyr Ala Trp Lys Met Val Gln Asp Thr Ser Phe
590 595 600
Ile Leu Cys Ile Val Val Ile Gln Pro Glu Ile Pro Val Lys Gln
605 610 615
Leu Lys Asn Leu Asn Thr Val Pro Ser Ser Lys Leu Leu Tyr His
620 625 630
Arg Leu Asp Leu Leu Gly Gln Pro Ser Ala Cys Leu His Phe Lys
635 640 645
Gln Leu Ala Thr Leu Glu Ser Pro Thr Ile Met Leu Ser Ala Gly
650 655 660
Ser Phe Ser Ser Pro Tyr Glu His Leu Ser Gln Pro Glu Thr Lys
665 670 675
Arg Met Val Glu His Tyr Thr Ala Tyr Leu Ser Asp Asn Thr Arg
680 685' 690
Leu Ile Ala Asn Pro Gly Leu Lys Phe Ser Val Arg Asn Glu Val
695 700 705
Met Ala Thr Ser His Val Thr Asp Glu Trp Met Thr Gln Met Glu
710 ~ 715 720
Met Ser Ser Leu Asn Thr Tyr Ile Val Arg Arg Tyr Ile Ala Thr
725 730 735
Pro Asn Gly Val Leu Arg Ile Tyr Pro Gly Ser Leu Met Asp Lys
740 745 750
Ala Phe Asp Pro Thr Arg Arg Gln Trp Tyr Leu His Ala Va1 Ala
755 760 765
Asn Pro Gly Leu I1e Ser Leu Thr G1y Pro Tyr Leu Asp Val Gly
770 775 780
Gly Ala Gly Tyr Val Val Thr Ile Ser His Thr Ile His Ser Ser
785 790 795
Ser Thr Gln Leu Ser Ser Gly His Thr Val Ala Val Met Gly Ile
800 805 810
Asp Phe Thr Leu Arg Tyr Phe Tyr Lys Val Leu Met Asp Leu Leu
815 820 825
Pro Val Cys Asn Gln Asp Gly Gly Asn Lys Ile Arg Cys Phe Ile
830 835 840
Met Glu Asp Arg Gly Tyr Leu Val Ala His Pro Thr Leu Ile Asp
845 850 855
pro Lys Gly His Ala Pro Val Glu Gln Gln His Ile Thr His Lys
860 865 870
17/71
CA 02422497 2003-03-14
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Glu Pro Leu Val Ala Asn Asp Ile Leu Asn His Pro Asn Phe Val
875 880 885
Lys Lys Asn Leu Cys Asn Ser Phe Ser Asp Arg Thr Val Gln Arg
890 895 900
Phe Tyr Lys Phe Asn Thr Ser Leu Ala Gly Asp Leu Thr Asn Leu
905 910 915
Val His Gly Ser His Cys Ser Lys Tyr Arg Leu Ala Arg Ile Pro
920 925 930
Gly Thr Asn Ala Phe Val Gly Ile Val Asn Glu Thr Cys Asp Ser
935 940 945
Leu Ala Phe Cys Ala Cys Ser Met Val Asp Arg Leu Cys Leu Asn
950 955 960
Cys His Arg Met Glu Gln Asn Glu Cys Glu Cys Pro Cys Glu Cys
965 970 975
Pro Leu Glu Va1 Asn Glu Cys Thr Gly Asn Leu Thr Asn Ala Glu
980 985 990
Asn Arg Asn Pro Ser Cys Glu Val His Gln Glu Pro Val Thr Tyr
995 1000 1005
Thr Ala Ile Asp Pro Gly Leu G1n Asp Ala Leu His Gln Cys Val
1010 1015 1020
Asn Ser Arg Cys Ser Gln Arg Leu Glu Ser Gly Asp Cys Phe Gly
1025 1030 1035
Val Leu Asp Cys Glu Trp Cys Met Val Asp Ser Asp Gly Lys Thr
1040 1045 1050
His Leu Asp Lys Pro Tyr Cys Ala Pro Gln Lys Glu Cys Phe Gly
1055 1060 1065
Gly IIe Val Gly Ala Lys Ser Pro Tyr Val Asp Asp Met Gly AIa
1070 1075 1080
Ile Gly Asp Glu Val Ile Thr Leu Asn Met Ile Lys Ser Ala Pro
1085 1090 1095
Val Gly Pro Val Ala Gly Gly Ile Met Gly Cys Ile Met Val Leu
1100 1105 1110
Val Leu Ala Val Tyr Ala Tyr Arg His Gln IIe His Arg Arg Ser
1115 1120 1125
His Gln His Met Ser Pro Leu Ala Ala Gln Glu Met Ser Val Arg
1130 1135 1240
Met Ser Asn Leu Glu Asn Asp Arg Asp Glu Arg Asp Asp Asp Ser
1145 1150 1155
His Glu Asp Arg Gly Ile Ile Ser Asn Thr Arg Phe Ile Ala Ala
1160, 1165 1170
Val Ile Glu Arg His Ala His Ser Pro Glu Arg Arg Arg Arg Tyr
1175 1180 1185
Trp Gly Arg Ser Gly Thr Glu Ser Asp His Gly Tyr Ser Thr Met
1190 1195 1200
Ser Pro Gln Glu Asp Ser Glu Asn Pro Pro Cys Asn Asn Asp Pro
1205 1210 1215
Leu Ser AIa Gly Val Asp Val G1y Asn His Asp GIu Asp Leu Asp
1220 1225 1230
Leu Asp Thr Pro Pro Gln Thr Ala Ala Leu Leu Ser His Lys Phe
1235 1240 1245
His His Tyr Arg Ser His His Pro Thr Leu His His Ser His His
1250 1255 1260
Leu Gln Ala Ala Val Thr Val His Thr Val Asp Ala GIu Cys
1265 1270
<210> 9
18/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
<211> 595
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6113039CD1
<400> 9
Met Lys Phe Phe Ser Tyr Ile Leu Val Tyr Arg Arg Phe Leu Phe
1 5 10 15
Val Val Phe Thr Val Leu Val Leu Leu Pro Leu Pro Tle Val Leu
20 25 30
His Thr Lys Glu Ala Glu Cys Ala Tyr Thr Leu Phe Val Val Ala
35 40 45
Thr Phe Trp Leu Thr Glu Ala Leu Pro Leu Ser Val Thr Ala Leu
50 55 60
Leu Pro Ser Leu Met Leu Pro Met Phe Gly Ile Met Pro Ser Lys
65 70 75
Lys Val Ala Ser Ala Tyr Phe Lys Asp Phe His Leu Leu Leu Ile
80 85 90
Gly Val Ile Cys Leu Ala Thr Ser Ile Glu Lys Trp Asn Leu His
95 100 105
Lys Arg Ile Ala Leu Lys Met Val Met Met Val Gly Val Asn Pro
110 115 120
Ala Trp Leu Thr Leu Gly Phe Met Ser Ser Thr Ala Phe Leu Ser
125 130 135
Met Trp Leu Ser Asn Thr Ser Thr Ala Ala Met Val Met Pro Ile
140 145 150
Ala Glu Ala Val Val Gln Gln Ile Ile Asn Ala Glu Ala Glu Val
155 160 165
Glu Ala Thr Gln Met Thr Tyr Phe Asn Gly Ser Thr Asn His Gly
170 175 180
Leu Glu Ile Asp Glu Ser Val Asn Gly His Glu Ile Asn Glu Arg
185 190 195
Lys Glu Lys Thr Lys Pro Val Pro Gly Tyr Asn Asn Asp Thr Gly
200 205 210
Lys Ile Ser Ser Lys Val Glu Leu Glu Lys Asn Ser Gly Met Arg
215 220 225
Thr Lys Tyr Arg Thr Lys Lys Gly His Val Thr Arg Lys Leu Thr
230 235 ,240
Cys Leu Cys Ile A1a Tyr Ser Ser Thr Ile Gly Gly Leu Thr Thr
245 250 255
Ile Thr Gly Thr Ser Thr Asn Leu Ile Phe Ala Glu Tyr Phe Asn
260 265 270
Thr Arg Tyr Pro Asp Cys Arg Cys Leu Asn Phe Gly Ser Trp Phe
275 280 285
Thr Phe Ser Phe Pro Ala Ala Leu Ile Ile Leu Leu Leu Ser Trp
290 295 300
Ile Trp Leu Gln Trp Leu Phe Leu Gly Phe Asn Phe Lys Glu Met
305 310 315
Phe Lys Cys Gly Lys Thr Lys Thr Val Gln Gln Lys Ala Cys Ala
320 325 330
Glu Val Ile Lys Gln Glu Tyr Gln Lys Leu G1y Pro Ile Arg Tyr
335 340 345
Gln Glu IIe Val Thr Leu Val Leu Phe Ile Ile Met Ala Leu Leu
19/71
CA 02422497 2003-03-14
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350 355 360
Trp Phe Ser Arg Asp Pro Gly Phe Val Pro Gly Trp Ser Ala Leu
365 370 375
Phe Sex Glu Tyr Pro Gly Phe Ala Thr Asp Ser Thr Val Ala Leu
380 385 390
Leu Ile Gly Leu Leu Phe Phe Leu Ile Pro Ala Lys Thr Leu Thr
395 400 405
Lys Thr Thr Pro Thr Gly Glu IIe Val Ala Phe Asp Tyr Ser Pro
410 415 420
Leu Ile Thr Trp Lys Glu Phe Gln Ser Phe Met Pro Trp Asp Ile
425 430 435
Ala Ile Leu Val Gly Gly Gly Phe Ala Leu Ala Asp Gly Cys Glu
440 445 450
Glu Ser Gly Leu Ser Lys Trp Ile Gly Asn Lys Leu Ser Pro Leu
455 . 460 465
Gly Sex Leu Pro Ala Trp Leu Ile Ile Leu Tle Ser Ser Leu Met
470 475 480
Val Thr Ser Leu Thr Glu Val Ala Ser Asn Pro Ala Thr Ile Thr
485 490 495
Leu Phe Leu Pro Ile Leu Ser Pro Leu Ala Glu Ala Ile His Val
500 505 5l0
Asn Pro Leu Tyr Ile Leu Ile Pro Ser Thr Leu Cys Thr Ser Phe
515 520 525
Ala Phe Leu Leu Pro Val Ala Asn Pro Pro Asn Ala Ile Val Phe
530 535 540
Ser Tyr G1y His Leu Lys Val Ile Asp Met Val Lys A1a Gly Leu
545 550 555
Gly Val Asn Ile Val Gly Val Ala Val VaI Met Leu Gly Ile Cys
560 565 570
Thr Trp Ile Val Pro Met Phe Asp Leu Tyr Thr Tyr Pro Ser Trp
575 580 585
Ala Pro Ala Met Ser Asn Glu Thr Met Pro
590 595
<210> 10
<211> 475
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7101782CD1
<400'> 10
Met Ser Pro Glu Val Thr Cys Pro Arg Arg G1y His Leu Pro Arg
1 5 10 15
Phe His Pro Arg Thr Trp Val Glu Pro Val Val Ala Ser Ser Gln
20 25 30
Val Ala A1a Ser Leu Tyr Asp Ala Gly Leu Leu Leu Val Val Lys
35 40 45
Ala Ser Tyr GIy Thr G1y Gly Ser Ser Asn His Ser Ala Ser Pro
50 55 60
Ser Pro Arg Gly Ala Leu G1u Asp Gln Gln Gln Arg Ala Ile Ser
65 70 75
Asn Phe Tyr Ile Ile Tyr Asn Leu Val Val Gly Leu Ser Pro Leu
80 85 90
20!71
CA 02422497 2003-03-14
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Leu Ser Ala Tyr Gly Leu Gly Trp Leu Ser Asp Arg Tyr His Arg
95 100 105
Lys Ile Ser Ile Cys Met Ser Leu Leu G1y Phe Leu Leu Ser Arg
110 115 120
Leu Gly Leu Leu Leu Lys Val Leu Leu Asp Trp Pro Val Glu Val
125 130 135
Leu Tyr Gly Ala Ala Ala Leu Asn Gly Leu Phe Gly Gly Phe Ser
140 145 150
Ala Phe Trp Ser Gly Val Met Ala Leu Gly Ser Leu Gly Ser Ser
155 160 165
Glu Gly Arg Arg Ser Val Arg Leu Ile Leu I1e Asp Leu Met Leu
170 175 180
Gly Leu Ala Gly Phe Cys Gly Ser Met Ala Ser Gly His Leu Phe
185 190 195
Lys Gln Met Ala Gly His Ser Gly Gln Gly Leu Ile Leu Thr Ala
200 205 210
Cys Ser Val Ser Cys Ala Ser Phe Ala Leu Leu Tyr Ser Leu Leu
215 220 225
Val Leu Lys Val Pro Glu Ser Val Ala Lys Pro Ser Gln Glu Leu
230 235 240
Pro Ala Val Asp Thr Val Ser Gly Thr Val Gly Thr Tyr Arg Thr
245 250 255
Leu Asp Fro Asp Gln Leu Asp Gln Gln Tyr Ala Val Gly His Pro
260 265 270
Pro Ser Pro Gly Lys Ala Lys Pro His Lys Thr Thr Ile Ala Leu
275 280 285
Leu Phe Val Gly Ala Ile Ile Tyr Asp Leu Ala Val Val Gly Thr
290 295 300
Val Asp Val Ile Pro Leu Phe Val Leu Arg Glu Pro Leu Gly Trp
305 310 315
Asn Gln Val Gln Val Gly Tyr Gly Met Ala Ala Gly Tyr Thr Ile
320 325 330
Phe Ile Thr Ser Phe Leu Gly Val Leu Val Phe Ser Arg Cys Phe
335 340 345
Arg Asp Thr Thr Met Ile Met Ile Gly Met Val Ser Phe Gly Ser
350 355 360
Gly Ala Leu Leu Leu Ala Phe Val Lys Glu Thr Tyr Met Phe Tyr
365 370 375
Ile Ala Arg A1a Val Met Leu Phe Ala Leu Ile Pra Val Thr Thr
380 385 390
Ile Arg Ser Ala Met Ser Lys Leu Ile Lys Gly Ser Ser Tyr Gly
395 400 405
Lys Val Phe Val Ile Leu Gln Leu Ser Leu Ala Leu Thr Gly Val
410 415 420
Val Thr Ser Thr Leu Tyr Asn Lys Ile Tyr G1n Leu Thr Met Asp
425 430 435
Met Phe Val Gly Ser Cys Phe Ala Leu Ser Ser Phe Leu Ser Phe
440 445 450
Leu Ala Ile Ile Pro Tle Ser Ile Val Ala Tyr Lys Gln Val Pro
455 460 465
Leu Ser Pro Tyr Gly Asp Ile Ile Glu Lys
470 475
<210> 11
<211> 927
<212> PRT
21/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473036CD1
<400> 11
Met Gln Pro Ala Arg G1y Pro Leu Ala Ser Glu Pro Arg Thr Val
1 5 10 15
Leu Val Leu Arg Phe Cys Ala Ser Leu Met Glu Met Lys Leu Pro
20 25 30
Gly Gln Glu Gly Phe Glu Ala Ser Ser Ala Pro Arg Asn Ile Pro
35 40 45
Ser Gly Glu Leu Asp Ser Asn Pro Asp Pro Gly Thr Gly Pro Ser
50 55 60
Pro Asp Gly Pro Ser Asp Thr Glu Ser Lys Glu Leu G1y Val Pro
' 65 70 75
Lys Asp Pro Leu Leu Phe Ile Gln Leu Asn Glu Leu Leu Gly Trp
80 85 90
Pro Gln Ala Leu Glu Trp Arg Glu Thr Gly Arg Trp Val Leu Phe
95 100 105
Glu Glu Lys Leu Glu Val Ala Ala Gly Arg Trp Ser Ala Pro His
110 115 120
Val Pro Thr Leu Ala Leu Pro Ser Leu Gln Lys Leu Arg Ser Leu
125 130 135
Leu Ala Glu Gly Leu Val Leu Leu Asp Cys Pro Ala Gln Ser Leu
140 145 150
Leu Glu Leu Val Gly Ser Thr His Pro Arg Lys Ala Ser Asp Asn
155 160 165
Glu Glu Ala Pro Leu Arg Glu Gln Cys Gln Asn Pro Leu Arg Gln
170 175 180
Lys Leu Pro Pro Gly Ala Glu Ala Gly Thr Val Leu Ala Gly Glu
185 190 195
Leu Gly Phe Leu A1a Gln Pro Leu Gly Ala Phe Val Arg Leu Arg
200 205 210
Asn Pro Val Val Leu Gly Ser Leu Thr Glu Val Ser Leu Pro Ser
215 220 225
Arg Phe Phe Cys Leu Leu Leu Gly Pro Cys Met Leu Gly Lys Gly
230 235 240
Tyr His Glu Met Gly Arg Ala Ala Ala Val Leu Leu Ser Asp Pro
245 250 255
Gln Phe Gln Trp Ser Val Arg Arg Ala Ser Asn Leu His Asp Leu
260 265 270
Leu Ala Ala Leu Asp Ala Phe Leu Glu Glu Val Thr Val Leu Pro
275 280 285
Pro G1y Arg Trp Asp Pro Thr Ala Arg Ile Pro Pro Pro Lys Cys
290 295 300
Leu Pro Ser Gln His Lys Arg Leu Pro Ser Gln Gln Arg Glu Ile
305 310 315
Arg Gly Pro Ala Val Pro Arg Leu Thr Ser Ala Glu Asp Arg His
320 325 330
Arg His Gly Pro His Ala His Ser Pro Glu Leu Gln Arg Thr Gly
335 340 345
Ser Asp Phe Leu Asp Ala Leu His Leu Gln Cys Phe Ser Ala Val
350 355 360
Leu Tyr Tle Tyr Leu Ala Thr Val Thr Asn Ala Ile Thr Phe Gly
22/71
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365 370 375
Gly Leu Leu Gly Asp Ala Thr Asp Gly Ala Gln Gly Val Leu Glu
380 385 390
Ser Phe Leu Gly Thr Ala Val Ala Gly Ala Ala Phe Cys Leu Met
395 400 405
Ala Gly Gln Pro Leu Thr Ile Leu Ser Ser Thr Gly Pro Val Leu
410 415 420
Val Phe Glu Arg Leu Leu Phe Ser Phe Ser Arg Asp Tyr Ser Leu
425 430 435
Asp Tyr Leu Pro Phe Arg Leu Trp Val Gly Ile Trp Val Ala Thr
440 445 ~ 450
Phe Cys Leu Val Leu Val Ala Thr Glu Ala Ser Val Leu Val Arg
455 460 465
Tyr Phe Thr Arg Phe Thr Glu Glu Gly Phe Cys Ala Leu Ile Ser
470 475 480
Leu Ile Phe Ile Tyr Asp Ala Val Gly Lys Met Leu Asn Leu Thr
485 490 495
His Thr Tyr Pro Ile Gln Lys Pro Gly Sex Ser Ala Tyr Gly Cys
500 505 510
Leu Cys Gln Tyr Pro Gly Pro Gly Gly Asn Glu Ser Gln Trp Ile
515 520 525
Arg Thr Arg Pro Lys Asp Arg Asp Asp Ile Val 5er Met Asp Leu
530 535 540
Gly Leu Ile Asn Ala Ser Leu Leu Pro Pro Pro Glu Cys Thr Arg
545 550 555
Gln Gly Gly His Pro Arg Gly Pro Gly Cys His Thr Val Pro Asp
560 565 570
Ile Ala Phe Phe Ser Leu Leu Leu Phe Leu Thr Ser Phe Phe Phe
575 580 585
Ala Met Ala Leu Lys Cys Val Lys Thr Ser Arg Phe Phe Pro Ser
590 595 600
Val Val Arg Lys Gly Leu Ser Asp Phe Ser Ser Val Leu Ala Ile
605 610 615
Leu Leu Gly Cys Gly Leu Asp Ala Phe Leu Gly Leu Ala Thr Pro
620 625 630
Lys Leu Met Val Pro Arg Glu Phe Lys Pro Thr Leu Pro Gly Arg
635 640 645
Gly Trp Leu Val Ser Pro Phe Gly Ala Asn Pro Trp Trp Trp Ser
650 655 660
Val Al,a Ala Ala Leu Pro Ala Leu Leu Leu Ser Ile Leu Ile Phe
665 670 675
Met Asp GIn Gln Ile Thr Ala Val Ile Leu Asn Arg Met Glu Tyr
680 685 690
Arg Leu Gln Lys Gly Ala Gly Phe His Leu Asp Leu Phe Cys Val
695 700 705
Ala Va1 Leu Met Leu Leu Thr Ser Ala Leu Gly Leu Pro Trp Tyr
710 715 720
Val Ser Ala Thr Val Ile Ser Leu Ala His Met Asp Ser Leu Arg
725 730 735
Arg G1u Ser Arg Ala Cys Ala Pro Gly Glu Arg Pro Asn Phe Leu
740 745 750
Gly Ile Arg Glu Gln Arg Leu Thr Gly Leu Val Val Phe Ile Leu
755 760 765
Thr Gly Ala Ser IIe Phe Leu Ala Pro Val Leu Lys Phe Ile Pro
770 775 780
Met Pro Val Leu Tyr Gly Ile Phe Leu Tyr Met Gly Va1 A1a Ala
23/71
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785 790 795
Leu Ser Ser Ile Gln Phe Thr Asn Arg Val Lys Leu Leu Leu Met
800 805 810
Pro Ala Lys His Gln Pro Asp Leu Leu Leu Leu Arg His Val Pro
815 820 825
Leu Thr Arg Val His Leu Phe Thr Ala Ile Gln Leu Ala Cys Leu
830 835 840
Gly Leu Leu Trp Ile Ile Lys Ser Thr Pro Ala Ala Ile Ile Phe
845 850 855
Pro Leu Met Leu Leu Gly Leu Val G1y Val Arg Lys Ala Leu Glu
860 865 870
Arg Val Phe Ser Pro Gln Glu Leu Leu Trp Leu Asp Glu Leu Met
875 880 885
Pro Glu G1u Glu Arg Ser Ile Pro Glu Lys Gly Leu Glu Pro Glu
890 895 900
His Ser Phe Ser Gly Ser Asp Ser Glu Asp Ser Glu Leu Met Tyr
905 910 915
Gln Pro Lys Ala Pro Glu Ile Asn Ile Ser Val Asn
920 925
<210> 12
<211> 516
<212> PRT
<213> Homo sapiens
<220>
<221> misC_feature
<223> Incyte ID No: 7476943CD1
<400> 12
Met Pro Ser Gly Ser His Trp Thr Ala Asn Ser Ser Lys Ile Ile
1 5 10 15
Thr Trp Leu Leu Glu Gln Pro Gly Lys Glu Glu Lys Arg Lys Thr
20 25 30
Met Ala Lys Val Asn Arg Ala Arg Ser Thr Ser Pro Pro Asp Gly
35 40 45
Gly Trp Gly Trp Met Ile Val Ala Gly Cys Phe Leu Val Thr Ile
50 55 60
Cys Thr Arg Ala Val Thr Arg Cys Ile 5er Ile Phe Phe Val Glu
65 70 75
Phe Gln Thr Tyr Phe Thr Gln Asp Tyr Ala Gln Thr Ala Trp Ile
80 85 90
His Ser Ile Val Asp Cys Val Thr Met Leu Cys Ala Pro Leu Gly
95 100 105
Ser Val Val Ser Asn His Leu Ser Cys Gln Val Gly Ile Met Leu
110 115 120
Gly Gly Leu Leu Ala Ser Thr Gly Leu Ile Leu Ser Ser Phe Ala
125 130 135
Thr Ser Leu Lys His Leu Tyr Leu Thr Leu G3y Val Leu Thr Gly
140 145 150
Leu Gly Phe Aha Leu Cys Tyr Ser Pro Ala Ile Ala Met Val Gly
155 160 165
Lys Tyr Phe Ser Arg Arg Lys Ala Leu Ala Tyr Gly Ile Ala Met
170 175 180
Ser Gly Ser Gly Ile Gly Thr Phe Ile Leu Ala Pro Val Val Gln
185 190 195
24/71
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Leu Leu Ile Glu Gln Phe Ser Trp Arg Gly Ala Leu Leu Ile Leu
200 205 210
Gly Gly Phe Val Leu Asn Leu Cys Val Cys Gly Ala Leu Met Arg
215 220 225
Pro Ile Thr Leu Lys Glu Asp His Thr Thr Pro Glu Gln Asn His
230 235 240
Val Cys Arg Thr Gln Lys Glu Asp Ile Lys Arg Val Ser Pro Tyr
245 250 255
Ser Ser Leu Thr Lys Glu Trp Ala Gln Thr Cys Leu Cys Cys Cys
260 265 270
Leu Gln Gln Glu Tyr Ser Phe Leu Leu Met Ser Asp Phe Val Val
275 280 285
Leu Ala Val Ser Val Leu Phe Met Ala Tyr Gly Cys Ser Pro Leu
290 295 300
Phe Val Tyr Leu Val Pro Tyr Ala Leu Ser Val Gly Val Ser His
305 310 315
Gln Gln Ala Ala Phe Leu Met Ser Ile Leu Gly Val Ile Asp Ile
320 325 330
Ile Gly Asn Ile Thr Phe Gly Trp Leu Thr Asp Arg Arg Cys Leu
335 340 345
Lys Asn Tyr Gln Tyr Val Cys Tyr Leu Phe Ala Val Gly Met Asp
350 355 360
Gly Leu Cys Tyr Leu Cys Leu Pro Met Leu Gln Ser Leu Pro Leu
365 370 375
Leu Val Pro Phe Ser Cys Thr Phe Gly Tyr Phe Asp Gly Ala Tyr
380 385 390
Val Thr Leu Ile Pro Val Val Thr Thr Glu Ile Val Gly Thr Thr
395 400 405
Ser Leu Ser Ser Ala Leu Gly Val Val Tyr Phe Leu His Ala Val
410 415 420
Pro Tyr Leu Val Ser Pro Pro Ile Ala Gly Arg Leu Val Asp Thr
425 430 435
Thr Gly Ser Tyr Thr Ala Ala Phe Leu Leu Cys Gly Phe Ser Met
440 445 450
Ile Phe 5er Ser Val Leu Leu Gly Phe Ala Arg Leu Ile Lys Arg
455 460 465
Met Arg Zys Thr Gln Leu Gln Phe Ile Ala Lys Glu Ser Asp Pro
470 475 480
Lys Leu Gln Leu Trp Thr Asn Gly Ser Va1 Ala Tyr Ser Val Ala
485 490 495
Arg Glu Leu Asp Gln Lys His Gly Glu Pro Val Ala Thr Ala Val
500 505 510
Pro G1y Tyr Ser Leu Thr
515
<210> 13
<211> 514
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 8003355CD1
<400> 13
Met His Gly Gly G1n Gly Pro Leu Leu Leu Leu Leu Leu Leu Ala
25/71
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1 5 10 15
Val Cys Leu Gly Ala Gln Gly Arg Asn Gln Glu Glu Arg Leu Leu
20 25 30
Ala Asp Leu Met Gln Asn Tyr Asp Pro Asn Leu Arg Pro Ala G1u
35 40 45
Arg Asp Ser Asp Val Val Asn Val Ser Leu Lys Leu Thr Leu Thr
50 55 60
Asn Leu Ile Ser Leu Asn Glu Arg Glu Glu Ala Leu Thr Thr Asn
65 70 75
Val Trp Ile Glu Val Gln Trp Cys Asp Tyr Arg Leu Arg Arg Asp
80 85 90
Pro Arg Asp Tyr Glu Gly Leu Trp Val Leu Arg Val Pro Ser Thr
95 100 105
Met Val Trp Arg Pro Asp Ile Val Leu Glu Asn Asn Ala Asp Gly
110 115 120
Val Phe Glu Val Ala Leu Tyr Cys Asn Val Leu Val Ser Pro Asp
125 130 135
Gly Cys Ile Tyr Trp Leu Pro Pro Ala Ile Phe Arg Ser Ala Cys
140 145 150
Ser Ile Ser Val Thr Tyr Phe Pro Phe Asp Trp Gln Asn Cys Ser
155 160 165
Leu Ile Phe Gln Ser Gln Thr Tyr Ser Thr Asn Glu Ile Asp Leu
170 175 180
Gln Leu Ser Gln Glu Asp Gly Gln Thr Ile Glu Trp Ile Phe Ile
185 190 195
Asp Pro Glu Ala Phe Thr Glu Asn Gly Glu Trp Ala Ile Gln His
200 205 210
Arg Pro Ala Lys Met Leu Leu Asp Pro Ala Ala Pro Ala Gln Glu
215 220 225
Ala Gly His Gln Lys Val Val Phe Tyr Leu Leu Ile Gln Arg Lys
230 235 240
Pro Leu Phe Tyr Val Ile Asn Ile Ile Ala Pro Cys Val Leu Ile
245 250 255
Ser Ser Val Ala Ile Leu Ile His Phe Leu Pro Ala Lys Ala Gly
260 265 270
Gly Gln Lys Cys Thr Val Ala Ile Asn Va1 Leu Leu Ala Gln Thr
275 280 285
Val Phe Leu Phe Leu Val Ala Lys Lys Val Pro Glu Thr Ser Gln
290 295 300
Ala Val Pro Leu Ile Ser Lys Tyr Leu Thr Phe Leu Leu Val Val
305 310 315
Thr Ile Leu Ile Val Val Asn Ala Val Val Val Leu Asn Val Ser
320 325 330
Leu Arg Ser Pro His Thr His Ser Met Ala Arg Gly Val Phe Leu
335 340 345
Arg Leu Leu Pro Gln Leu Leu Arg Met His Val Arg Pro Leu Ala
350 355 360
Pro Ala Ala Val Gln Asp Thr Gln 5er Arg Leu Gln Asn Gly Ser
365 370 375
Ser Gly Trp Ser Ile Thr Thr Gly Glu Glu Val Ala Leu Cys Leu
380 385 390
Pro Arg Ser Glu Leu Leu Phe Gln Gln Trp Gln Arg Gln Gly Leu
395 400 405
Val Ala Ala Ala Leu G1u Lys Leu Glu Lys Gly Pro Glu Leu Gly
410 415 420
Leu Ser Gln Phe Cys Gly Sex Leu Lys Gln Ala Ala Pro Ala Tle
26/71
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425 430 435
Gln Ala Cys Val Glu Ala Cys Asn Leu Ile Ala Cys Ala Arg His
440 445 450
Gln Gln Ser His Phe Asp Asn Gly Asn Glu Glu Trp Phe Leu Val
455 460 465
Gly Arg Val Leu Asp Arg Val Cys Phe Leu Ala Met Leu Ser Leu
470 475 480
Phe Ile Cys Gly Thr Ala Gly Ile Phe Leu Met Ala His Tyr Asn
485 490 495
Arg Val Pro Ala Leu Pro Phe Pro Gly Asp Pro Arg Pro Tyr Leu
500 505 510
Pro Ser Pro Asp
<210> 14
<211> 691
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3116448CD1
<400> 14
Met Glu Leu Arg 5er Thr Ala Ala Pro Arg Ala Glu Gly Tyr Ser
1 5 10 15
Asn Val Gly Phe Gln Asn Glu Glu Asn Phe Leu Glu Asn GIu Asn
20 25 30
Thr Ser Gly Asn Asn Ser Ile Arg Ser Arg Ala Val Gln Ser Arg
35 40 45
Glu His Thr Asn Thr Lys Gln Asp Glu Glu Gln Val Thr Val Glu
50 55 60
Gln Asp Ser Pro Arg Asn Arg Glu His Met Glu Asp Asp Asp Glu
65 70 75
Glu Met Gln Gln Lys Gly Cys Leu Glu Arg Arg Tyr Asp Thr Val
80 85 90
Cys Gly Phe Cys Arg Lys His Lys Thr Thr Leu Arg His Ile Ile
95 100 105
Trp Gly Ile Leu Leu Ala Gly Tyr Leu Val Met Val Ile Ser Ala
110 115 120
Cys Val Leu Asn Phe His Arg Ala Leu Pro Leu Phe Val Ile Thr
125 130 135
Val Ala Ala Ile Phe Phe Val Val Trp Asp His Leu Met Ala Lys
140 145 150
Tyr Glu His Arg Ile Asp Glu Met Leu Ser Pro Gly Arg Arg Leu
155 160 265
Leu Asn Ser His Trp Phe Trp Leu Lys Trp Val Ile Trp Ser Ser
170 175 180
Leu Va1 Leu Ala Val Ile Phe Trp Leu Ala Phe Asp Thr Ala Lys
185 190 195
Leu Gly Gln Gln Gln Leu Val Ser Phe Gly Gly Leu Ile Met Tyr
200 205 210
Ile Val Leu Leu Phe Leu Phe Ser Lys Tyr Pro Thr Arg Val Tyr
215 220 225
Trp Arg Pro Val Leu Trp Gly Ile G1y Leu Gln Phe Leu Leu Gly
230 235 240
27/71
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Leu Leu Ile Leu Arg Thr Asp Pro Gly Phe Ile Ala Phe Asp Trp
245 250 255
Leu Gly Arg Gln Val Gln Thr Phe Leu Glu Tyr Thr Asp Ala Gly
260 265 270
Ala Ser Phe Gly Phe Gly Glu Lys Tyr Lys Asp His Phe Phe Gly
275 280 285
Phe Lys Val Leu Ala Ile Val Val Phe Phe Ser Thr Val Met Ser
290 295 300
Met Leu Tyr Tyr Leu Gly Leu Met Gln Trp Tle Ile Arg Lys Val
305 310 315
Gly Trp Ile Met Leu Val Thr Thr Gly Ser Ser Pro Ile Glu Ser
320 325 330
Val Val Ala Ser Gly Asn Ile Phe Val G1y Gln Thr Glu Ser Pro
335 340 345
Leu Leu Val Arg Pro Tyr Leu Pro Tyr Ile Thr Lys Ser Glu Leu
350 355 360
His A1a Ile Met Thr Ala Gly Phe Ser Thr Ile Ala Gly Ser Val
365 370 375
Leu Gly Ala Tyr Ile Ser Phe Gly Val Pro Ser Ser His Leu Leu
380 385 390
Thr Ala Ser Val Met Ser Ala Pro A1a Ser Leu Ala Ala Ala Lys
395 400 405
Leu Phe Trp Pro G1u Thr Glu Lys Pro Lys Ile Thr Leu Lys Asn
410 415 420
Ala Met Lys Met Glu Ser Gly Asp Ser Gly Asn Leu Leu Glu Ala
425 430 435
Ala Thr Gln Gly Ala Ser Ser Ser Ile Ser Leu Val Ala Asn Ile
440 445 450
Ala Val Asn Leu Ile Ala Phe Leu Ala Leu Leu Ser Phe Met Asn
455 460 465
Ser Ala Leu Ser Trp Phe Gly Asn Met Phe Asp Tyr Pro Gln Leu
470 475 480
Ser Phe Glu Leu I1e Cys Ser Tyr Tle Phe Met Pro Phe Ser Phe
485 490 495
Met Met Gly Val G1u Trp Gln Asp Ser Phe Met Vah Ala Arg Leu
500 505 510
Ile Gly Tyr Lys Thr Phe Phe Asn Glu Phe Val Ala Tyr Glu His
515 520 525
Leu Ser Lys Trp Ile His Leu Arg Lys Glu Gly Gly Pro Lys Phe
530 535 540
Val Asn Gly Val Gln Gln Tyr Ile Ser Ile Arg Ser Glu Ile Ile
545 550 555
Ala Thr Tyr Ala Leu Cys G1y Phe Ala Asn Ile Gly Ser Leu Gly
560 565 570
Ile Val Ile Gly Gly Leu Thr Ser Met Ala Pro Ser Arg Lys Arg
575 580 585
Asp Ile Ala Ser Gly Ala Val Arg Ala Leu Ile Ala Gly Thr Val
590 595 600
A1a Cys Phe Met Thr Ala Cys Ile Ala Gly Ile Leu 5er Ser Thr
605 610 615
Pro Val Asp Ile Asn Cys His His Val Leu Glu Asn Ala Phe Asn
620 625 630
Ser Thr Phe Pro Gly Asn Thr Thr Lys Val Ile Ala Cys Cys Gln
635 640 645
Ser Leu Leu Ser Ser Thr Val Ala Lys Gly Pro Gly Glu Val Ile
650 655 660
28/71
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Pro Gly Gly Asn His Ser Leu Tyr Ser Leu Lys Gly Cys Cys Thr
665 670 675
Leu Leu Asn Pro Ser Thr Phe Asn Cys Asn Gly Ile Ser Asn Thr
680 685 690
Phe
<210> 15
<211> 342
<222> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 622868CD1
<400> 15
Met Lys Ser Arg Thr Trp Ala Ser Val His Leu His Ser Phe Phe
1 5 10 15
AIa Val Gly Thr Leu Leu Val Ala Leu Thr Gly Tyr Leu Val Arg
20 25 30
Thr Trp Trp Leu Tyr Gln Met Ile Leu Ser Thr Val Thr Val Pro
35 40 45
Phe Ile Leu Cys Cys Trp Val Leu Pro Glu Thr Pro Phe Trp Leu
50 55 60
Leu Ser Glu Gly Arg Tyr Glu Glu Ala Gln Lys Ile Val Asp Ile
65 70 75
Met Ala Lys Trp Asn Arg Ala Ser Ser Cys Lys Leu Ser Glu Leu
80 85 90
Leu Ser Leu Asp Leu Gln Gly Pro Val Ser Asn Ser Pro Thr Glu
95 100 105
Val Gln Lys His Asn Leu Ser Tyr Leu Phe Tyr Asn Trp Ser Ile
110 115 120
Thr Lys Arg Thr Leu Thr Val Trp Leu Ile Trp Phe Thr Gly Ser
125 130 135
Leu Gly Phe Tyr Ser Phe Ser Leu Asn Sex Val Asn Leu Gly G1y
140 145 150
Asn Glu Tyr Leu Asn Leu Phe Leu Leu Gly Val Val Glu Ile Pro
155 160 165
Ala Tyr Thr Phe Val Cys Ile A1a Thr Asp Lys Val Gly Arg Arg
170 175 180
Thr Va1 Leu Ala Tyr Ser Leu Phe Cys Ser Ala Leu Ala Cys Gly
185 190 195
Val Val Met Val Ile Pro Gln Lys His Tyr Ile Leu Gly Val Val
200 205 210
Thr Ala Met Val Gly Lys Phe Ala Ile Gly Ala Ala Phe Gly Leu
215 220 225
Ile Tyr Leu Tyr Thr Ala Glu Leu Tyr Pro Thr Ile Val Arg Ser
230 235 240
Leu Ala Val Gly Ser Gly 5er Met Val Cys Arg Leu Ala Ser Ile
245 250 255
Leu Ala Pro Phe Ser Val Asp Leu Ser Ser Ile Trp Ile Phe Ile
260 265 270
Pro Gln Leu Phe Val Gly Thr Met Ala Leu Leu Ser G1y Val Leu
275 280 285
Thr Leu Lys Leu Pro Glu Thr Leu Gly Lys Arg Leu Ala Thr Thr
29/71
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290 295 300
Trp Glu Glu Ala Ala Lys Leu Glu Ser Glu Asn Glu Ser Lys Ser
305 320 315
Ser Lys Leu Leu Leu Thr Thr Asn Asn Ser Gly Leu Glu Lys Thr
320 325 330
Glu Ala Ile Thr Pro Arg Asp 5er Gly Leu Gly Glu
335 340
<210> 16
<211> 791
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7476494CD1
<400> 16
Met Gly His Phe Glu Lys Gly Gln His Ala Leu Leu Asn Glu Gly
1 5 10 15
Glu Glu Asn Glu Met Glu Ile Phe Gly Tyr Arg Thr Gln Gly Cys
20 25 30
Arg Lys Ser Leu Cys Leu Ala Gly Ser Ile Phe Ser Phe Gly Ile
35 40 45
Leu Pro Leu Val Phe Tyr Trp Arg Pro Ala Trp His Val Trp Ala
50 55 60
His Cys Val Pro Cys Ser Leu Gln Glu Ala Asp Thr Val Leu Leu
65 70 75
Arg Thr Thr Val Arg Cys Ile Lys Val Gln Lys Ile Arg Tyr Val
80 85 90
Trp Asn Tyr Leu Glu Gly Gln Phe Gln Lys Ile Gly Ser Leu Glu
95 100 105
Asp Trp Leu Ser Ser Ala Lys Ile His Gln Lys Phe Gly Ser Gly
110 115 120
Leu Thr Arg Glu Glu Gln Glu Ile Arg Arg Leu Met Cys Gly Pro
125 130 135
Asn Thr Ile Asp Val Glu Val Thr Pro Ile Trp Lys Leu Leu I1e
140 145 150
Lys Glu Val Leu Asn Pro Phe Tyr Ile Phe Gln Leu Phe Ser Val
155 160 165
Cys Leu Trp Phe Ser Glu Asp Tyr Lys Glu Tyr Ala Phe Ala Ile
170 175 180
I1e Ile Met Ser Ile Ile Ser Ile Ser Leu Thr Val Tyr Asp Leu
185 190 195
Arg Glu Gln Ser Val Lys Leu His His Leu Val Glu Ser His Asn
200 205 210
Ser Ile Thr Val Ser Val Cys Gly Arg Lys Ala Gly Val Gln Glu
215 220 225
Leu Glu Ser Arg Val Leu Val Pro Gly Asp Leu Leu Ile Leu Thr
230 235 240
Gly Asn Lys Val Leu Met Pro Cys Asp Ala Val Leu Ile Glu Gly
245 250 255
Ser Cys Val Val Asp Glu Gly Met Leu Thr Gly Glu Ser Ile Pro
260 265 270
Val Thr Lys Thr Pro Leu Pro Lys Met Asp Ser Ser Val Pro Trp
275 280 285
30/71
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Lys Thr Gln Ser Glu Ala Asp Tyr Lys Arg His Val Leu Phe Cys
290 295 300
Gly Thr Glu Val Ile Gln Ala Lys Ala Ala Cys Ser Gly Thr Val
305 310 315
Arg Ala Val Val Leu G1n Thr Gly Phe Asn Thr Ala Lys Gly Asp
320 325 330
Leu Val Arg Ser Ile Leu Tyr Pro Lys Pro Val Asn Phe Gln Leu
335 340 345
Tyr Arg Asp AIa Ile Arg Phe Leu Leu Cys Leu Val Gly Thr Ala
350 355 360
Thr Ile Gly Met Ile Tyr Thr Leu Cys Val Tyr Val Leu Ser Gly
365 370 375
Glu Pro Pro Glu Glu Val Val Arg Lys Ala Leu Asp Val Ile Thr
380 385 390
Ile Ala Val Pro Pro Ala Leu Pro Ala Ala Leu Thr Thr GIy Tle
395 400 405
Ile Tyr Ala Gln Arg Arg Leu Lys Lys Arg Gly Ile Phe Cys Ile
410 415 420
Ser Pro Gln Arg Ile Asn Val Cys Gly Gln Leu Asn Leu Val Cys
425 430 435
Phe Asp Lys Thr Gly Thr Leu Thr Arg Asp Gly Leu Asp Leu Trp
440 445 450
Gly Val Val Ser Cys Asp Arg Asn Gly Phe Gln Glu Val His 5er
455 460 465
Phe Ala Ser Gly Gln Ala Leu Pro Trp Gly Pro Leu Cys Ala Ala
470 475 480
Met Ala Ser Cys His Ser Leu Ile Leu Leu Asp Gly Thr Ile Gln
485 490 495
Gly Asp Pro Leu Asp Leu Lys Met Phe Glu Ala Thr Thr Trp Glu
500 505 520
Met Ala Phe Ser Gly Asp Asp Phe His I1e Lys Gly Val Pro Ala
515 520 525
His Ala Met Va1 Val Lys Pro Cys Arg Thr Ala Ser Gln Val Pro
530 535 540
Val Glu Gly Ile Ala Ile Leu His Gln Phe Pro Phe Ser Ser Ala
545 550 555
Leu Gln Arg Met Thr Val Ile Val Gln Glu Met Gly Gly Asp Arg
560 565 570
Leu Ala Phe Met Lys Gly Ala Pro Glu Arg Val Ala Ser Phe Cys
575 580 585
Gln Pro Glu Thr Val Pro Thr Ser Phe Val Ser Glu Leu Gln Ile
590 595 600
Tyr Thr Thr Gln GIy Phe Arg Val Ile Ala Leu Ala Tyr Lys Lys
605 610 615
Leu Glu Asn Asp His His Ala Thr Thr Leu Thr Arg G1u Thr Val
620 625 630
Glu Ser Asp Leu Tle Phe Leu Gly Leu Leu Ile Leu Glu Asn Arg
635 640 645
Leu Lys Glu Glu Thr Lys Pro Val Leu Glu Glu Leu IIe Ser Ala
650 655 660
Arg Ile Arg Thr Val Met Ile Thr Gly Asp Asn Leu Gln Thr Ala
665 670 675
Ile Thr Val Ala Arg Lys Ser Gly Met Val Ser Glu Ser Gln Lys
680 685 690
Val Ile Leu Ile Glu Ala Asn Glu Thr Thr Gly Ser Ser Ser Ala
695 700 705
31/71
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Ser Ile Ser Trp Thr Leu Val Glu Glu Lys Lys His Ile Met Tyr
710 715 720
Gly Asn Gln Asp Asn Tyr Ile Asn Ile Arg Asp Glu Val Ser Asp
725 730 735
Lys Gly Arg Glu Gly Ser Tyr His Phe Ala Leu Thr Gly Lys Ser
740 745 750
Phe His Val Ile Ser Gln His Phe Ser Ser Leu Leu Pro Lys Ile
755 760 7&5
Leu Ile Asn Gly Thr Ile Phe Ala Arg Met Ser Pro Gly Gln Lys
770 775 780
Ser Ser Leu Val Glu Glu Phe Gln Lys Leu Glu
785 790
<210> 27
<211> 1108
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7477260CD1
<400> 17
Met Val Thr Gly Gly Gln His His Pro Gly Ala Gly Leu Ser Phe
1 5 10 15
Thr G1u Leu Glu Asn Thr Phe Pro Leu Cys Leu Pro Pro Thr Pro
20 25 30
Phe Leu Leu Ala Leu Trp Ser Ser Cys Leu Pro Trp Asp Thr Gln
35 40 45
Gln Thr Cys Cys Pro 5er Phe Ala Gly Ser Pro Ala Ala G1u Gln
50 55 60
Leu Gln Asp Ile Leu Gly Glu Glu Asp Glu A1a Pro Asn Pro Thr
65 70 75
Leu Phe Thr Glu Met Asp Thr Leu Gln His Asp Gly Asp Gln Met
80 85 90
Glu Trp Lys Glu Ser Ala Arg Trp Ile Lys Phe Glu Glu Lys Val
95 100 105
Glu Glu Gly Gly Glu Arg Trp Ser Lys Pro His Val Ser Thr Leu
110 115 120
Ser Leu His Ser Leu Phe Glu Leu Arg Thr Cys Leu G1n Thr Gly
125 130 135
Thr Val Leu Leu Asp Leu Asp Ser Gly Ser Leu Pro Gln Ile Ile
140 145 150
Asp Asp Val I1e Glu Lys Gln I1e Glu Asp G1y Leu Leu Arg Pro
155 160 165
Glu Leu Arg Glu Arg Val Ser Tyr Val Leu Leu Arg Arg His Arg
170 175 180
His Gln Thr Lys Lys Pro Ile His Arg Ser Leu Ala Asp Ile Gly
185 190 195
Lys Ser Val Ser Thr Thr Asn Arg Ser Pro Ala Arg Ser Pro Gly
200 205 210
Ala G1y Pro Ser Leu His His Ser Thr Glu Asp Leu Arg Met Arg
215 220 225
Gln Ser Ala Asn Tyr Gly Arg Leu Cys His Ala Gln Ser Arg Ser
230 235 240
Met Asn Asp Ile Ser Leu Thr Pro Asn Thr Asp Gln Arg Lys Asn
32/71
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245 250 255
Lys Phe Met Lys Lys Ile Pro Lys Asp Ser Glu Ala Ser Asn Val
260 265 270
Leu Val Gly Glu Val Asp Phe Leu Asp Gln Pro Phe Ile Ala Phe
275 280 285
Val Arg Leu Ile Gln Ser Ala Met Leu Gly Gly Val Thr Glu Val
290 295 300
Pro Val Pro Thr Arg Phe Leu Phe Ile Leu Leu Gly Pro Ser Gly
305 310 315
Arg Ala Lys Ser Tyr Asn Glu Ile Gly Arg Ala Ile Ala Thr Leu
320 325 330
Met Val Asp Asp Leu Phe Ser Asp Val Ala Tyr Lys Ala Arg Asn
335 340 345
Arg Glu Asp Leu Ile Ala Gly Ile Asp G1u Phe L2u Asp Glu Val
350 355 360
Ile Val Leu Pro Pro Gly Glu Trp Asp Pro Asn Ile Arg Ile Glu
365 370 375
Pro Pro Lys Lys Val Pro Ser Ala Asp Lys Arg Lys Ser Leu Phe
380 385 390
Ser Leu Ala Glu Leu Gly Gln Met Asn Gly Ser Val Gly Gly Gly
395 400 405
Gly Gly Ala Pro Gly Gly Gly Asn Gly Gly Gly Gly Gly Gly Gly
410 415 420
Ser Gly Gly GIy AIa Gly Ser Gly Gly Ala Gly Gly Thr Ser Ser
425 430 435
Gly Asp Asp Gly Glu Met Pro Ala Met His Glu Ile Gly Glu Glu
440 445 450
Leu Ile Trp Thr Gly Arg Phe Phe Gly Gly Leu Cys Leu Asp Ile
455 460 465
Lys Arg Lys Leu Pro Trp Phe Pro Ser Asp Phe Tyr Asp Gly Phe
470 475 480
His Ile Gln Ser Ile Ser Ala Ile Leu Phe Ile Tyr Leu Gly Cys
485 490 495
Ile Thr Asn Ala Ile Thr Phe G1y Gly Leu Leu Gly Asp Ala Thr
500 505 510
Asp Asn Tyr Gln Gly Val Met Glu Ser Phe Leu Gly Thr Ala Met
515 520 525
A1a Gly Ser Leu Phe Cys Leu Phe Ser Gly Gln Pro Leu Ile Ile
530 535 540
Leu Ser Ser Thr G1y Pro Ile Leu Ile Phe Glu Lys Leu Leu Phe
545 550 555
Asp Phe Ser Lys Gly Asn Gly Leu Asp Tyr Met Glu Phe Arg Leu
560 565 570
Trp Ile Gly Leu His Ser A1a Val Gln Cys Leu Ile Leu Val Ala
575 580 585
Thr Asp Ala Ser Phe Ile Ile Lys Tyr Ile Thr Arg Phe Thr Glu
590 595 600
G1u Gly Phe Ser Thr Leu Ile Ser Phe Ile Phe Ile Tyr Asp Ala
605 610 615
Ile Lys Lys Met Ile Gly A1a Phe Lys Tyr Tyr Pro Ile Asn Met
620 625 630
Asp Phe Lys Pro Asn Phe Ile Thr Thr Tyr Lys Cys Glu Cys Val
635 640 645
Ala Pro Asp Thr Gly Asp Leu Asn Thr Thr Val Phe Asn Ala Ser
650 655 660
Ala Pro Leu Ala Pro Asp Thr Asn Ala Ser Leu Tyr Asn Leu Leu
33/71
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665 670 675
Asn~Leu Thr Ala Leu Asp Trp Ser Leu Leu Ser Lys Lys Glu Cys
680 685 690
Leu Ser Tyr Gly Gly Arg Leu Leu Gly Asn Ser Cys Lys Phe Ile
695 700 705
Pro Asp Leu Ala Leu Met Ser Phe Ile Leu Phe Phe Gly Thr Tyr
710 715 720
Ser Met Thr Leu Thr Leu Lys Lys Phe Lys Phe Ser Arg Tyr Phe
725 730 735
Pro Thr Lys Val Arg Ala Leu Val Ala Asp Phe Ser Ile Val Phe
740 745 750
Ser Ile Leu Met Phe Cys Gly Ile Asp Ala Cys Phe Gly Leu Glu
755 760 765
Thr Pro Lys Leu His Val Pro Ser Val Ile Lys Pro Thr Arg Pro
770 775 780
Asp Arg Gly Trp Phe Val Ala Pro Phe Gly Lys Asn Pro Trp Trp
785 790 795
Val Tyr Pro Ala Ser Ile Leu Pro Ala Leu Leu Val Thr Ile Leu
800 805 810
Ile Phe Met Asp Gln Gln Ile Thr Ala Val Ile Val Asn Arg Lys
815 820 825
Glu Asn Lys Leu Lys Lys Ala Ala Gly Tyr His Leu Asp Leu Phe
830 835 840
Trp Val Gly Ile Leu Met Ala Leu Cys Ser Phe Met Gly Leu Pro
845 850 855
Trp Tyr Val Ala Ala Thr Val Ile Ser Ile Ala His Ile Asp Ser
860 865 870
Leu Lys Met Glu Thr Glu Thr Ser Ala Pro Gly G1u Gln Pro Gln
875 880 885
Phe Leu G1y Val Arg Glu Gln Arg Val Thr Gly Ile Ile Val Phe
890 ~ 895 900
Ile Leu Thr Gly Ile Ser Val Phe Leu Ala Pro Ile Leu Lys Cys
905 910 915
Ile Pro Leu Pro Val Leu Tyr Gly Val Phe Leu Tyr Met Gly Val
920 925 930
Ala Ser Leu Asn Gly 21e Gln Phe Trp Glu Arg Cys Lys Leu Phe
935 940 945
Leu Met Pro Ala Lys His Gln Pro Asp His Ala Phe Leu Arg His
950 955 960
Val Pro Leu Arg Arg Ile His Leu Phe Thr Leu Val Gln Ile Leu
965 i 970 975
Cys Leu Ala Val Leu Trp Ile Leu Lys Ser Thr Val Ala Ala Ile
980 985 990
Ile Phe Pro Val Met Ile Leu Gly Leu Ile Ile Val Arg Arg Leu
995 1000 1005
Leu Asp Phe Ile Phe Ser Gln His Asp Leu Ala Trp Ile Asp Asn
1010 1015 1020
Ile Leu Pro Glu Lys Glu Lys Lys Glu Thr Asp Lys Lys Arg Lys
1025 1030 2035
Arg Lys Lys Gly Ala His Glu Asp Cys Asp Glu Glu Glu Lys Asp
1040 1045 1050
Leu Pro Val Gly Val Thr His Ser Asp Ser Ser Phe Ser Asp Thr
1055 1060 1065
Glu Leu Asp Arg Ser Tyr Ser Arg Asn Pro Val Phe Met Val Pro
1070 1075 1080
Gln Val Lys Ile Glu Met Glu Ser Asp Tyr Asp Phe Thr Asp Met
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1085 1090 1095
Asp Lys Tyr Arg Arg Glu Thr Asp Ser Glu Thr Thr Leu
1100 1105
<210> 18
<211> 480
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1963058CD1
<400> 18
Met Gly Pro Gly Pro Pro Ala Al.a Gly Ala Ala Pro Ser Pro Arg
1 5 10 15
Pro Leu Ser Leu Val Ala Arg Leu Ser Tyr Ala Val Gly His Phe
20 25 30
Leu Asn Asp Leu Cys Ala Ser Met Trp Phe Thr Tyr Leu Leu Leu
35 40 45
Tyr Leu His Ser Val Arg Ala Tyr Ser Ser Arg Gly Ala'Gly Leu
50 55 60
Leu Leu Leu Leu Gly Gln Val Ala Asp Gly Leu Cys Thr Pro Leu
65 70 75
Val Gly Tyr Glu Ala Asp Arg Ala Ala Ser Cys Cys Ala Arg Tyr
80 85 90
Gly Pro Arg Lys Ala Trp His Leu Val Gly Thr Val Cys Val Leu
95 100 105
Leu Ser Phe Pro Phe Ile Phe Ser Pro Cys Leu Gly Cys Gly Ala
110 115 120
Ala Thr Pro Glu Trp Ala Ala Leu Leu Tyr Tyr Gly Pro Phe Ile
225 230 135
Val Ile Phe Gln Phe Gly Trp Ala Ser Thr Gln Ile Ser His Leu
240 145 250
Ser Leu Ile Pro Glu Leu Val Thr Asn Asp His Glu Lys Val Glu
255 160 165
Leu Thr Ala Leu Arg Tyr A1a Phe Thr Val Val Ala Asn Tle Thr
170 175 180
Val Tyr Gly Ala Ala Trp Leu Leu Leu His Leu Gln Gly Ser Ser
185 190 195
Arg Val Glu Pro Thr Gln Asp Ile Ser Ile Ser Asp Gln Leu Gly
200 205 210
Gly Gln Asp Val Pro Val Phe Arg Asn Leu Ser Leu Leu Val Val
215 220 225
Gly Val Gly Ala Val Phe Ser Leu Leu Phe His Leu Gly Thr Arg
230 235 240
Glu Arg Arg Arg Pro His Ala Glu Glu Pro Gly Glu His Thr Pro
245 250 255
Leu Leu Ala Pro Ala Thr A1a G1n Pro Leu Leu Leu Trp Lys His
260 265 270
Trp Leu Arg Glu Pro A1a Phe Tyr Gln Val Gly Ile Leu Tyr Met
275 280 285
Thr Thr Arg Leu Ile Val Asn Leu Ser Gln Thr Tyr Met Ala Met
290 295 300
Tyr Leu Thr Tyr Ser Leu His Leu Pro Lys Lys Phe Ile Ala Thr
305 310 315
35/71
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Ile Pro Leu Val Met Tyr Leu Ser Gly Phe Leu Ser Ser Phe Leu
320 325 330
Met Lys Pro Ile Asn Lys Cys Ile Gly Arg Asn Met Thr Tyr Phe
335 340 345
Ser Gly Leu Leu Val Ile Leu Ala Phe Ala Ala Trp Val Ala Leu
350 355 360
Ala Glu Gly Leu Gly Val Ala Val Tyr Ala Ala Ala Val Leu Leu
365 370 375
Gly Ala Gly Cys Ala Thr Ile Leu Val Thr Ser Leu Ala Met Thr
380 385 390
Ala Asp Leu Ile Gly Pro His Thr Asn Ser Gly Ala Phe Val Tyr
395 400 405
Gly Ser Met Ser Phe Leu Asp Lys Val Ala Asn Gly Leu AIa Val
410 415 420
Met Ala Ile Gln Ser Leu His Pro Cys Pro Ser Glu Leu Cys Cys
425 430 435
Arg Ala Cys Val 5er Phe Tyr His Trp Ala Met Val A1a Val Thr
440 445 450
Gly Gly Val Gly Val Ala Ala Ala Leu Cys Leu Cys Ser Leu Leu
455 460 465
Leu Trp Pro Thr Arg Leu Arg Arg Trp Asp Arg Asp Ala Arg Pro
470 475 480
<210> Z9
<221> 382
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2395967CD1
<400> 29
Met Ser Glu Phe Trp Leu Tle Ser Ala Pro Gly Asp Lys Glu Asn
1 5 10 25
Leu Gln Ala Leu Glu Arg Met Asn Thr Val Thr Ser Lys Ser Asn
20 25 30
Leu Ser Tyr Asn Thr Lys Phe Ala Tle Pro Asp Phe Lys Val Gly
35 40 45
Thr Leu Asp Ser Leu Val Gly Leu Ser Asp Glu Leu Gly Lys Leu
50 55 60
Asp Thr Phe A2a Glu Ser Leu Ile Arg Arg Met Ala Gln Ser Va2
65 70 75
Val Glu Val Met Glu Asp Ser Lys Gly Lys Val Gln Glu His Leu
80 85 90
Leu Ala Asn Gly Val Asp Leu Thr Ser Phe Val Thr His Phe Glu
'95 100 205
Trp Asp Met Ala Lys Tyr Pro Val Lys Gln Pro Leu Val Ser Val
210 215 220
Val Asp Thr Ile A1a Lys Gln Leu Ala Gln Ile Glu Met Asp Leu
125 130 135
Lys Ser Arg Thr Ala Ala Tyr Asn Thr Leu Lys Thr Asn Leu Glu
140 145 150
Asn Leu Glu Lys Lys Ser Met Gly Asn Leu Phe Thr Arg Thr Leu
155 160 165
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Ser Asp Ile Val Ser Lys Glu Asp Phe Val Leu Asp Ser Glu Tyr
170 175 180
Leu Val Thr Leu Leu Val Ile Val Pro Lys Pro Asn Tyr Ser Gln
185 190 195
Trp Gln Lys Thr Tyr Glu Ser Leu Ser Asp Met Val Val Pro Arg
200 205 210
Ser Thr Lys Leu Ile Thr Glu Asp Lys Glu Gly Gly Leu Phe Thr
215 220 225
Val Thr Leu Phe Arg Lys Val Ile Glu Asp Phe Lys Thr Lys Ala
230 235 240
Lys Glu Asn Lys Phe Thr Val Arg Glu Phe Tyr Tyr Asp Glu Lys
245 250 255
Glu Ile Glu Arg Glu Arg Glu Glu Met Ala Arg Leu Leu Ser Asp
260 265 270
Lys Lys Gln Gln Tyr Gly Pro Leu Leu Arg Trp Leu Lys Val Asn
275 280 285
Phe Ser Glu Ala Phe Ile Ala Trp Ile His Ile Lys Ala Leu Arg
290 295 300
Val Phe Val Glu Ser Val Leu Arg Tyr Gly Leu Pro Val Asn Phe
305 310 315
Gln Ala Val Leu Leu Gln Pro His Lys Lys Ser Ser Thr Lys Arg
320 325 330
Leu Arg Glu Val Leu Asn Ser Val Phe Arg His Leu Asp Glu Val
335 340 345
Ala Ala Thr Ser Ile Leu Asp Ala Ser Val Glu Ile Pro Gly Leu
350 355 360
Gln Leu Asn Asn Gln Asp Tyr Phe Pro Tyr Val Tyr Phe His Ile
365 370 375
Asp Leu Ser Leu Leu Asp
380
<210> 20
<211> 484
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3586648CD1
<400> 20
Met Tyr Thr Ser His Glu Asp Ile G1y Tyr Asp Phe Glu Asp Gly
1 5 10 15
Pro Lys Asp Lys Lys Thr Leu Lys Pro His Pro Asn Ile Asp Gly
20 25 30
Gly Trp Ala Trp Met Met Val Leu Ser Ser Phe Phe Val His Ile
35 40 45
Leu Ile Met G1y Ser Gln Met Ala Leu Gly Val Leu Asn Val Glu
50 55 60
Trp Leu Glu Glu Phe His G1n Ser Arg Gly Leu Thr Ala Trp Val
65 70 75
Ser Ser Leu Ser Met Gly Ile Thr Leu Ile Val Gly Pro Phe Ile
80 85 90
Gly Leu Phe I1e Asn Thr Cys Gly Cys Arg Gln Thr Ala Ile Ile
95 100 105
Gly Gly Leu Val Asn Ser Leu Gly Trp Va1 Leu Ser Ala Tyr Ala
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110 115 120
Ala Asn Val His Tyr Leu Phe Ile Thr Phe Gly Val Ala Ala Gly
125 130 135
Leu Gly Ser Gly Met Ala Tyr Leu Pro Ala Val Val Met Val Gly
140 145 150
Arg Tyr Phe Gln Lys Arg Arg Ala Leu Ala Gln Gly Leu Ser Thr
155 160 165
Thr Gly Thr Gly Phe Gly Thr Phe Leu Met Thr Val Leu Leu Lys
170 175 180
Tyr Leu Cys Ala Glu Tyr Gly Trp Arg Asn Ala Met Leu Ile Gln
185 190 195
Gly Ala Val Ser Leu Asn Leu Cys Val Cys Gly Ala Leu Met Arg
200 205 210
Pro Leu Ser Pro Gly Lys Asn Pro Asn Asp Pro Gly Glu Lys Asp
215 220 225
Va1 Arg Gly Leu Pro Ala His Ser Thr Glu Ser Val Lys Ser Thr
230 235 240
Gly Gln Gln Gly Arg Thr Glu Glu Lys Asp Gly Gly Leu Gly Asn
245 250 255
Glu Glu Thr Leu Cys Asp Leu Gln Ala Gln Glu Cys Pro Asp GIn
260 265 270
Ala Gly His Arg Lys Asn Met Cys Ala Leu Arg Ile Leu Lys Thr
275 280 285
Val Ser Trp Leu Thr Met Arg Val Arg Lys Gly Phe Glu Asp Trp
290 295 300
Tyr Ser Gly Tyr Phe Gly Thr Ala Ser Leu Phe Thr Asn Arg Met
305 310 315
Phe Val Ala Phe Ile Phe Trp Ala Leu Phe Ala Tyr Ser Ser Phe
320 325 330
Val Ile Pro Phe Ile His Leu Pro G1u Ile VaI Asn Leu Tyr Asn
335 340 345
Leu Ser Glu Gln Asn Asp Val Phe Pro Leu Thr Ser Ile T-le Ala
350 355 360
Ile Va1 His Ile Phe Gly Lys Val Ile Leu Gly Val Ile Ala Asp
365 370 375
Leu Pro Cys Ile Ser Val Trp Asn Val Phe Leu Leu A1a Asn Phe
380 385 390
Thr Leu Va1 Leu Ser Ile Phe Ile Leu Pro Leu Met His Thr Tyr
395 400 405
Ala Gly Leu Ala Val I1e Cys Ala Leu Ile Gly Phe Ser Ser Gly
410 425 420
Tyr Phe Ser Leu Met Pro Val Val Thr G1u Asp Leu Val Gly Ile
425 430 435
Glu His Leu Ala Asn A1a Tyr Gly Ile Ile Ile Cys Ala Asn Gly
440 445 450
Ile Ser Ala Leu Leu Gly Pro Pro Phe Ala Gly Lys Leu Ser Glu
455 460 465
Val Leu Arg Ala Gln Ser Ala Cys Thr Tyr Gly Ala Leu Cys Tyr
470 475 480
Lys Val Pro Asp
<210> 21
<211> 736
<212> PRT
<213> Homo sapiens
38/71
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<220>
<221> misc_feature
<223> Incyte ID No:~7473396CD1
<400> 21
Met Gln Asn Ile Thr Lys Glu Phe Gly Thr Phe Lys Ala Asn Asp
1 5 10 15
Asn Ile Asn Leu Gln Val Lys Ala Gly Glu Ile His Ala Leu Leu
20 25 30
Gly Glu Asn Gly Ala Gly Lys Ser Thr Leu Met Asn Val Leu Ser
35 40 45
Gly Leu Leu Glu Pro Thr Ser Gly Lys Ile Leu Met Arg Gly Lys
50 55 60
Glu Val Gln Ile Thr~Ser Pro Thr Lys Ala Asn Gln Leu Gly Ile
65 70 75
Gly Met Val His Gln His Phe Met Leu Val Asp Ala Phe Thr Val
80 85 90
Thr Glu Asn Ile Val Leu Gly Ser Glu Pro Ser Arg A1a Gly Met
95 100 105
Leu Asp His Lys Lys Ala Arg Lys Glu Ile Gln Lys Val Ser Glu
110 115 120
Gln Tyr Gly Leu Ser Val Asn Pro Asp Ala Tyr Val Arg Asp Ile
125 130 135
Ser Val GIy Met Glu Gln Arg Val Glu Ile Leu Lys Thr Leu Tyr
140 145 150
Arg Gly Ala Asp Val Leu Ile Phe Asp Glu Pro Thr Ala Val Leu
155 160 165
Thr Pro Gln Glu Ile Asp Glu Leu Ile Val Ile Met Lys Glu Leu
170 175 180
Val Lys Glu GIy Lys Ser Ile Ile Leu IIe Thr His Lys Leu Asp
185 190 195
Glu Ile Lys Ala Val Ala Asp Arg Cys Thr Val Ile Arg Arg Gly
200 205 210
Lys Gly Ile Gly Thr Val Asn Val Lys Asp Val Thr Ser Gln Gln
215 220 225
Leu Ala Asp Met Met Val Gly Arg Ala VaI Ser Phe Lys Thr Met
230 235 240
Lys Lys GIu Ala Lys Pro Gln Glu Val Val Leu Ser Ile G1u Asn
245 250 255
Leu Val Val Lys Glu Asn Arg Gly Leu Glu Ala Val Lys Asn Leu
260 265 270
Asn Leu Glu VaI Arg AIa GIy Glu Val Leu Gly Ile Ala Gly Ile
275 280 285
Asp Gly Asn Gly Gln Ser Glu Leu Ile Gln Ala Leu Thr Gly Leu
290 295 300
Arg Lys Ala Glu Ser Gly His Ile Lys Leu Lys Gly Glu Asp Ile
305 310 315
Thr Asn Lys Lys Pro Arg Lys Ile Thr Glu His Gly Val Gly His
320 325 330
Val Pro Glu Asp Arg His Lys Tyr GIy Leu Val Leu Asp Met Thr
335 340 345
Leu Ser Glu Asn Ile Ala Leu Gln Thr Tyr His Gln Lys Pro Tyr
350 355 360
Ser Lys Asn Gly Met Leu Asn Tyr Ser Val Ile Asn Glu His Ala
365 370 375
Arg Glu Leu Ile Glu Glu Tyr Asp Val Arg Thr Thr Asn Glu Leu
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380 385 390
Val Pro Ala Lys Ala Leu Ser Gly Gly Asn Gln Gln Lys Ala Ile
395 400 405
Ile Ala Arg Ile Val Asp Arg Asp Pro Asp Leu Leu Ile Val Ala
410 415 420
Asn Pro Thr Arg Gly Leu Asp Val Gly Glu Phe Val Ala Val Thr
425 430 435
Gly Val Ser Gly Ser Gly Lys Ser Thr Leu Val Asn Ser Ile Leu
440 445 450
Lys Lys Ser Leu Ala Gln Lys Leu Asn Lys Asn Ser Ala Lys Pro
455 460 465
Gly Lys Phe Lys Thr Ile Ser Gly Tyr Glu Ser Ile Glu Lys Ile
470 475 480
Ile Asp Ile Asp Gln Ser Pro Ile Gly Arg Thr Pro Arg Ser Asn
485 490 495
Pro Ala Thr Tyr Thr Ser Val Phe Asp Asp Ile Arg Gly Leu Phe
500 505 510
Ala Gln Thr Asn Glu Ala Lys Met Arg Gly Tyr Lys Lys G1y Arg
515 520 525
Phe Ser Phe Asn Val Lys Gly Gly Arg Cys Glu Ala Cys Arg Gly
530 535 540
Asp Gly Ile Ile Lys Ile Glu Met His Phe Leu Pro Asp Val Tyr
545 550 555
Val Pro Cys Glu Val Cys His Gly Lys Arg Tyr Asn Ser Glu Thr
560 565 570
Leu Glu Val His Tyr Lys Gly Lys Ser Ile Ala Asp Ile Leu Glu
575 580 585
Met Thr Val Glu Asp Ala Val Glu Phe Phe Lys His Ile Pro Lys
590 595 600
I1e His Arg Lys Leu Gln Thr Ile Val Asp Val Gly Leu Gly Tyr
605 610 . 615
Val Thr Met Gly Gln Pro Ala Thr Thr Leu Ser Gly Gly Glu Ala
620 625 630
Gln Arg Met Lys Leu Ala Ser Glu Leu His Lys Ile Ser Asn Gly
635 640 645
Lys Asn Phe Tyr Ile Leu Asp Glu Pro Thr Thr Gly Leu His Ser
650 655 660
Asp Asp Ile Ala Arg Leu Leu His Val Leu Gln Arg Leu Val Asp
665 670 675
Ala Gly Asn Thr Val Leu Val Ile Glu His Asn Leu Asp Val Ile
680 685 690
Lys Thr Ala Asp Tyr IIe Ile Asp Leu Gly Pro GIu Gly GIy Glu
695 700 705
Gly Gly Gly Thr I1e Leu Thr Thr Gly Thr Pro G1u Glu Ile Ile
710 715 720
Asn VaI Lys Glu Ser Tyr Thr Gly His Tyr Leu Lys Lys Ile Met
725 730 735
Val
<210> 22
<211> 465
<212> PRT
<213> Homo sapiens
<220>
40/71
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<221> misc_feature
<223> Incyte ID No: 7476283CD1
<400> 22
Met G1y Pro Leu Lys Ala Phe Leu Phe Ser Pro Phe Leu Leu Arg
1 5 10 15
Ser Gln Ser Arg Gly Val Arg Leu Val Phe Leu Leu Leu Thr Leu
20 25 30
His Leu Gly Asn Cys Val Asp Lys Ala Asp Asp Glu Asp Asp Glu
35 40 45
Asp Leu Lys Val Asn Lys Thr Trp Val Leu Ala Pro Lys Ile His
50 55 60
Glu Gly Asp Ile Thr Gln Ile Leu Asn Ser Leu Leu Gln Gly Tyr
65 70 75
Asp Asn Lys Leu Arg Pro Asp Ile Gly Val Arg Pro Thr Val Ile
80 85 90
Glu Thr Asp Val Tyr Val Asn Ser Ile Gly Pro Va1 Asp Pro Ile
95 100 105
Asn Met Glu Tyr Thr Ile Asp Tle Ile Phe Ala Gln Thr Trp Phe
110 115 120
Asp Ser Arg Leu Lys Phe Asn Ser Thr Met Lys Val Leu Met Leu
125 130 135
Asn Ser Asn Met Val Gly Lys Ile Trp I1e Pro Asp Thr Phe Phe
140 145 150
Arg Asn Ser Arg Lys Ser Asp Ala His Trp Ile Thr Thr Pro Asn
155 160 165
Arg Leu Leu Arg Ile Trp Asn Asp Gly Arg Val Leu Tyr Thr Leu
170 175 280
Arg Leu Thr Ile Asn Ala Glu Cys Tyr Leu G1n Leu His Asn Phe
185 290 295
Pro Met Asp Glu His Ser Cys Pro Leu Glu Phe Ser Ser Asp Gly
200 205 210
Tyr Pro Lys Asn Glu Ile Glu Tyr Lys Trp Lys Lys Pro Ser Val
215 220 225
Glu Val Ala Asp Pro Lys Tyr Trp Arg Leu Tyr Gln Phe Ala Phe
230 235 240
Val Gly Leu Arg Asn Ser Thr Glu Ile Thr His Thr Ile Ser Gly
245 250 255
Asp Tyr Val Ile Met Thr Ile Phe Phe Asp Leu Ser Arg Arg Met
260 265 270
Gly Tyr Phe Thr Ile Gln Thr Tyr Ile Pro Cys Ile Leu Thr Val
275 280 285
Val Leu Ser Trp Val Ser Phe Trp Ile Asn Lys Asp Ala Val Pro
290 295 300
Ala Arg Thr Ser Leu Gly Ile Thr Thr Val Leu Thr Met Thr Thr
305 310 315
Leu Ser Thr Ile Ala Arg Lys Ser Leu Pro Lys Val Ser Tyr Val
320 325 330
Thr Ala Met Asp Leu Phe Val Ser Val Cys Phe Tle Phe Val Phe
335 340 345
Ala Ala Leu Met Glu Tyr Gly Thr Leu His Tyr Phe Thr Ser Asn
350 355 360
Gln Lys Gly Lys Thr Ala Thr Lys Asp Arg Lys Leu Lys Asn Lys
365 370 375
Ala Ser Met Thr Pro Gly Leu His Pro Gly Ser Thr Leu Ile Pro
380 385 390
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Met Asn Asn Ile Ser Val Pro Gln Glu Asp Asp Tyr Gly Tyr Gln
395 400 405
Cys Leu Glu Gly Lys Asp Cys Ala Ser Phe Phe Cys Cys Phe Glu
410 415 420
Asp Cys Arg Thr Gly Ser Trp Arg Glu Gly Arg Ile His Ile Arg
425 430 435
zle Ala Lys Ile Asp Ser Tyr Ser Arg Ile Phe Phe Pro Thr Ala
440 445 450
Phe Ala Leu Phe Asn Leu Val Tyr Trp Val Gly Tyr Leu Tyr Leu
455 460 465
<210> 23
<211> 235
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7477105CD1
<400> 23
Met Gly Ser Val Gly Ser Gln Arg Leu Glu Glu Pro 5er Val Ala
1 5 10 15
Gly Thr Pro Asp Pro Gly Val Val Met Ser Phe Thr Phe Asp Ser
20 25 30
His Gln Leu Glu Glu Ala Ala Glu Ala Ala Gln Gly Gln Gly Leu
35 40 45
Arg Ala Arg Gly Val Pro Ala Phe Thr Asp Thr Thr Leu Asp Glu
50 55 60
Pro Val Pro Asp Asp Arg Tyr His Ala Ile Tyr Phe Ala Met Leu
65 70 75
Leu Ala G1y Val Gly Phe Leu Leu Pro Tyr Asn Ser Phe Ile Thr
80 85 90
Asp Val Asp Tyr Leu His His Lys Tyr Pro Gly Thr Ser Ile Val
95 100 105
Phe Asp Met Ser Leu Thr Tyr Ile Leu Val Ala Leu Ala Ala Val
120 115 120
Leu Leu Asn Asn Val Leu Val~Glu Arg Leu Thr Leu His Thr Arg
125 130 135
Ile Thr Ala Gly Tyr Leu Leu Ala Leu G1y Pro Leu Leu Phe Ile
140 145 150
Ser Ile Cys Asp Val Trp Leu Gln Leu Phe Ser Arg Asp Gln Ala
155 160 165
Tyr Ala 21e Asn Leu Ala Ala Val Gly Thr Val Ala Phe Gly Cys
170 175 180
Thr Val Gln Gln Ser Ser Phe Tyr Gly His Arg Leu AIa Gln Pro
185 190 195
Pro Pro Gly Thr Pro Pro His Glu Leu Trp Ser Pro Glu Arg Arg
200 205 210
Gly A1a Ala Pro His Leu Val Thr Leu Arg Ala Ser Pro Ser Val
215 220 225
Leu Ile Leu Arg Asp Cys Phe Ser Gln Thr
230 235
<210> 24
42/71
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<211> 662
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7482079CD1
<400> 24
Met Leu Lys Gln Ser Glu Arg Arg Arg Ser Trp Ser Tyr Arg Pro
1 5 10 15
Trp Asn Thr Thr Glu Asn Glu Gly Ser Gln His Arg Arg Ser Ile
20 25 30
Cys Ser Leu Gly Ala Arg Ser Gly Ser Gln Ala Sex Ile His Gly
35 40 45
Trp Thr Glu Gly Asn Tyr Asn Tyr Tyr Ile Glu Glu Asp Glu Asp
50 55 60
Gly Glu Glu Glu Asp Gln Trp Lys Asp Asp Leu Ala Glu Glu Asp
65 70 75
Gln Gln Ala Gly Glu Va1 Thr Thr Ala Lys Pro Glu Gly Pro Ser
80 85 90
Asp Pro Pro Ala Leu Leu Ser Thr Leu Asn Val Asn Va1 Gly Gly
95 100 105
His Ser Tyr Gln Leu Asp Tyr Cys Glu Leu Ala Gly Phe Pro Lys
110 115 , 120
Thr Arg Leu Gly Arg Leu Ala Thr Ser Thr Ser Arg Ser Arg Gln
125 130 135
Leu Ser Leu Cys Asp Asp Tyr Glu Glu Gln Thr Asp Glu Tyr Phe
140 145 150
Phe Asp Arg Asp Pro Ala Val Phe Gln Leu Val Tyr Asn Phe Tyr
255 160 1&5
Leu Ser Gly Val Leu Leu Val Leu Asp Gly Leu Cys Pro Arg Arg
170 175 180
Phe Leu Glu Glu Leu Gly Tyr Trp Gly Val Arg Leu Lys Tyr Thr
185 190 195
Pro Arg Cys Cys Arg Ile Cys Phe Glu Glu Arg Arg Asp Glu Leu
200 205 210
Ser Glu Arg Leu Lys Ile Gln His Glu Leu Arg Ala Gln Ala Gln
215 220 225
Va1 Glu Glu Ala Glu Glu Leu Phe Arg Asp Met Arg Phe Tyr Gly
230 235 240
Pro Gln Arg Arg Arg Leu Trp Asn Leu Met GIu Lys Pro Phe Ser
245 250 255
Ser Val Ala Ala Lys Ala Ile Gly Val Ala Ser Ser Thr Phe Val
260 265 270
Leu Val Ser Val Val Ala Leu Ala Leu Asn Thr VaI Glu Glu Met
275 280 285
Gln Gln His Ser Gly Gln Gly Glu Gly Gly Pro Asp Leu Arg Pro
290 295 300
Ile Leu Glu His Val G1u Met Leu Cys Met Gly Phe Phe Thr Leu
305 310 315
Glu Tyr Leu Leu Arg Leu Ala Ser Thr Pro Asp Leu Arg Arg Phe
320 325 330
Ala Arg Ser Ala Leu Asn Leu Val Asp Leu Val Ala Ile Leu Pro
335 340 345
Leu Tyr Leu GIn Leu Leu Leu Glu Cys Phe Thr Gly GIu Gly His
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350 355 360
Gln Arg Gly Gln Thr Val Gly Ser Val Gly Lys Val Gly Gln Val
365 370 375
Leu Arg Val Met Arg Leu Met Arg Ile Phe Arg Ile Leu Lys Leu
380 385 390
Ala Arg His Ser Thr Gly Leu Arg Ala Phe Gly Phe Thr Leu Arg
395 400 405
Gln Cys Tyr Gln Gln Val Gly Cys Leu Leu Leu Phe Ile Ala Met
410 415 420
Gly Ile Phe Thr Phe Ser Ala Ala Val Tyr Ser Val Glu His Asp
425 430 435
Val Pro Ser Thr Asn Phe Thr Thr Ile Pro His Ser Trp Trp Trp
440 445 450
Ala Ala Va1 Ser Thr Phe Ala Leu Gly Phe Pro Ile Leu Phe Pro
455 460 465
Ser Pro Va1 Ser Cys Ser Ser Leu Pro Trp Leu Ser Ala Thr Arg
470 475 480
Leu Trp Leu Leu Ile Leu Val Phe Pro Pro Thr Pro Asn Arg Arg
485 490 495
Ile Gln Leu Thr Lys Arg Arg Trp Met Ser Lys Val Val Glu Arg
500 505 510
Glu Leu Ser Arg Ser Val Asn Ser Ser Ser His Met Ser Met Ala
515 520 525
Val Ala Lys Asn Lys Arg Glu Asn Ala Ser Pro Ile Met Gln Thr
530 535 540
Leu His Lys Phe Leu Phe Met Ala Phe Ala Gln Pro Ile Gly Gln
545 550 555
Ser Lys Ser His Gly Gln Ala Ala Ser Gln Arg Ala Gly Gln Val
560 565 570
Ser Ile Ser Thr Val Gly Tyr Gly Asp Met Tyr Pro Glu Thr His
575 580 585
Leu Gly Arg Phe Phe Ala Phe Leu Cys Ile Ala Phe Gly Ile Ile
590 595 600
Leu Asn Gly Met Pro Ile Ser Ile Leu Tyr Asn Lys Phe Ser Asp
605 6l0 615
Tyr Tyr Ser Lys Leu Lys Ala Tyr Glu Tyr Thr Thr Ile Arg Arg
620 625 630
Glu Arg Gly Glu Val Asn Phe Met Gln Arg Ala Arg Lys Lys Ile
635 640 645
Ala Glu Cys Leu Leu Gly Ser Asn Pro Gln Leu Thr Pro Arg G1n
650 655 660
Glu Asn
<210> 25
<211> 371
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No. 55145506CD1
<400> 25
Met Asn Asp Glu Asp Tyr Ser Thr Ile Tyr Asp Thr Ile Gln Asn
1 5 10 15
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Glu Arg Thr Tyr Glu Val Pro Asp Gln Pro Glu Glu Asn Glu Ser
20 25 30
Pro His Tyr Asp Asp Val His Glu Tyr Leu Arg Pro Glu Asn Asp
35 40 45
Leu Tyr Ala Thr Gln Leu Asn Thr His Glu Tyr Asp Phe Val Ser
50 55 60
Val Tyr Thr Ile Lys Gly Glu Glu Thr Ser Leu Ala Ser Val Gln
65 70 75
Ser Glu Asp Arg Gly Tyr Leu Leu Pro Asp Glu Ile Tyr Ser Glu
80 85 ' 90
Leu Gln Glu Ala His Pro Gly Glu Pro Gln Glu Asp Arg Gly Ile
95 100 105
Ser Met Glu Gly Leu Tyr Ser Ser Ala Gln Asp Gln Gln Leu Cys
110 115 120
Ala Ala Glu Leu Gln Glu Asn Gly Ser Val Met Lys Glu Asp Leu
125 130 135
Pro Ser Pro Ser Ser Phe Thr Ile Gln His Ser Lys Ala Phe Ser
140 145 150
Thr Thr Lys Tyr Ser Cys Tyr Ser Asp Ala Glu Gly Leu Glu G1u
155 160 165
Lys Glu Gly Ala His Met Asn Pro Glu Ile Tyr Leu Phe Val Lys
170 175 180
Ala Gly Ile Asp Gly Glu Ser Ile Gly Asn Cys Pro Phe Ser Gln
185 190 195
Arg Leu Phe Met Ile Leu Trp Leu Lys Gly Val Val Phe Asn Val
200 205 210
Thr Thr Val Asp Leu Lys Arg Lys Pro Ala Asp Leu His Asn Leu
215 220 22S
Ala Pro Gly Thr His Pro Pro Phe Leu Thr Phe Asn Gly Asp Val
230 235 240
Lys Thr Asp Val Asn Lys Ile Glu Glu Phe Leu Glu Glu Thr Leu
245 250 255
Thr Pro Glu Lys Tyr Pro Lys Leu Ala Ala Lys His Arg Glu Ser
260 265 270
Asn Thr AIa Gly Ile Asp Ile Phe Ser Lys Phe Ser Ala Tyr Ile
275 280 285
Lys Asn Thr Lys Gln Gln Asn Asn Ala Ala Leu Glu Arg Gly Leu
290 295 300
Thr Lys A1a Leu Lys Lys Leu Asp Asp Tyr Leu Asn Thr Pro Leu
305 310 315
Pro G1u Glu Ile Asp AIa Asn Thr Cys Gly Glu Asp Lys Gly Ser
320 325 330
Arg Arg Lys Phe Leu Asp Gly Asp Glu Leu Thr Leu Ala Asp Cys
335 340 345
Asn Leu Leu Pro Lys Leu His Val Val Lys Thr His Leu Leu Thr
350 355 360
Ser 5er Sex Asn Phe Leu Arg Asn Lys Tyr His
365 370
<210> 26
<211> 468
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
45/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
<223> Incyte ID No: 5950519CD1
<400> 26
Met Arg Gly Ser Pro Gly Asp Ala Glu Arg Arg Gln Arg Trp Gly
Z 5 ~ 10 15
Arg Leu Phe Glu Glu Leu Asp Ser Asn Lys Asp Gly Arg Val Asp
20 25 30
Val His Glu Leu Arg Gln Gly Leu Ala Arg Leu Gly Gly Gly Asn
35 40 45
Pro Asp Pro Gly Ala Gln Gln Gly Ile Ser Ser Glu Gly Asp Ala
50 55 60
Asp Pro Asp Gly Gly Leu Asp Leu Glu Glu Phe Ser Arg Tyr Leu
65 70 75
Gln Glu Arg Glu Gln Arg Leu Leu Leu Met Phe His Ser Leu Asp
80 85 90
Arg Asn Gln Asp Gly His Ile Asp Val Ser Glu Ile Gln Gln Ser
95 100 105
Phe Arg Ala Leu Gly Ile Ser Ile Ser Leu Glu Gln Ala Glu Lys
110 115 120
Tle Leu His Ser Met Asp Arg Asp Gly Thr Met Thr Ile Asp Trp
125 230 235
G1n Glu Trp Arg Asp His Phe Leu Leu His Ser Leu Glu Asn Val
140 245 250
Glu Asp Val Leu Tyr Phe Trp Lys His Ser Thr Val Leu Asp Ile
155 160 265
Gly Glu Cys Leu Thr Va1 Pro Asp Glu Phe Ser Lys Gln Glu Lys
170 175 280
Leu Thr Gly Met Trp Trp Lys Gln Leu Val A1a Gly Ala Val Ala
185 190 ~ 195
Gly Ala Val Ser Arg Thr Gly Thr Ala Pro Leu Asp Arg Leu Lys
200 205 210
Val Phe Met GIn Val His Ala Ser Lys Thr Asn Arg Leu Asn Ile
215 220 225
Leu Gly Gly Leu Arg Ser Met Va1 Leu Glu Gly Gly Ile Arg Ser
230 235 240
Leu Trp Arg Gly Asn Gly Ile Asn Val Leu Lys Ile Ala Pro Glu
245 250 255
Ser Ala Ile Lys Phe Met Ala Tyr Glu Gln Tle Lys Arg Ala Ile
260 265 270
Leu Gly Gln Gln Glu Thr Leu His Val Gln Glu Arg Phe Val Ala
275 280 285
Gly Ser Leu Ala Gly A1a Thr Ala Gln Thr Tle Ile Tyr Pro Met
290 295 300
Glu Val Leu Lys Thr Arg Leu Thr Leu Arg Arg Thr Gly Gln Tyr
305 310 315
Lys G1y Leu Leu Asp Cys Ala Arg Arg Tle Leu Glu Arg Glu Gly
320 325 330
Pro Arg Ala Phe Tyr Arg Gly Tyr Leu Pra Asn Val Leu Gly Ile
335 340 345
Ile Pro Tyr Ala Gly Ile Asp Leu Ala Val Tyr Glu Thr Leu Lys
3S0 355 360
Asn Trp Trp Leu Gln Gln Tyr Ser His Asp Ser Ala Asp Pro Gly
365 370 375
Ile Leu Val Leu Leu A1a Cys Gly Thr Ile Ser Ser Thr Cys Gly
380 385 390
Gln Ile Ala 5er Tyr Pro Leu Ala Leu Val Arg Thr Arg Met Gln
46/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
395 400 405
Ala Gln Ala Ser Ile Glu Gly Gly Pro Gln Leu Ser Met Leu Gly
410 415 420
Leu Leu Arg His Tle Leu Ser Gln Glu Gly Met Arg G1y Leu Tyr
425 430 435
Arg GIy Ile Ala Pro Asn Phe Met Lys VaI Ile Pro Ala Val Ser
440 445 450
Ile Ser Tyr Val Val Tyr Glu Asn Met Lys Gln Ala Leu Gly Va1
455 460 465
Thr Ser Arg
<210> 27
<221> 2229
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1687189CB1
<400> 27
gcctgagcgg ccgaactcgg cagctccaac ccaactcggc ttaactccgc ctcaccgagc 60
ccagtccaag actctgtgct ccctaggttt gcaacagctc tctgatcatc ttcttcaatt 120
cctgctagga tgccgtggca agcatttcgc agatttggtc aaaagctggt acgcagacgt 180
acactggagt caggcatggc tgagactcgc cttgccagat gcctaagcac cctggattta 240
gtggccctgg gtgtgggcag cacattgggt gcaggcgtgt atgtcctagc tggcgaggtg 300
gccaaagata aagcagggcc atccattgtg atctgctttt tggtggctgc cctgtcttct 360
gtgttggctg ggctgtgcta tgcggagttt ggtgcccggg ttccccgttc tggttcggca 420
tatctctaca gctatgtcac tgtgggtgaa ctctgggcct tcaccactgg ctggaacctc 480
atcctctcct atgtcattgg tacagccagt gtggcccggg cctggagctc tgcttttgac 540
aacctgattg ggaaccacat ctctaagact ctgcaggggt ccattgcact gcacgtgccc 600
catgtccttg cagaatatcc agatttcttt gctttgggcc tcgtgttgct gctcactgga 660
ttgttggctc tcggggctag tgagtcggcc ctggttacca aagtgttcac aggcgtgaac 720
cttttggttc ttgggttcgt catgatctct ggcttcgtta agggggacgt gcacaactgg 780
aagctcacag aagaggacta cgaattggcc atggctgaac tcaatgacac ctatagcttg 840
ggtcctctgg gctctggagg atttgtgcct ttcggcttcg agggaattct ccgtggagca 900
gcgacctgtt tctatgcatt tgttggtttc gactgtattg ctaccactgg agaagaagcc 960
cagaatcccc agcgttccat cccgatgggc attgtgatct cactgtctgt ctgctttttg 1020
gcgtattttg ctgtctcttc tgcactcacc ctgatgatgc cttactaeca gcttcagcct 1080
gagagccctt tgcctgaggc atttctctac attggatggg ctcctgcccg ctatgttgtg 1140
gctgttggct ccctctgtgc tctttctacc agcctcctgg gctccatgtt ccccatgcct 1200
cgggtgatct acgcgatggc agaggatggc ctcctgttcc gtgtacttgc teggatccac 1260
accggcacac gcaccccaat catagccacc gtggtctctg gcattattgc agcattcatg 1320
gcattcctct tcaaactcac tgatcttgtg gacctcatgt caattgggac cctgcttgct 1380
tactccctgg tgtcgatttg tgttctcatc ctcaggtatc aacctgatca ggagacaaag 1440
actggggaag aagtggagtt gcaggaggag gcaataacta ctgaatcaga gaagttgacc 1500
ctatggggac tatttttccc actcaactcc atccccactc cactctctgg~ccaaattgtc 1560
tatgtttgtt cctcattgct tgctgtcctg ctgactgctc tttgcctggt gctggcccag 1620
tggtcagttc cattgctttc tggagacctg ctgtggactg cagtggttgt gctgctcctg 1680
ctgctcatta ttgggatcat tgtggtcatc tggagacagc cacagagctc cactcccctt 1740
cactttaagg tgcctgcttt gcctctcctc ccactaatga gcatctttgt gaatatttac 1800
cttatgatgc agatgacagc tggtacctgg gcccgatttg gggtctggat gctgattggc 2860
tttgctatct acttcggcta tgggatccag cacagcctgg aagagattaa gagtaaccaa 2920
ccctcacgca agtctagagc caaaactgta gaccttgatc ccggcactct ctatgtccac 1980
tcagtttgac atcgtcacac ctaaatgctg tctggtcccc tgcacaataa tggagagtac 2040
47/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
tcctgacccc agtgacagct agccctcccc tgtgatggtg gtggtggata ctaatacagt 2100
tctgtacgat gtgaaggatg tgtctttgct atttcttgtc tattttaacc cgtctgcttc 2160
taaatgatgt ctagctgctt accaacttta aaaaatgata ttaaaagaaa gtagaaaaat 2220
aaaaaaaaa 2229
<210> 28
<211> 7610
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Tncyte ID No: 7078207CB1
<400> 28
gcgccccgcc cccgcgcggg cgatgcccag cggcgcggcg ggctgcgggg cccggcgggg 60
cgcgcagagg agcgggccgc ggcgctgagg cggcggagcg tggccccgcc atgggcttcc 220
tgcaccagct gcagctgctg ctctggaaga acgtgacgct caaacgccgg agcccgtggg 180
tcctggcctt cgagatcttc atccccctgg tgctgttctt tatcctgctg gggctgcgac 240
agaagaagcc caccatctcc gtgaaggaag tctccttcta cacagcggcg cccctgacgt 300
ctgccggcat cctgcctgtc atgcaatcgc tgtgcccgga cggccagcga gacgagttcg 360
gcttcctgca gtacgccaac tccacggtca cgcagctgct tgagcgcctg gaccgcgtgg 420
tggaggaagg caacctgttt gacccagcgc ggcccagcct gggctcagag ctcgaggccc 480
tacgccagca tctggaggcc ctcagtgcgg gcccgggcac ctcggggagc cacctggaca 540
gatccacagt gtcttccttc tctctggact cggtggccag aaacccgcag gagctctggc 600
gtttcctgac gcaaaacttg tcgctgccca atagcacggc ccaagcactc ttggccgccc 660
gtgtggaccc gcccgaggtc taccacctgc tctttggtcc ctcatctgcc ctggattcac 720
agtctggcct ccacaagggt caggagccct ggagccgcct agggggcaat cccctgttcc 780
ggatggagga gctgctgctg gctcctgccc tcctggagca gctcacctgc acgccgggct 840
cgggggagct gggccggatc ctcactgtgc ctgagagtca gaagggagcc ctgcagggct 900
accgggatgc tgtctgcagt gggcaggctg ctgcgcgtgc caggcgcttc tctgggctgt 960
ctgctgagct ccggaaccag ctggacgtgg ccaaggtctc ccagcagctg ggcctggatg 1020
cccccaacgg ctcggactcc tcgccacagg cgccaccccc acggaggctg caggcgcttc 2080
tgggggacct gctggatgcc cagaaggttc tgcaggatgt ggatgtcctg tcggccctgg 1140
CCCtgCtaCt gCCCCagggt gCCtgCdCtg gCCggaCCCC CggaCCCCCa gccagtggtg 1200
cgggtggggc ggccaatggc actggggcag gggcagtcat gggccccaac gccaccgctg 2260
aggagggcgc accctctgct gcagcactgg ccaccccgga cacgctgcag ggccagtgct 1320
cagccttcgt acagctctgg gccggcctgc agcccatctt gtgtggcaac aaccgcacca 2380
ttgaacccga ggcgctgcgg cggggcaaca tgagctccct gggcttcacg agcaaggagc 1440
agcggaacct gggcctcctc gtgcacctca tgaccagcaa ccccaaaatc ctgtacgcgc 1500
ctgcgggctc tgaggtcgac cgcgtcatcc tcaaggccaa cgagactttt gcttttgtgg 1560
gcaacgtgac tcactatgcc caggtctggc tcaacatctc ggcggagatc cgcagcttcc 1620
tggagcaggg caggctgcag caacacctgc gctggctgca gcagtatgta gcagagctgc 1680
ggctgcaccc cgaggcactg aacctgtcac tggatgagct gccgccggcc ctgagacagg 1740
acaacttctc gctgcccagt ggcatggccc tcctgcagca gctggatacc attgacaacg 1800
cggcctgcgg ctggatccag ttcatgtcca aggtgagcgt ggacatcttc aagggcttcc 1860
ccgacgagga gagcattgtc aactacaccc tcaaccaggc ctaccaggac aacgtcactg 1920
tttttgccag tgtgatcttc cagacccgga aggacggctc gCtCCCgCCt cacgtgcact 1980
acaagatccg ccagaactcc agcttcaccg agaaaaccaa cgagatccgc cgcgectact 2040
ggcggcctgg gcccaatact ggcggccgct tctacttcct ctacggcttc gtctggatcc 2100
aggacatgat ggagcgcgcc atcatcgaca cttttgtggg gcacgacgtg gtggagccag 2160
gcagctacgt gcagatgttc ccctacccct gctacacacg cgatgacttc ctgtttgtca 2220
ttgagcacat gatgccgctg tgcatggtga tctcctgggt ctactccgtg gccatgacca 2280
tccagcacat cgtggcggag aaggagcacc ggctcaagga ggtgatgaag accatgggcc 2340
tgaacaacgc ggtgcactgg gtggcctggt tcatcaccgg ctttgtgcag ctgtccatct 2400
ccgtgacagc actcaccgcc atcctgaagt acggccaggt gcttatgcac agccacgtgg 2460
48/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
tcatcatctg gctcttcctg gcagtctacg cggtggccac catcatgttc tgcttcctgg 2520
tgtctgtgct gtactccaag gccaagctgg cctcggcctg cggtggcatc atctacttcc 2580
tgagctacgt gccctacatg tacgtggcga tccgagagga ggtggcgcat gataagatca 2640
cggccttcga gaagtgcatc gcgtccetca tgtccacgac ggcctttggt ctgggctcta 2700
agtacttcgc gctgtatgag gtggccggcg tgggcatcca gtggcacacc ttcagccagt 2760
ccccggtgga gggggacgac ttcaacttgc tcctggctgt caccatgctg atggtggacg 2820
ccgtggtcta tggcatcctc acgtggtaca ttgaggctgt gcacccaggc atgtacgggc 2880
tgccccggcc ctggtacttc ccactgcaga agtcctactg gctgggcagt gggcggacag 2940
aagcctggga gtggagctgg ccgtgggcac gcaccccccg cctcagtgtc atggaggagg 3000
accaggcctg tgccatggag agccggcgct ttgaggagac ccgtggcatg gaggaggagc 3060
ccacccacct gcctctggtt gtctgcgtgg acaaactcac caaggtctac aaggacgaca 3120
agaagctggc cctgaacaag ctgagcctga acctctacga gaaccaggtg gtctccttct 3180
tgggccacaa cggggcgggc aagaccacca ccatgtccat cctgaccggc ctgttccctc 3240
caacgtcggg ttccgccacc atctacgggc acgacatccg cacggagatg gatgagatcc 3300
gcaagaacct gggcatgtgc ccgcagcaca atgtgctctt tgaccggctc acggtggagg 3360
aacacctctg gttctactca cggctcaaga gcatggctca ggaggagatc cgcagagaga 3420
tggacaagat gatcgaggac ctggagctct ccaacaaacg gcactcactg gtgcagacat 3480
tgtcgggtgg catgaagcgc aagctgtccg tggccatcgc cttcgtgggc ggctctcgcg 3540
ccatcatcct ggacgagccc acggcgggcg tggaccccta cgcgcgccgc gccatctggg 3600
acctcatcct gaagtacaag ccaggccgca ccatccttct gtccacccac cacatggatg 3660
aggctgacct gcttggggac cgcattgcca tcatctccca tgggaagctc aagtgctgcg 3720
gctccccgct cttcctcaag ggcacctatg gcgacgggta ccgcctcacg ctggtcaagc 3780
ggcccgccga gccggggggc ccccaagagc cagggctggc atccagcccc ccaggtcggg 3840
ccccgctgag cagctgctcc gagctccagg tgtcccagtt catccgcaag catgtggcct 3900
cctgcctgct ggtctcagac acaagcacgg agctctccta catcctgccc agcgaggccg 3960
ccaagaaggg ggctttcgag cgcctcttcc agcacctgga gcgcagcctg gatgcactgc 4020
acctcagcag cttcgggctg atggacacga ccctggagga agtgttcctc aaggtgtcgg 4080
aggaggatca gtcgctggag aacagtgagg ccgatgtgaa ggagtccagg aaggatgtgc 4140
tccctggggc ggagggcccg gcgtctgggg agggtcacgc tggcaatctg gcccggtgct 4200
cggagctgac ccagtcgcag gcatcgctgc agtcggcgtc atctgtgggc tctgcccgtg 4260
gcgacgaggg agctggctac accgacgtct atggcgacta ccgccccctc tttgataacc 4320
cacaggaccc agacaatgtc agcctgcaag aggtggaggc agaggccctg tcgagggtcg 4380
gccagggcag ccgcaagctg gacggcgggt ggctgaaggt gcgccagttc cacgggctgc 4440
tggtcaaacg cttccactgc gcccgccgca actccaaggc actcttctcc cagatcttgc 4500
tgccagcctt cttcgtctgc gtggccatga ccgtggccct gtccgtcccg gagattggtg 4560
atctgccccc gctggtcctg tcaccttccc agtaccacaa ctacacccag ecccgtggca 4620
atttcatccc ctacgccaac gaggagcgcc gcgagtaccg gctgcggcta tcgcccgacg 4680
ccagccccca gcagctcgtg agcacgttcc ggctgccgtc gggggtgggt gccacctgcg 4740
tgctcaagtc tcccgccaac ggctcgctgg ggcccacgtt gaacctgagc agcggggagt 4800
cgcgcctgct ggcggctcgg ttcttcgaca gcatgtgtct ggagtccttc acacaggggc 4860
tgccactgtc caatttcgtg ccacccccac cctcgcccgc cccatctgac tcgccagcgt 4920
ccccggatga ggacctgcag gcctggaacg tctccctgcc gcccaccgct gggccagaaa 4980
tgtggacgtc ggcaccctcc ctgccgcgcc tggtacggga gcccgtccgc tgcacctgct 5040
ctgcgcaggg caccggcttc tcctgcccca gcagtgtggg cgggcacccg ccccagatgc 5100
gggtggtcac aggcgacatc ctgaccgaca tcaccggcca caatgtctct gagtacctgc 5160
tcttcacctc cgaccgcttc cgactgcacc ggtatggggc catcaccttt ggaaacgtcc 5220
tgaagtccat cccagcctca tttggcacca gggccccacc catggtgcgg aagatcgcgg 5280
tgcgcagggc tgcccaggtt ttctacaaca acaagggcta tcacagcatg cccacctacc 5340
tcaacagcct caacaacgcc atcctgcgtg ccaacctgcc caagagcaag ggcaacccgg 5400
cggcttacgg catcaccgtc accaaccacc ccatgaataa gaccagcgcc agcctctccc 5460
tggattacct gctgcagggc acggatgtcg tcatcgccat cttcatcatc gtggccatgt 5520
ccttcgtgcc ggccagcttc gttgtcttcc tcgtggccga gaagtccacc aaggccaagc 5580
atctgcagtt tgtcagcggc tgcaacccca tcatctactg gctggcgaac tacgtgtggg 5640
acatgctcaa ctacctggtc cccgctacct gctgtgtcat catcctgttt gtgttcgacc 5700
tgccggccta cacgtcgccc accaacttcc ctgccgtcct ctccctcttc ctgctctatg 5760
ggtggtccat cacgcccatc atgtacccgg cctccttctg gttcgaggtc cccagctccg 5820
49/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
cctacgtgtt cctcattgtc atcaatctct tcatcggcat caccgccacc gtggccacct 5880
tcctgctaca gctcttcgag cacgacaagg acctgaaggt tgtcaacagt tacctgaaaa 5940
gctgcttcct cattttcccc aactacaacc.tgggccacgg gctcatggag atggcctaca 6000
acgagtacat caacgagtac tacgccaaga ttggccagtt tgacaagatg aagtccccgt 6060
tcgagtggga cattgtcacc cgcggactgg tggccatggc ggttgagggc gtcgtgggct 6120
tcctcctgac catcatgtgc cagtacaact tcctgcggcg gccacagcgc atgcctgtgt 6180
ctaccaagcc tgtggaggat gatgtggacg tggccagtga gcggcagcga gtgctccggg 6240
gagacgccga caatgacatg gtcaagattg agaacctgac caaggtctac aagtcccgga 6300
agattggccg tatcctggcc gttgaccgcc tgtgcctggg tgtgcgtcct ggcgagtgct 6360
tcgggctcct gggcgtcaac ggtgcgggca agaccagcac cttcaagatg ctgaccggcg 6420
acgagagcac gacggggggc gaggccttcg tcaatggaca cagcgtgctg aaggagctgc 6480
tccaggtgca gcagagcctc ggctactgcc cgcagtgtga cgcgctgttc gacgagctca 6540
cggcccggga gcacctgcag ctgtacacgc ggctgcgtgg gatctcctgg aaggacgagg 6600
cccgggtggt gaagtgggct ctggagaagc tggagctgac caagtacgca gacaagccgg 6660
ctggcaccta cagcggcggc aacaagcgga agctctccac ggccatcgcc ctcattgggt 6720
acccagcctt catcttcctg gacgagccca ccacaggcat ggaccccaag gcccggcgct 6780
tcctctggaa cctcatcctc gacctcatca agacagggcg ttcagtggtg ctgacatcac 6840
acagcatgga ggagtgcgag gcgctgtgca cgcggctggc catcatggtg aacggtcgcc 6900
tgcggtgcct gggcagcatc cagcacctga agaaccggtt tggagatggc tacatgatca 6960
cggtgcggac caagagcagc cagagtgtga aggacgtggt gcggttcttc aaccgcaact 7020
tcccggaagc catgctcaag gagcggcacc acacaaaggt gcagtaccag ctcaagtcgg 7080
agcacatctc gctggcccag gtgttcagca agatggagca ggtgtctggc gtgctgggca 7140
tcgaggacta ctcggtcagc cagaccacac tggacaatgt gttcgtgaac tttgccaaga 7200
agcagagtga caacctggag cagcaggaga cggagccgcc atccgcactg cagtcccctc 7260
tcggctgctt gctcagcctg ctccggcccc ggtctgcccc cacggagctc cgggcacttg 7320
tggcagacga gcccgaggac ctggacacgg aggacgaggg cctcatcagc ttcgaggagg 7380
agcgggccca gctgtccttc aacacggaca cgctctgctg accacccaga gctgggccag 7440
ggaggacacg ctccactgac cacccagagc tgggccaggg actcaacaat ggggacagaa 7500
gtcccccagt gcctgccagg gcctggagtg gaggttcagg accaaggggc ttctggtcct 7560
ccagcccctg tactcggcca tgtCCtgCgg tcactgcggt tgccggccct 7610
<210> 29
<211> 2219
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Tncyte ID No: 1560619CB1
<400> 29
ggcagcatga gccgatcacc cctcaatccc agccaactcc gatcagtggg ctcccaggat 60
gCCCtggCCC ccttgcctcc aCCtgCtCCC CdgaatCCCt CCaCCCdCtC ttgggaccct 120
ttgtgtggat ctctgccttg gggcctcagc tgtcttctgg ctctgcagca tgtcttggtc 180
atggcttctc tgctctgtgt ctcccacctg ctcctgcttt gcagtctctc cccaggagga 240
ctctcttact ccccttctca gctcctggcc tccagcttct tttcatgtgg tatgtctacc 300
atcctgcaaa cttggatggg cagcaggctg cctcttgtcc aggctccatc cttagagttc 360
cttatccctg ctctggtgct gaccagccag aagctacccc gggccatcca gacacctgga 420
aactcctccc tcatgctgca cctttgtagg ggacctagct gccatggcct ggggcactgg 480
aacacttctc tccaggaggt gtccggggca gtggtagtat ctgggctgct gcagggcatg 540
atggggctgc tggggagtcc cggccacgtg ttcccccact gtgggcccct ggtgctggct 600
cccagcctgg ttgtggcagg gctctctgcc cacagggagg tagcccagtt ctgcttcaca 660
cactgggggt tggccttgct ggttatcctg ctcatggtgg tctgttctca gcacctgggc 720
tcctgccagt ttcatgtgtg cccctggagg cgagcttcaa cgtcatcaac tcacactcct 780
ctccctgtct tccggctcct ttcggtgctg atcccagtgg cctgtgtgtg gattgtttct 840
gcctttgtgg gattcagtgt tatcccccag gaactgtctg cccccaccaa ggcaccatgg 900
50/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
atttggctgc ctcacccagg tgagtggaat tggcctttgc tgacgcccag agctctggct 960
gcaggcatct ccatggcctt ggcagcctcc accagttccc tgggctgcta tgccctgtgt 1020
ggccggctgc tgcatttgcc tcccccacct ccacatgcct gcagtcgagg gctgagcctg 1080
gaggggctgg gcagtgtgct ggccgggctg ctgggaagcc ccatgggcac tgcatccagc 1140
ttccccaacg tgggcaaagt gggtcttatc caggctggat ctcagcaagt ggctcactta 1200
gtggggctac tctgcgtggg gcttggactc tcccccaggt tggctcagct cctcaccacc 1260
atcccactgc ctgttgttgg tggggtgctg ggggtgaccc aggctgtggt tttgtctgct 1320
ggattctcca gcttctacct ggctgacata gactctgggc gaaatatctt cattgtgggc 1380
ttctccatct tcatggcctt gctgctgcca agatggtttc gggaagcccc agtcctgttc 2440
agcacaggct ggagcccctt ggatgtatta ctgcactcac tgctgacaca gcccatcttc 1500
ctggctggac tctcaggctt cctactagag aacacgattc ctggcacaca gcttgagcga 1560
ggcctaggtc aagggctacc atctcctttc actgcccaag aggctcgaat gcctcagaag 1620
cccagggaga aggctgctca agtgtacaga cttcctttcc ccatccaaaa cctctgtccc 1680
tgcatccccc agcctctcca ctgcctctgc ccactgcctg aagaccctgg ggatgaggaa 1740
ggaggctcct ctgagccaga agagatggca gacttgctgc ctggctcagg ggagccatgc 1800
cctgaatcta gcagagaagg gtttaggtcc cagaaatgac cagaacgcct acttctgccc 1860
tggttaattt agccctaact ctcatctgct ggagagtcag ctcccaaact gttctttctt 1920
gtaggcagag gatatgtgtg tgtgtattac atgggactgt ctagaggttc catttcccaa 1980
tagggtgggt tgcctttcct tgtcttaatt aggcctaact gttccagagc agaggccatg 2040
atttagtgga ccatgaatga ttgagatttt gcctgtgtac tatcaatgcc acttgaacca 2100
cagcattcac tttaatactt actgagcatc tcccatgtgc aaggtcctgg aactacaggg 2160
ataagacagg gtccatgccg tctcaaggca tttacggttt aaaaagacct ttgtaatta 2219
<210> 30
<211> 1280
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2614283CB1
<400> 30
ccgcaggagc cgggccggag tgagcgcacc tcgcggggcc cctcggggca ggtgggtgag 60
cgccacccgg agtcccgcgc gcaactttca gggcgcactc ggcggggcgg ctgcgcggct 120
gccgggactc ggcgcgggac tgcatggagg ccaaggagaa gcagcatctg ttggacgcca 180
ggccggcaat ccggtcatac acgggatctc tgtggcagga aggggctggc tggattcctc 240
tgccccgacc tggcctggac ttgcaggcca ttgagctggc tgcccagagc aaccatcact 300
gccatgctca gaagggtcct gacagtcact gtgaccccaa gaaggggaag gcccagcgcc 360
agctgtatgt agcctctgcc atctgcctgt tgttcatgat cggagaagtc gttggtgggt 420
acctggcaca cagcttggct gtcatgactg acgcagcaca cctgctcact gactttgcca 480
gcatgctcat cagcctcttc tccctctgga tgtcctcccg gccagccacc aagaccatga 540
actttggctg gcagagagct gagatcttgg gagccctggt ctctgtactg tccatctggg 600
tcgtgacggg ggtactggtg tacctggctg tggagcggct gatctctggg gactatgaaa 660
ttgacggggg gaccatgctg atcacgtcgg gctgcgctgt ggctgtgaac atcataatgg 720
ggttgaccct tcaccagtct ggccatgggc acagccacgg caccaccaac cagcaggagg 780
agaaccccag cgtccgagct gccttcatcc atgtgatcgg cgactttatg cagagcatgg 840
gtgtcctagt ggcagcctat attttatact tcaagccaga atacaagtat gtagacccca 900
tctgcacctt cgtcttctcc atcctggtcc tggggacaac cttgaccatc ctgagagatg 960
tgatcctggt gttgatggaa gggaccccca agggcgttga cttcacagct gttcgtgatc 1020
tgctgctgtc ggtggagggg gtagaagccc tgcacagcct gcatatctgg gcactgacgg 1080
tggcccagcc tgttctgtct gtccacatcg ccattgctca gaatacagac gcccaggctg 1140
tgctgaagac agccagcagc cgcctccaag ggaagttcca cttccacacc gtgaccatcc 1200
agatcgagga ctactcggag gacatgaagg actgtcaggc atgccagggc ccctcagact 1260
gactgctcag ccaggcacca 1280
51/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
<210> 31
<211> 2727
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2667691CB1
<400> 31
cagtagtggt gggacggcac tagctgctgg ggcctgccgc cccgggagtg gctgcagcag 60
cgccaggaat cgaggatggt aaaatgaccc aggggaagaa gaagaaacgg gccgcgaacc 120
gcagtatcat gctggccaag aagatcatca ttaaggacgg aggcacgcct caaggaatag 180
gttctcctag tgtctatcat gcagttatcg tcatcttttt ggagtttttt gcttggggac 240
tattgacagc acccaccttg gtggtattac atgaaacctt tcctaaacat acatttctga 300
tgaacggctt aattcaagga gtaaagggtt tgttgtcatt ccttagtgcc ccgcttattg 360
gtgctctttc tgatgtttgg ggccgaaaat ccttcttgct gctaacggtg tttttcacat 420
gtgccccaat tcctttaatg aagatcagcc catggtggta ctttgctgtt atctctgttt 480
ctggggtttt tgcagtgact ttttctgtgg tatttgcata cgtagcagat ataacccaag 540
agcatgaaag aagtatggct tatggactgg tttcagcaac atttgctgca agtttagtca 600
ccagtcctgc aattggagct tatcttggac gagtatatgg ggacagcttg gtggtggtct 660
tagctacagc aatagctttg ctagatattt gttttatcct tgttgctgtg ccagagtcgt 720
tgcctgagaa aatgcggcca gcatcctggg gagcacccat ttcctgggaa caagctgacc 780
cttttgcgtc cttaaaaaaa gtcggccaag attccatagt gctgctgatc tgcattacag 840
tgtttctctc ctacctaccg gaggcaggcc aatattccag ctttttttta tacctcagac 900
agataatgaa attttcacca gaaagtgttg cagcgtttat agcagtcctt ggcattcttt 960
ccattattgc acagaccata gtcttgagtt tacttatgag gtcaattgga aataagaaca 1020
ccattttact gggtctagga tttcaaatat tacagttggc atggtatggc tttggttcag 1080
aaccttggat gatgtgggct gctggggcag tagcagccat gtctagcatc acctttcctg 1140
ctgtcagtgc acttgtttca cgaactgctg atgctgatca acagggtgtc gttcaaggaa 1200
tgataacagg aattcgagga ttatgcaatg gtctgggacc ggccctctat ggattcattt 1260
tctacatatt ccatgtggaa cttaaagaac tgccaataac aggaacagac ttgggaacaa 1320
acacaagccc tcagcaccac tttgaacaga attccatcat ccctggccct cccttcctat 1380
ttggagcctg ttcagtactg ctggctctgc ttgttgcctt gtttattccg gaacatacca 1440
atttaagctt aaggtccagc agttggagaa agcactgtgg cagtcacagc catcctcata 1500
atacacaagc gccaggagag gccaaagaac ctttactcca ggacacaaat gtgtgacgac 1560
tgaaatcagg aagatttttc tatcagcacc caggtcttag ttttcacctc tagttctgga 1620
tgtacattcc atttccatcc acagtgtact ttaagattgt cttaagaaat gtatctgcat 2680
gaactccgtg ggaactaaag gaagtgggaa cttagaacca gacagttttc caaagatgtt 1740
acaatttctt ttgaaaaacc ttttgtttat tagcaccaat ttcttgccac taagctattt 1800
gttttattat acatccttta attaaaaact atatatgtaa cttcttagat attagcaaat 1860
gtctctgcta ccatttcctt aaggtgttga gctttaactc tatgctgact cagtgagaca 1920
cagtaggtag tatggttgtg gacctatttg ttttaacatt gtaaaatttt gagtcagatt 1980
ttaatattgt aaaatcttgg gtcaaataat tcaaagcctt aatgcagatg cactaaaaca 2040
aagaaatggt aaatgaattg tttgcattta aaaaaaaaaa ctcttaagaa aactgtacta 2100
aatctgaatc atgttttgag cttgtttgca gtacttttaa acattattca ctactgtttt 2160
tgaagtgaga aagtatcagc catttagcat ttaagttggg gtatttagag cctgtaatct 2220
aaatgctggc tcaaatttat tccccagcta cttcttatac cactattctt ttaatgtttg 2280
cataatcata agcacctcaa cacttgaata cataatctaa aaattatata gtaaagctgg 2340
tagccttgaa aatgtcagtg tgatatctat tatgtagata aatatatata gtggcctttc 2400
aggactgtca cagtaacact ttatttacag agctaatgtt tgtcctaaat tttcaggacc 2460
ctagaggaga gctttataca attaccgatg tgaatttctc taaagtgtat atttttgtgt 2520
ccagttatat tatttaaaaa agtgttactt tgtaaaaatt gtatataaag aactgtatag 2580
tttacactgt tttcatcttg tgtgtggtta ttgcttaatg ctttttaaac ttggaacact 2640
cactatggtt aaataaggtc ttaaaagaaa tgtaaatatt ctgttaataa agttaaatat 2700
tttaatgatt ttttttttaa aaaaaaa 2727
52/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
<210> 32
<211> 1631
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3211415CB1
<400> 32
ttgcgcttga atgttcgttg actggcccgt cggttttaca aggcccggac aagcgctggg 60
gattcccgtt tgaggcgtca ctactgtcac tgccatcacc ccacggagcc acttctagag 120
gggagtagac ccggcccttc gccgggcaga gaagatgttg cccctgtcca tcaaagacga 180
tgaatacaaa ccacccaagt tcaatttgtt cggcaagatc tcgggctggt ttaggtctat 240
actgtccgac aagacttccc ggaacctgtt tttcttcctg tgcctgaacc tctctttcgc 300
ttttgtggaa ctactctacg gcatctggag caactgctta ggcttgattt ccgactcttt 360
tcacatgttt ttcgatagca ctgccatttt ggctggactg gcagcttctg ttatttcaaa 420
atggagagat aatgatgctt tctcctatgg gtatgttaga gcggaagttc tggctggctt 480
tgtcaatggc ctatttttga tcttcactgc tttttttatt ttctcagaag gagttgagag 540
agcattagcc cctccagatg tccaccatga gagactgctt cttgtttcca ttcttgggtt 600
tgtggtaaac ctaataggaa tatttgtttt caaacatgga ggtcatggac attctcatgg 660
ctctggtggc cacggacaca gtcattccct ctttaatggt gctctagatc aggcacatgg 720
ccatgtcgat cattgccata gccatgaagt gaaacatggt gctgcacata gccatgatca 780
tgctcatgga catggacact ttcattctca tgatggcccg tccttaaaag aaacaacagg 840
acccagcaga cagattttac aaggtgtatt tttacatatc ctagcagata cacttggaag 900
tattggtgta attgcttctg ccatcatgat gcaaaatttt ggtctgatga tagcagatcc 960 ~'
tatctgttca attcttatag ccattcttat agttgtaagt gttattcctc ttttaagaga 1020
atctgttgga atattaatgc agagaactcc tcccctatta gaaaatagtc tgcctcagtg 1080
ctatcagagg gtacagcagt tgcaaggagt ttacagttta caggaacagc'acttctggac 1140
tttatgttct gacgtttatg ttgggacctt gaaattaata gtagcacctg atgctgatgc 1200
taggtggatt ttaagccaaa cacataatat ttttactcag gctggagtga gacagctcta 1260
cgtacagatt gactttgcag ccatgtagtg aatggaaaga aattatgcac cttttatgga 1320
ccaaattttt ctggcccaac ccgatgagat gaagcatttc aaacttgagg agaagagaga 1380
ctgcaggacg aggtggacag aaaaaccgtc agtaacaccg aggacatcta aaatctagtg 1440
atgaacacga tgacaaccaa agtagcacaa gagaagaggc aagtcacagg gaggggtttg 1500
ggaaccttat gtcacgctta agaactggga tgggaggttt taaacaaaaa aaaaaaaaaa 1560
aaaaggggcg gccgccgact attgaaccct tcgcccgggg aataaattcc ggcccggtac 1620
ctcgaggggg g
1631
<210> 33
<211> 2673
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4739923CB1
<400> 33
ggacatttta aaagggccgg agattgcggg cgtcagtggc catggcggat acagcgacta 60
cagcatcggc ggcggcggct agtgccgcta gcgcctcgag cgatgcacct cctttccaac 120
tgggcaaacc ccgcttccag cagacgtcct tctatggccg cttcaggcac ttcttggata 180
tcatcgaccc tcgcacactc tttgtcactg agagacgtct cagagaggct gtgcagctgc 240
tggaggacta taagcatggg accctgcgcc cgggggtcac caatgaacag ctctggagtg 300
cacagaaaat caagcaggct attctacatc cggacaccaa tgagaagatc ttcatgccat 360
ttagaatgcc aggttatatt ccttttggga cgccaattgt agtcggtctt ctcttgccca 420
53/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
accagacact ggcatccact gtcttctggc agtggctgaa ccagagccac aatgcctgtg 480
tcaactatgc aaaccgcaat gcgaccaagc cttcacctgc atccaagttc atccagggat 540
acctgggagc tgtcatcagc gccgtctcca ttgctgtggg ccttaatgtc ctggttcaga 600
aagccaacaa gctcacccca gccacccgcc ttctcatcca gaggtttgtg ccgttccctg 660
ctgtagccag tgccaatatc tgcaatgtgg tcctgatgcg gtacggggag ctggaggaag 720
ggattgatgt cctggacagc gatggcaacc tcgtgggctc ctccaagatc gcagcccgac 780
acgccctgct ggagacggcg ctgacgcgag tggtcctgcc catgcccatc ctggtgctac 840
ccccgatcgt catgtccatg ctggagaaga cggctctcct gcaggcacgc ccccggctgc 900
tcctccctgt gcaaagcctc gtgtgcctgg cagccttcgg cctggccctg ccgctggcca 960
tcagcctctt cccgcaaatg tcagagattg aaacatccca attagagccg gagatagccc 1020
aggccacgag cagccggaca gtggtgtaca acaaggggtt gtgagtgtgg tcagcggcct 1080
ggggacggag cactgtgcag ccggggagct gaggggcagg gccgtagact cacggctgca 1140
cctgcaggga gcagcacgcc aaccccagca gtcctgggcc ccctgggaga gtgctcaacc 1200
tacagtggag ggagactgac ccattcacat tttaacatag gcaagaggag ttctaacaca 1260
tttcgtacaa aaaaataatc aagtgcattt ctgggcctta tgtggggttg tcaaaactcc 1320
actcagcaca attatgtgtg aagctgaaaa attgtagagt gcccatgggg tagaagtaga 1380
atccttttat actttggttc cttttttatt tttatttttt atcagaatca aatctgagcc 1440
ttagtttcag ctgaccagaa gtggcaggag gacaggtgga ggcgagccag attaggcctg 1500
gagttgggct ggtttggtgg ccaggctggt aaatttagga tattacaatg gccagcccag 1560
atggctccct gggctggcat ggggagggga gagaaggtgg tctgcacccc acaggatgaa 1620
ctagccatga ctagggtcct caggcagtgg cccagggaat cagggagcac tggaggcctc 1680
tgcaagattc tgtgggcagc tggctctgaa tgtagccagc ccacatccat tccagagctg 1740
cagaaccagt ccctctgagt gaagtccagt gaccctggag ctagggtccc ccttcgtgga 1800
gctctaactg attcagggcc cctgaagtga~ccccagctcc aggcaggaaa ccccgagaag 1860..
gaatggtgct tggcaggaac catggacctg cacttggcct cttcgggaag atcctccttc 1920
caggccccag gctggatgct gggttctggg gctgagagtg gggctagact gggctggctg 1980
ccttctgcgg agcttctcca gccaccaagg ctgcctgccc catcccatcc ctttctaagc 2040
aggaaggtct catgcctgag aatgcctagg ccagctcctt agacacctat cagagaagca 2100
gcatcaacct aggagcagtg ggccctggct ctgtcactaa atggcagtga gatgtcagac 2160'
aaattcattc ccttctctca gcctccactc cctgtctgaa accagaagac tggatcaagg 2220
ggttcccact ggctcctcca gcaagacctc gtctttgctt gtcctgctca gatgctggtc 2280
atcctgggca tgtccccagt gtggactctg gactgggaag ggggcaggcc cctttggacc 2340
tgcagttggc ctcagcagaa ggccttgcct tgtgtatgtg actccatatc ccgggagcag 2400
ttgacctttg ccaaacactt tacagttctg gaggaggagg taacatagat gcctgggcct 2460
gatggtgggg ccatacccat gtgtcgcctc tcactctggc agcctcagag gccccttgct 2520
gctggctccc atctccctcc catttgcaga ccaggaagga agagcaagct gtacaaaggg 2580
aagcagagcc tggggtgggt gtgagcaggg tgacccctca tctgaaaggc ccaaaccagg 2640
gggaagcacc agcctcagtg cagccccctc ctg 2673
<210> 34
<211> 3958
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 55030459CB1
<400> 34
atggcccgcc agccggagga agaggagacg gccgtggccc gggcgcggcg gccgcccctc 60
tggctgctct gcctggtcgc gtgctggctc ctgggcgccg gggccgaagc cgacttctcc 120
atcctggacg aggcgcaagt gctggcgagc cagatgcgga ggctggcggc cgaggagctg 180
ggggtcgtca ccatgcagcg gatattcaac tcctttgttt acactgagaa aatctcaaat 240
ggagaaagtg aagtacagca gctagccaaa aaaatccgag agaagttcaa ccgttacttg 300
gatgtggtca atcggaacaa gcaagttgta gaagcatcct atacggctca cctaacctct 360
cccctaactg caattcaaga ctgctgtact atcccacctt ccatgatgga attcgatggg 420
54/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
aactttaata ccaatgtgtc tagaacaatt agttgtgatc gactttctac tactgttaat 480
agccgggcct tcaatccagg acgagactta aattcagttc ttgcagacaa cctgaaatcc 540
aaccctggaa ttaagtggca atatttcagt tcagaagaag gaattttcac tgttttccca 600
gcacacaagt tccggtgtaa gggcagctac gaacaccgca gtagacccat ctacgtctct 660
acagtccggc cgcagtcaaa gcacatagta gtgattctgg accacggggc ttcagtcaca 720
gacactcagc ttcagattgc caaggacgct gctcaggtca tcctcagcgc catcgatgaa 780
catgacaaga tttctgtgtt aactgtggca gataccgtcc ggacttgctc actagaccag 840
tgctataaga ccttcttgtc tccagccacc agtgagacaa aaaggaaaat gtccaccttt 900
gttagcagcg tgaagtcttc agacagtcct acccagcacg cagtgggatt ccaaaaggca 960
tttcagctga ttcgaagtac aaacaataac acaaagttcc aagcaaatac agacatggtc 1020
atcatttacc tgtcagctgg cattacatca aaggactctt cggaagaaga taaaaaagcg 1080
actctccaag tcatcaatga agaaaatagc tttctaaaca actctgtaat gattctcacc 1140
tatgccctca tgaacgatgg ggtgactggt ttgaaagagc tggcttttct gagggatcta 1200
gctgaacaga attcagggaa gtacggtgtg ccagaccgga cggccttgcc tgtgattaag 1260
ggcagcatga tggtgctgaa tcagttgagc aacctggaga ccacagtggg caggttctac 1320
acaaaccttc ccaaccggat gattgatgaa gccgtcttca gcctgccctt ctctgatgag 1380
atgggagatg gtttgataat gactgtgagt aaaccctgtt attttggaaa cctacttctg 1440
ggaattgtag gtgtggacgt gaatctggct tacattcttg aagacgtgac gtattaccaa 1500
gactctttgg cttcctatac ttttctcata gacgacaaag gatatacact tatgcaccca 1560
tctcttacca ggccatattt attgtcagag cccccacttc atactgacat catacattat 1620
gaaaatattc caaaatttga attagttcgg caaaatatcc taagcctccc tctgggcagc 1680
cagattatcg cagtccctgt gaactcatcc ctgtcttggc acataaacaa gctgagagaa 1740
actggaaagg aagcctacaa tgttagctat gcctggaaga tggtacaaga cacttccttt 1800
attctgtgta ttgtggtgat acaaccagaa atacctgtga aacaactgaa gaacctcaac 1860
actgttccca gcagcaagct gctgtaccac cggctggatc tccttggcca gcccagtgct 1920'
tgcctccact tcaaacagct ggcaacccta gaaagtccca ccatcatgct gtctgctggc 1980
agcttttcct ccccctatga gcacctcagc cagccagaga caaagcgcat ggtagagcac 2040
tacaccgcct atctcagcga caacacccgc ctcattgcta acccgggcct caaattctct 2100
gtcagaaatg aagtaatggc taccagccac gtcacagatg aatggatgac acaaatggaa 2160
atgagtagcc tgaacactta cattgtccgc cgttacatag caacacccaa tggcgtcctc 2220
agaatttatc ctggttccct catggacaaa gcatttgatc ccactaggag acaatggtat 2280
ctccatgcag tagctaatcc agggttgatt tctttgactg gtccttactt agatgttgga 2340
ggagctggtt atgttgtgac aatcagtcac acaattcatt catccagtac acagctgtct 2400
tctgggcaca ctgtggctgt gatgggcatt gacttcacac tcagatactt ctacaaagtt 2460
ctgatggacc tattacctgt ctgtaaccaa gatggtggca acaaaataag gtgcttcata 2520
atggaggaca ggggttatct ggtggcgcac ccgactctca tcgaccccaa aggacatgca 2580
cctgtggagc agcagcacat cacccacaag gagcccctgg tagcaaatga tatcctcaac 2640
caccccaact ttgtaaagaa aaacctgtgc aacagcttca gtgacagaac ggtccagagg 2700
ttttataaat tcaacaccag ccttgcgggg gatttgacga accttgtgca tggcagccac 2760
tgttccaaat acagattagc aaggatccca ggaaccaacg cgtttgttgg cattgtcaac 2820
gaaacctgcg actctcttgc cttctgtgcc tgcagcatgg tggaccgact ctgtctcaac 2880
tgtcaccgaa tggaacaaaa tgaatgtgaa tgtccttgtg agtgccctct agaggtcaat 2940
gagtgcactg gcaacctcac caatgcagag aaccgaaacc ccagctgcga ggtccaccag 3000
gagccggtga catacacagc tattgaccct ggcctgcaag atgctcttca ccagtgtgtc 3060
aacagcaggt gcagtcagag gctggaaagt ggggactgtt ttggggtgct ggattgtgaa 3120
tggtgcatgg tggacagtga tggaaagact cacctggaca aaccctactg tgccccccag 3180
aaagaatgct tcggggggat tgtgggagcc aaaagtccct acgttgatga catgggagca 3240
ataggtgatg aggtgatcac attaaacatg attaaaagcg cccctgtggg tcctgtggct 3300
ggagggatca tgggatgcat catggtcttg gtcctggcgg tgtatgccta ccgccaccag 3360
attcatcgcc ggagccatca gcatatgtct cctcttgctg cccaagaaat gtcagtgcgt 3420
atgtccaacc tggagaatga cagagatgaa agggacgacg acagccacga agacagaggc 3480
atcatcagca acactcggtt tatagctgcg gtcatcgaac gacatgcaca cagtccagaa 3540
agaaggcgcc gctactgggg tcgatcagga acagaaagtg atcatggtta cagcaccatg 3600
agcccacagg aggacagtga aaatcctcca tgcaacaatg accccttgtc agccggggtc 3660
gatgtgggaa accatgatga ggacttagac ctggataccc cccctcagac tgctgcccta 3720
ctaagtcaca agttccacca ctaccggtca caccacccta cacttcatca tagccaccac 3780
55/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
ttacaggcgg ccgtcacggt acacactgtc gatgcagaat gctaacaatc tcctcacctc 3840
cacgccaaga tgagatctgg gagctacaga atgttctgga aagaaaaaga accggcttaa 3900
aacccacaag cagagacctc ccttgtgttt gtgctttgtg cagagttgtt tgagtcat 3958
<210> 35
<211> 2000
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6113039CB1
<400> 35
gctcaggaca atgaaattct tcagttacat tctggtttat cgccgatttc tcttcgtggt 60
tttcactgtg ttggttttac tacctctgcc catcgtcctc cacaccaagg aagcagaatg 120
tgcctacaca ctctttgtgg tcgccacatt ttggctcaca gaagcattgc ctctgtcggt 180
aacagctttg ctacctagtt taatgttacc catgtttggg atcatgcctt ctaagaaggt 240
ggcatctgct tatttcaagg attttcactt actgctaatt ggagttatct gtttagcaac 300
atccatagaa aaatggaatt tgcacaagag aattgctctg aaaatggtga tgatggttgg 360
tgtaaatcct gcatggctga cgctggggtt catgagcagc actgcctttt tgtctatgtg 420
gctcagcaac acctcgacgg ctgccatggt gatgcccatt gcggaggctg tagtgcagca 480
gatcatcaat gcagaagcag aggtcgaggc cactcagatg acttacttca acggatcaac 540
caaccacgga ctagaaattg atgaaagtgt taatggacat gaaataaatg agaggaaaga 600
gaaaacaaaa ccagttccag gatacaataa tgatacaggg aaaatttcaa gcaaggtgga 660
gttggaaaag aactcaggca tgagaaccaa atatcgaaca aagaagggcc acgtgacacg 720
taaacttacg tgtttgtgca ttgcctactc ttctaccatt ggtggactga caacaatcac 780
tggtacctcc accaacttga tctttgcaga gtatttcaat acacgctatc ctgactgtcg 840
ttgcctcaac tttggatcat ggtttacgtt ttccttccca gctgccctta tcattctact 900
cttatcctgg atctggcttc agtggctttt cctaggattc aattttaagg agatgttcaa 960
atgtggcaaa accaaaacag tccaacaaaa agcttgtgct gaggtgatta agcaagaata 1020
ccaaaagctt gggccaataa ggtatcaaga aattgtgacc ttggtcctct tcattataat 1080
ggctctgcta tggtttagtc gagaccccgg atttgttcct ggttggtctg cacttttttc 1140
agagtaccct ggttttgcta cagattcaac tgttgcttta cttatagggc tgctattctt 1200
tcttatccca gctaagacac tgactaaaac tacacctaca ggagaaattg ttgcttttga 1260
ttactctcca ctgattactt ggaaagaatt ccagtcattc atgccctggg atatagccat 1320
tcttgttggt ggagggtttg ccctggcaga tggttgtgag gagtctggat tatctaagtg 1380
gataggaaat aaattatctc ctctgggttc attaccagca tggctaataa ttctgatatc 1440
ttctttgatg gtgacatctt taactgaggt agccagcaat ccagctacca ttacactctt 1500
tctcccaata ttatctccat tggccgaagc cattcatgtg aaccctcttt atattctgat 1560
accttctact ctgtgtactt catttgcatt cctcctacca gtagcaaatc cacccaatgc 1620
tattgtcttt tcatatggtc atctgaaagt cattgacatg gttaaagctg gacttggtgt 1680
caacattgtt ggtgttgctg tggttatgct tggcatatgt acttggattg tacccatgtt 1740
tgacctctac acttaccctt cgtgggctcc tgctatgagt aatgagacca tgccataata 1800
agcacaaaat ttctgactat cttgcggtaa tttctggaag acattaatga ttgactgtaa 1860
aatgtggctc taaataacta atgacacaca tttaaatcag ttatggtgta gctgctgcaa 1920
ttcccgtgaa tacccgaaac ctgctgttat aactcagagt ccatatttgt tattgcagtg 1980
caactaaaga gcatctatgt 2000
<210> 36
<211> 1997
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
56J71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
<223> Incyte ID No: 7101781CB1
<400> 36
ctgctcgcca ctggccggcg cgctcccggc gcacggagca cactcgcgct cccggcgeac 60
ggagcacact cgcgctccgg gactgaaacc tgagcagccg tagcagccga atttgggagc 120
atatccttgt cactgcagcc agaaagccct tcgatcccca tcagagaggt cacatgagcc 180
ccgaggtcac ctgcccgcgg aggggccacc tgcctcgctt ccacccgagg acctgggttg 240
agcccgtggt ggcatcgtcc caggtggctg cctccctcta cgatgcgggg ctactcctcg 300
tggtgaaggc gtcctacgga accggaggct cctccaacca cagtgccagc ccatcgcccc 360
ggggggctct agaggaccaa cagcagagag ccatctccaa tttctacatt atctacaacc 420
ttgtggtggg cctgtccccc ctgctgtccg cctacgggct gggatggctc agcgaccgct 480
accaccgaaa gatctccatc tgcatgtcgc tgctgggctt cctgctctcc cgcctcgggc 540
tgctgctcaa ggtgctgctg gactggccag tggaggtgct gtacggggcg gcggcgctga 600
acgggctatt cggcggcttc tccgccttct ggtccggggt catggcgctg ggatcgctgg 660
gctcctccga gggccgccgc tctgtgcgcc tcatcctcat tgacctgatg ctgggcttgg 720
cggggttctg cgggagcatg gcttccgggc atctcttcaa gcagatggct gggcactctg 780
ggcagggcct gatactgacg gcctgcagcg tgagctgtgc ctcgtttgcc ctgctctaca 840
gccttttggt gctaaaggtc cctgagtcgg tggccaaacc cagccaggag ctccccgccg 900
tggataccgt gtctggcacg gttggcacat accgcactct ggatcctgat cagttggacc 960
aacagtatgc agtggggcac cctccatctc ctggaaaagc aaaaccccat aaaaccacca 1020
ttgccttgct ctttgtgggt gctatcatat atgacctggc ggtggtgggc acagtggacg 1080
tgatccctct ttttgtgctg agggagcctc tcggttggaa ccaagtgcag gtgggctatg 114D
gtatggctgc agggtacacc atcttcatca ccagcttcct gggtgtcctg gtcttctccc 1200
gctgctttcg ggacaccacc atgatcatga ttgggatggt ctcctttggg tcaggagccc 1260
tcctcttggc ttttgtgaaa gagacataca tgttctatat tgctcgagcc gtcatgctgt 1320
ttgctctcat ccccgtcaca accatccgat cagctatgtc caaactcata aagggctcct 1380
cttatggaaa ggtgttcgtc atactgcagc tgtccttggc tctgaccggc gtggtgacat 1440
ccaccttgta caacaagatc taccagctca ccatggacat gtttgtgggc tcctgctttg 1500
ctctctcctc ctttctctcc ttcctggcca tcattccaat tagcatcgtg gcctataaac 1560
aagtcccatt gtcaccatat ggagacatca tagagaaatg aagatgctta cctgcaggaa 1620
ctgaaaacat cagccatggc caggccccca gaagacaaaa gaagggacca gggaactggt 1680
gacctaagca acccactgct taagaaacct gcgttccagc cagagttggc ctcagaatga 1740
cctgctctgg ctcagggatc cctggtggat ggggaaaagc actttcctgg tgatggaaaa 1800
acgttctcag ctttaagaca cccccattag gcagacactg ggttctgtga cagcagagca 1860
tgaccttaag ggttacaggg aggctgcaca cagcagtccc aggccctgtg aggggcttca 1920
gactccagct acagcgagcc tgcccctttt cttcaaggga ctgtcttgag ggctccaaag 1980'
tatagctaac tagtcac 1997
<210> 37
<211> 3069
<212> DNA
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7473036CB1
<400> 37
atgcaaccag ccagagggcc cctggcttca gaacctagga ctgtactggt tctgagattc 60
tgtgcaagcc tcatggaaat gaagctgcca ggccaggaag ggtttgaagc ctccagtgct 120
cctagaaata ttccttcagg ggagctggac agcaaccctg accctggcac cggccccagc 180
cctgatggcc cctcagacac agagagcaag gaactgggag tacccaaaga ccctctgctc 240
ttcattcagc tgaatgagct gctgggctgg ccccaggcgc tggagtggag agagacaggc 300
aggtgggtac tgtttgagga gaagttggag gtggctgcag gccggtggag tgccccccac 360
gtgcccaccc tggcactgcc cagcctccag aagctccgca gcctgctggc cgagggcctt 420
gtactgctgg actgcccagc tcagagcctc ctggagctcg tgggctctac tcatccaaga 480
57/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
aaggcttctg acaatgagga agcccccctg agggaacagt gtcagaaccc cctgagacag 540
aagctacctc caggagctga ggcagggact gtgctggcag gggagctggg cttcctggca 600
cagccactgg gagcctttgt tcgactgcgg aaccctgtgg tactggggtc ccttactgag 660
gtgtccctcc caagcaggtt tttctgcctt ctcctgggcc cctgtatgct gggaaagggc 720
taccatgaga tgggacgggc agcagctgtc ctcctcagtg acccgcaatt ccagtggtca 780
gttcgtcggg ccagcaacct tcatgacctt ctggcagccc tggatgcatt cctagaggag 840
gtgacagtgc ttcccccagg tcggtgggac ccaacagccc ggattccccc gcccaaatgt 900
ctgccatctc agcacaaaag gcttccctcg caacagcggg agatcagagg tcccgccgtc 960
ccgcgcctga cctcggctga ggacaggcac cgccatgggc cacacgcaca cagcccggag 1020
ttgcagcgga ccggcagcga tttcttggac gccctgcatc tccagtgctt ctcggccgta 1080
ctctacattt acctggccac tgtcactaat gccatcactt ttgggggtct gctgggagat 1140
gccactgatg gtgcccaggg agtgctggaa agtttcctgg gcacagcagt ggctggagct 1200
gccttctgcc tgatggcagg ccagcccctc accattctga gcagcacggg gccagtgctg 1260
gtctttgagc gcctgctctt ctctttcagc agagattaca gcctggacta cctgcccttc 1320
cgcctatggg tgggcatctg ggtggctacc ttttgcctgg tgctggtggc cacagaggcc 1380
agtgtgctgg tgcgctactt cacccgcttc actgaggaag gtttctgtgc cctcatcagc 1440
ctcatcttca tctacgatgc tgtgggcaaa atgctgaact tgacccatac ctatcctatc 1500
cagaagcctg ggtcctctgc ctacgggtgc ctctgccaat acccaggccc aggaggaaat 1560
gagtctcaat ggataaggac aaggccaaaa gacagagacg acattgtaag catggactta 1620
ggcctgatca atgcatcctt gctgccgcca cctgagtgca cccggcaggg aggccaccct 1680
cgtggccctg gctgtcatac agtcccagac attgccttct tctcccttct cctcttcctt 1740
acttctttct tctttgctat ggccctcaag tgtgtaaaga ccagccgctt cttcccctct 1800
gtggtgcgca aagggctcag cgacttctcc tcagtcctgg ccatcctgct cggctgtggc 1860
cttgatgctt tcctgggcct agccacacca aagctcatgg tacccagaga gttcaagccc 1920
acactccctg ggcgtggctg gctggtgtca ccttttggag ccaacccctg gtggtggagt 1980
gtggcagctg ccctgcctgc cctgctgctg tctatcctca tcttcatgga ccaacagatc 2040
acagcagtca tcctcaaccg catggaatac agactgcaga agggagctgg cttccacctg 2100
gacctcttct gtgtggctgt gctgatgcta ctcacatcag cgcttggact gccttggtat 2160
gtctcagcca ctgtcatctc cctggctcac atggacagtc ttcggagaga gagcagagcc 2220
tgtgcccccg gggagcgccc caacttcctg ggtatcaggg aacagaggct gacaggcctg 2280
gtggtgttca tccttacagg agcctccatc ttcctggcac ctgtgctcaa gttcattcca 2340
atgcctgtgc tctatggcat cttcctgtat atgggggtgg cagcgctcag cagcattcag 2400
ttcactaata gggtgaagct gttgttgatg ccagcaaaac accagccaga cctgctactc 2460
ttgcggcatg tgcctctgac cagggtccac ctcttcacag ccatccagct tgcctgtctg 2520
gggctgcttt ggataatcaa gtctacccct gcagccatca tcttccccct catgttgctg 2580
ggccttgtgg gggtGC~aaa ggccctggag agggtcttct caccacagga actcctctgg 2640
ctggatgagc tgatgccaga ggaggagaga agcatccctg agaaggggct ggagccagaa 2700
cactcattca gtggaagtga cagtgaagat tcagagctga tgtatcagcc aaaggctcca 2760
gaaatcaaca tttctgtgaa ttagctggag taggagtctg ggagtggaga ccccaggaaa 2820
cagcatgagt tcacaggtgc ttactcagga agtcaggaca tttttggcct ttggcttaac 2880
ttccagatgc tcagtcggct tggggaagga ctgaagggca gctgccaaga cctcagttac 2940
ctcctgacct gagggtggag agtggcagga agcaagcatg tttgctgtgc acttaggaaa 3000
ggctggtgag ccagagggac tgatcaggcc ccattcactc tctactcatt aaaaggtcct 3060
gagccacaa 3069
<210> 38
<211> 2241
<222> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7476943CB1
<400> 38
gccggcggcc cccgctcccg gatccccagc gccctggcca agaagcttcc tcggctcccc 60
58/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
ctcttccctc tccctgacac ggttgtgcag agggcgcggt ggctcaggcc ctggcaacca 120
ccattctact ttttgtgtct atgagtttga ctaccctaag gacctcacat ggcgagtaac 180
ccatgggcca ggtagcgttc tatgccaacc ttgaatgcca tcaggaagtc actggacagc 240
aaactcttcc aagatcataa cttggctgtt ggagcaacct ggaaaagaag aaaaaagaaa 300
aaccatggca aaagtaaata gagctcggtc tacctcccct ccagatggag gctggggctg 360
gatgattgtg gctggctgtt tccttgttac catctgcaca cgggcagtca caagatgtat 420
ctcaattttt tttgtggagt tccagacata cttcactcag gattacgcac aaacggcatg 480
gatccattcc attgtagatt gtgtgaccat gctctgtgct ccacttggga gtgttgtcag 540
taaccattta tcctgtcaag tgggaatcat gctgggtggc ttgcttgcat ctactggact 600
catcctgagc tcatttgcca cgagtctgaa gcatctctac ctcactctgg gagttcttac 660
aggtcttgga tttgcacttt gttactctcc agctattgcc atggttggca agtacttcag 720
cagacggaaa gcccttgctt atggtatcgc catgtcagga agtggcattg gcaccttcat 780
cctggctcct gtggttcagc tccttattga acagttttcc tggcggggag ccttactcat 840
tcttgggggc tttgtcttga atctctgtgt atgtggtgcc ttgatgaggc caattactct 900
taaagaggac cacacaactc cagagcagaa ccatgtgtgt agaactcaga aagaagacat 960
taagcgggtg tctccctatt catctttgac caaagaatgg gcacagactt gcctctgttg 1020
ctgtttgcag caagagtaca gttttttact catgtcagac tttgttgtgt tagccgtctc 1080
cgttctgttt atggcttatg gctgcagccc tctctttgtg tacttggtgc cttatgcttt 1140
gagtgttgga gtgagtcatc agcaagctgc ttttcttatg tccatacttg gagtgattga 1200
cattattggc aatatcacat ttggatggct gaccgacaga aggtgtctga agaattacca 1260
gtatgtttgc tacctctttg ccgtgggaat ggatgggctc tgctatctct gcctcccaat 1320
gcttcaaagt ctccctctgc tcgtgccttt ctcttgtacc tttggctact ttgatggtgc 1380
ctatgtgact ttgatcccag tagtgaccac agagatagtg gggaccacct ctttgtcatc 1440
agcgcttggt gtggtatact tccttcacgc agtgccatac ttggtgagcc cacccatcgc 1500
aggacggctg gtagatacca ccggcagcta cactgcagca ttcctcctct gtggattttc 1560
aatgatattt agttctgtgt tgcttggctt tgctagactt ataaagagaa tgagaaaaac 1620
ccagttgcag ttcattgcca aagaatctga tcctaagctg cagctatgga ccaatggatc 1680
agtggcttat tctgtggcaa gagaattaga tcagaaacat ggggagcctg tggctacagc 1740
agtgcctggc tacagcctca catgaccaaa ggccttgagc cccagaatct tcaggtttga 1800
gagaggtggg gccaccagat tcttcatgtt tctgaaactt tttattttgg cagaaggatt 1860
gccttccaag gaaattatta ttattgtttt gttaacatat taatatttat aagggaaaac 1920
agcacataat aaggaaagct ggactagccc agagccttct catttgggat ttgtgctcat 1980
aactgaactc gtatctttgg tcaatgggca tagctctgta agaaatgtaa ggacacagct 2040
gatataatta gctgtaatta gggataattt caaagcataa ccaaagcaga tgacactggg 2100
cagcagcttt gttccagtct caggcccttc atgttccctc ctcagaaaga aatggaacat 2160
taacgtggta gctttggtta cttggtctgg ttagagaagg aggccagtga gtggggggtg 2220
aagtgaaaag caaataaagt a 2241
<210> 39
<211> 1593
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 8003355CB1
<400> 39
ccttggagct gttgtcccac ccctgtcact gcagagagct gaggcaccat gcatgggggc 60
caggggccgc tgctcctcct gctgctgctg gctgtctgcc tgggggccca gggccggaac 120
caggaggagc gcctgctcgc agacctgatg caaaactacg accccaacct gcggcccgcg 180
gaacgagact cggatgtggt caatgtcagc ctgaagctaa ccctcaccaa cctcatctcc 240
ctgaacgagc gagaggaagc cctcaccacc aatgtctgga tagaggtgca gtggtgcgac'300
tatcgcctgc gccgggatcc gcgagactac gaaggcctgt gggtgctgag ggtgccgtcc 360
accatggtgt ggcggccgga tatcgtgctg gagaacaacg cggacggtgt cttcgaggtg 420
gccctctact gcaatgtgct cgtgtcccct gacggctgta tctactggct gccgcctgcc 480
59!71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
atcttccgtt ccgcctgctc tatctcagtc acctacttcc ccttcgactg gcagaactgc 540
tcccttatct tccagtccca gacttacagc accaatgaga ttgatctgca gctgagtcag 600
gaagatggcc agaccatcga gtggattttc attgaccctg aggccttcac agagaatggg 660
gagtgggcca tccagcaccg accagccaag atgctcctgg acccagcggc gccagcccag 720
gaagcaggcc accagaaggt ggtgttctac ctgctcatcc agcgcaagcc cctcttctac 780
gtcatcaaca tcatcgcccc ctgtgtgctc atctcctctg tcgccatcct catccacttc 840
cttectgcca aggctggggg ccagaagtgt accgtcgcca tcaacgtgct cctggcccag 900
actgtcttcc tcttccttgt ggccaagaag gtgcctgaaa cctcccaggc ggtgccactc 960
atcagcaagt acctgacctt cctcctggtg gtgaccatcc tcattgtcgt gaatgctgtg 1020
gttgtgctca atgtctcctt gcggtctcca cacacacact ccatggcccg aggggtgttc 1080
ctgaggctct tgccccagct gctgaggatg cacgttcgcc cgctggcccc ggcagctgtg 1140
caggacaccc agtcccggct acagaatggc tcctcgggat ggtcgatcac aactggggag 1200
gaggtggccc tctgcctgcc tcgcagtgaa ctcctcttcc agcagtggca gcggcaaggg 1260
ctggtggcgg cagcgctgga gaagctagag aaaggcccgg agttagggct gagccagttc 1320
tgtggcagcc tgaagcaggc tgccccagcc atccaggcct gtgtggaagc ctgcaacctc 1380
attgcctgtg cccggcacca gcagagtcac tttgacaatg ggaatgagga gtggttcctg 1440
gtgggccgag tgctggaccg cgtctgcttc ctggccatgc tctcgctctt catctgtggc 1500
acagctggca tcttcctcat ggcccactac aaccgggtgc cggccctgcc attccctgga 1560
gatccacgcc cctacctgcc ctcaccagac tga 1593
<210> 40
<211> 2121
<212> DNA
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte ID No: 3116448CB1
<400> 40
gtacaaagga cctccagacc agagccagcc agcagcaaaa agagcatgga gctgaggagt 60
acagcagccc ccagagctga gggctacagc aacgtgggct tccagaatga agaaaacttt 120
cttgagaacg agaacacatc aggaaacaac tcaataagaa gcagagctgt gcaaagcagg 180
gagcacacaa acaccaaaca ggatgaagaa caggtcacag ttgagcagga ttctccaaga 240
aacagagaac acatggagga tgatgatgag gagatgcaac aaaaagggtg tttggaaagg 300
aggtatgaca cggtatgtgg tttctgtagg aaacacaaaa caactcttcg gcacatcatc 360
tggggcattt tattagcagg ttatctggtt atggtgattt cggcctgtgt gctgaacttt 420
cacagagccc ttcctctttt tgtgatcacc gtggctgcca tcttctttgt tgtctgggat 480
cacctgatgg ccaaatacga acatcgaatt gatgagatgc tgtctcctgg cagaaggctt 540
ctaaacagcc attggttctg gctgaagtgg gtgatctgga gctccctggt cctagcagtt 600
attttctggt tggcctttga cactgccaaa ttgggtcaac agcagctggt gtccttcggt 660
gggctcataa tgtacattgt cctgttattt ctattttcca agtacccaac cagagtttac 720
tggagacctg tcttatgggg aatcgggcta cagtttcttc ttgggctctt gattctaagg 780
actgaccctg gatttatagc ttttgattgg ttgggcagac aagttcagac ttttctggag 840
tacacagatg ctggtgcttc atttggcttt ggtgagaaat acaaagacca cttctttgga 900
tttaaggtcc tggcgatcgt ggttttcttc agcactgtga tgtccatgct gtactacctg 960
ggactgatgc agtggattat tagaaaggtt ggatggatca tgctagttac tacgggatca 1020
tctcctattg aatctgtagt tgcttctggc aatatatttg ttggacaaac ggagtctcca 1080
ctgctggtcc gaccatattt accttacatc accaagtctg aactccacgc catcatgacc 2140
gccgggttct ctaccattgc tggaagcgtg ctaggtgcat acatttcttt tggggttcca 1200
tcctcccact tgttaacagc gtcagttatg tcagcacctg cgtcattggc tgctgctaaa 1260
ctcttttggc ctgagacaga aaaacctaaa ataaccctca agaatgccat gaaaatggaa 1320
agtggtgatt cagggaatct tctagaagct gcaacacagg gagcatcctc ctccatctcc 1380
ctggtggcca acatcgctgt gaatctgatt gccttcctgg ccctgctgtc ttttatgaat 1440
tcagccctgt cctggtttgg aaacatgttt gactacccac agctgagttt tgagctaatc 1500
tgctcctaca tcttcatgcc cttttccttc atgatgggag tggaatggca ggacagcttt 1560
60/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
atggttgcca gactcatagg ttataagacc ttcttcaatg aatttgtggc ttatgagcac 1620
ctctcaaaat ggatccactt gaggaaagaa ggtggaccca aatttgtaaa cggtgtgcag 1680
caatatatat caattcgttc tgagataatc gccacttacg ctctctgtgg ttttgccaat 1740
atcgggtccc taggaatcgt gatcggcgga ctcacatcca tggctccttc cagaaagcgt 1800
gatatcgcct cgggggcagt gagagctctg attgcgggga ccgtggcctg cttcatgaca 1860
gcctgcatcg caggcatact ctccagcact cctgtggaca tcaactgcca tcacgtttta 1920
gagaatgcct tcaactccac tttccctgga aacacaacca aggtgatagc ttgttgccaa 1980
agtctgttga gcagcactgt tgccaagggt cctggtgaag tcatcccagg aggaaaccac 2040
agtctgtatt ctttgaaggg ctgctgcaca ttgttgaatc catcgacctt taactgcaat 2100
gggatctcta atacattttg a 2121
<210> 41
<211> 1225
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 622868CB1
<400> 41
aattcattgg catgaagtct cggacatggg cgtctgtcca tttgcattcc ttttttgcag 60
ttggaaccct gctggtggct ttgacaggat acttggtcag gacctggtgg ctttaccaga 120
tgatcctctc cacagtgact gtccccttta tcctgtgctg ttgggtgctc ccagagacac 180
ctttttggct tctctcagag ggacgatatg aagaagcaca aaaaatagtt gacatcatgg 240
ccaagtggaa cagggcaagc tcctgtaaac tgtcagaact tttatcactg gacctacaag 300
gtcctgttag taatagcccc actgaagttc agaagcacaa cctatcatat ctgttttata 360
actggagcat tacgaaaagg acacttaccg tttggctaat ctggttcact ggaagtttgg 420
gattctactc gttttccttg aattctgtta acttaggagg caatgaatac ttaaacctct 480
tcctcctggg tgtagtggaa attcccgcct acaccttcgt gtgcatcgcc acggacaagg 540
tcgggaggag aacagtcctg gcctactctc ttttctgcag tgcactggcc tgtggtgtcg 600
ttatggtgat cccccagaaa cattatattt tgggtgtggt gacagctatg gttggaaaat 660
ttgccatcgg ggcagcattt ggcctcattt atctttatac agctgagctg tatccaacca 720
ttgtaagatc gctggctgtg ggaagcggca gcatggtgtg tcgcctggcc agcatcctgg 780
cgccgttctc tgtggacctc agcagcattt ggatcttcat accacagttg tttgttggga 840
ctatggccct cctgagtgga gtgttaacac taaagcttcc agaaaccctt gggaaacggc 900
tagcaactac ttgggaggag gctgcaaaac tggagtcaga gaatgaaagc aagtcaagca 960
aattacttct cacaactaat aatagtgggc tggaaaaaac ggaagcgatt acccccaggg 1020
attctggtct tggtgaataa atgtgccatg cctgctgtct agcacctgaa atattattta 1080
ccctaatgcc tttgtattag aggaatctta ttctcatctc ccatatgttg tttgtatgtc 1140
tttttaataa attttgtaag aaaattttaa agcaaatatg ttataaaaga aataaaaact 1200
aagatgaaaa ttctcagttt taaaa 1225
<210> 42
<211> 2693
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7476494CB1
<400> 42
tcctttgaga cagctgctct gagagaatgc aataagcagg gagcagccag caattcctcc 60
tagcagaggg cgactcgtgg gaggagttca gtttgccaag tattgtcatt tgttgagaga 120
aggtgtgtgc tcaaggagga gttttaacct ggaggatcat taactctttt agtcagctga 180
61/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
ggagctgcgg tggctcggcg agttggagtt catcctggaa gcgtctgcac gacaaggtca 240
gggatgaggt gtggaataac tttttcatgg gacactttga gaagggccag cacgctctgc 300
tcaatgaagg agaagagaat gagatggaga tatttggcta tcggactcaa ggctgccgga 360
aaagtctctg ccttgccgga tccatcttct catttggaat cctccccttg gtgttttact 420
ggagaccagc atggcacgta tgggcacatt gtgtcccatg ttccttgcaa gaagcagaca 480
ctgtgttgct gaggacaacg gtgagatgca tcaaagtgca gaaaataaga tatgtttgga 540
actacttaga aggacagttc cagaaaattg gttctttgga agactggctc agttctgcca 600
agatacatca aaaatttgga tcaggcttga caagagaaga acaggagatt aggaggttaa 660
tgtgtgggcc taatactatc gatgttgaag ttacaccaat ttggaaactg ctcatcaagg 720
aggttctaaa tccattttat atatttcaac tcttcagtgt ctgtttgtgg tttagtgaag 780
actataagga atatgctttt gccatcataa tcatgtccat aatttccata tctttgacag 840
tatatgatct cagagagcaa tctgtaaaac tccaccatct cgtcgagtca cataatagca 900
ttacggtctc tgtatgtggg agaaaagctg gagttcaaga gctggaatca cgcgtcctgg 960
tgcctggaga tttattaatt ttgacaggga acaaagtgct aatgccatgt gatgccgttc 1020
tgattgaagg cagctgtgtg gtggatgaag gcatgctgac aggagaaagt attccagtca 1080
ccaaaactcc gttacccaag atggatagct ctgtgccctg gaaaacacag agtgaagcgg 1140
attacaagcg gcatgtcctc ttctgtggaa cagaggttat ccaggccaag gcagcttgct 1200
ctgggaccgt gagagccgtg gtactgcaga ctggattcaa cactgcaaag ggagaccttg 1260
tgagatccat tctctaccct aagccagtga attttcagtt gtacagggat gccatcaggt 1320
tcctcctgtg ccttgtagga acagccacca ttgggatgat ctatactctg tgtgtctatg 1380
tgcttagtgg ggaacctcca gaggaggtgg tgaggaaagc ccttgacgtc atcacaattg 1440
cggttcctcc ggctctacct gctgctctga ccacaggcat tatctatgcc cagaggaggc 1500
tgaagaagag aggcatcttc tgcattagcc cccagaggat caacgtatgt ggacagttaa 1560
accttgtctg ctttgacaag acaggcacct taacaaggga cggcttggac ctctggggag 1620
tcgtgtcctg tgataggaat ggctttcagg aagttcacag ctttgcctca ggccaggctt 1680
tgccatgggg cccactgtgt gcagcgatgg ccagctgcca ctctctgatc cttcttgatg 1740
ggaccatcca gggagaccct ctggacctca aaatgtttga agccaccacc tgggaaatgg 1800'
ctttttctgg ggacgatttc cacatcaagg gagtgccggc acatgccatg gtagttaagc 1860 '
cctgcagaac agccagccag gtcccagtgg aaggaattgc aatcctgcat cagttcccat 1920
tctcatcggc actgcaaaga atgacagtca ttgtccaaga gatgggaggt gaccgactgg 1980
cattcatgaa aggtgcacca gagagggtgg ccagcttttg ccaacctgag acagtaccca 2040
ctagttttgt tagcgaactt cagatttaca cgacacaggg cttccgagtc atagcactgg 2100
ectacaagaa gctggaaaat gaecatcacg ctactacctt gacgagggag acggtagaat 2160
cagacctgat atttctgggg ctgctgatct tggagaatcg attgaaggaa gagacaaaac 2220
ctgtcttgga agagctcatc tcagcccgga taaggactgt aatgatcaca ggtgacaatc 2280.
ttcagactgc aataacagtg gccagaaaat ctggaatggt ttctgaaagc cagaaagtca 2340
ttctcattga ggcaaatgaa accaccgggt cctcatcagc atctatatct tggacgttag 2400
tagaagagaa gaaacacatt atgtatggga atcaggacaa ttacattaac atcagggatg 2460
aagtctctga taaaggcaga gaaggaagtt accattttgc cctaactgga aaatcctttc 2520
atgttataag tcaacatttc agcagcctac tgccaaagat attgatcaat gggaccatct 2580
ttgcaagaat gtctcctggg cagaagtcca gtctggtgga agaatttcag aaactggagt 2640
aggttctttg ccagtgcagg tggcatgaac tgcatggagg cataacagtc agg 2693
<210> 43
<211> 3569
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7477260CB1
<400> 43
agaccagtgt tggaggatgg cttgcttggg gcccggtggg aagaagaacc ccctcgggtg 60
gtaagctgaa gttgggtcag agtgcttctc acttctctct tcagttctgg ttcttgctac 120
tgccctggct tcgaccattc ccccatcatt cttcactgcc agctgcaaga ccctggatct 180
62/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
gaatgcagac taaatctttt catctctttt atcttaaaga gtatctcgcc cacctctgat 240
tcatggttac aggaggccag catcacccag gagctggact tagttttaca gaattagaaa 300
atacttttcc cttgtgcttg cctcctactc catttctgtt ggccttgtgg tcctcctgcc 360
ttccatggga cactcagcag acctgctgcc cctcttttgc agggtcccca gctgctgagc 420
agctccagga catcctgggg gaggaagatg aggctcccaa ccccaccctc tttacagaga 480
tggatactct gcagcatgac ggagaccaga tggagtggaa ggagtcagcc aggtggataa 540
agtttgaaga aaaggtagag gaaggcggcg aacgctggag caagccccac gtgtccacac 600
tatccctgca cagcctcttc gagctccgta cctgcctgca gacggggacg gtgctgctgg 660
atttggacag tggctcctta ccacagatca tagatgatgt cattgagaag cagattgagg 720
atggtctcct gcggccagag ctccgggaga gggtcagtta cgtcctcctg aggaggcacc 780
gccaccaaac caagaagccc atccaccgct ccttagctga cattgggaag tcagtctcca 840
ccacaaatcg cagtcctgcc cggagccctg gtgctggccc gagtctacac cactccacgg 900
aagacctgcg gatgcggcag agtgcaaatt acggacgtct gtgtcatgcc cagagcagaa 960
gcatgaatga catttctctc accccaaaca cagaccagcg gaaaaacaaa ttcatgaaga 1020
agatccccaa ggactcagaa gcgtccaacg tgctcgtggg cgaggtggac ttcctagacc 1080
agccattcat cgcgttcgtg cgcctcatcc agtcggccat gctgggagga gtgaccgagg 1140
tgcctgtccc caccagattt ctgtttatac tactgggacc ttctgggaga gcaaaatcct 1200
acaatgaaat tggccgtgcc attgcaaccc tcatggtaga tgatctcttc agtgacgtgg 1260
cctacaaagc ccgcaatcgg gaagatctga tcgcaggaat tgatgaattt ctggatgagg 1320
tcatcgtcct tcctcctgga gaatgggacc caaatatccg gattgagccc cccaagaagg 1380
tgccctctgc tgacaagagg aaatctctgt tctccctagc agagctgggc cagatgaatg 1440
gctctgtggg aggaggcggc ggagctcctg gaggaggcaa tggaggtggt ggtggtggtg 1500
gcagtggcgg cggggctggc agtggcgggg ccggcggaac aagcagcggg gatgatggag 1560
agatgccagc catgcatgaa atcggggagg aacttatctg gacaggaagg ttcttcggtg 1620
gcctgtgtct ggatatcaag aggaagttgc cctggttccc aagtgacttc tatgatggct 1680°
tccacattca gtccatctct gccatcctat tcatctacct cggctgtatc accaacgcga 1740
tcacctttgg tgggcttctg ggggatgcca ccgacaatta tcagggagtg atggagagct 1800'
tcctgggcac tgccatggct ggctccttgt tctgcctctt ctcgggacag cctctcatca 1860
ttctcagcag cacggggccc atcctcatct ttgagaagct cctcttcgac ttcagcaaag 1920
gcaatggcct ggactacatg gagttccgcc tctggattgg cctacactca gctgtccagt 1980
gccttatcct agtggccaca gatgccagct ttatcatcaa atatatcacc cgcttcaccg 2040
aggagggctt ctccaccctt atcagcttca tcttcatcta cgatgccatc aagaagatga 2100
tcggtgcctt caagtactac cctatcaata tggacttcaa gccaaacttc atcactacct 2160
acaagtgcga gtgtgtcgcc cctgacacag gtgacctgaa tacaaccgtg ttcaatgctt 2220
cagccccatt ggcaccagac accaacgctt ctctgtacaa cctccttaac ctcacagcgt 2280
tggactggtc cctgctgagc aagaaggagt gtctgagcta cggtgggcgc ctgcttggga 2340
attcctgcaa gtttatccca gacctggcgc tcatgtcctt catccttttc tttgggacat 2400
actccatgac cctgaccctg aagaagttca aattcagccg ctattttcct accaaggtcc 2460
gggccctggt ggctgacttt tccattgttt tctccatcct gatgttctgt ggaatcgatg 2520
cctgttttgg cctagaaact cccaagctgc atgtgcccag tgtcatcaag ccaacgcggc 2580
ctgaccgagg ctggttcgtg gccccctttg ggaagaaccc gtggtgggta tacccagcaa 2640
gcatcctgcc cgccctgctg gtgaccatcc tgatcttcat ggaccagcag atcactgccg 2700
tcattgtcaa ccggaaggag aacaaactga agaaggctgc cggctaccat ctggacctgt 2760
tctgggtggg catcctcatg gctttgtgct cctttatggg gctcccctgg tacgtggctg 2820
ccacggtcat ctccatcgcc cacatcgaca gcctcaagat ggagacagag accagtgccc 2880
ctggggagca gccccagttt ctgggagtca gggaacagag agtaaccggc atcatcgtct 2940
tcatcctgac gggaatctct gtcttcctgg ctcccatcct aaagtgtatc cccctgccgg 3000
tgctgtacgg agtcttcctc tacatgggcg tggcctccct gaatggcatc cagttctggg 3060
aacgctgcaa gctcttcctg atgccagcca agcaccagcc ggaccatgcc ttcctgcggc 3120
acgtgccgct gcgccggatc cacctcttca ccctggtgca gatcctctgc ctggcggtgc 3180
tctggatcct caaatccacg gtggctgcca tcatcttccc ggtcatgatc ctgggcctca 3240
tcatcgttcg aaggcttctg gatttcatct tttcccagca cgacctggcc tggattgaca 3300
acatcctccc agagaaggaa aaaaaggaga cagacaagaa gaggaagaga aaaaaagggg 3360
cccacgagga ctgtgatgag gaggaaaaag atcttccagt tggagttact cactctgatt 3420
cttccttcag tgacacagaa cttgaccgaa gctactcacg gaacccagtg ttcatggtgc 3480
cacaggtgaa gatagagatg gagtcagact atgacttcac agacatggat aaataccgaa 3540
63/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
gagaaactga cagtgagacc accctctag 3569
<210> 44
<211> 3920
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1963058CB1
<400> 44
cggacgcggc ggacgtgggt gagggcgcgg ccgtaagaga gcgggacgcg gggtgcccgg 60
cgcgtggtgg gggtccccgg cgcctgcccc cacggcaccc aagaaggcct ggccagggta 120
ccctccgcgg agcccggggg tggggggcgc ggggccggcg ccgcgatggg cccgggaccc 180
ccagcggccg gagcggcgcc gtccccgcgg ccgctgtccc tggtggcgcg gctgagctac 240
gccgtgggcc acttcctcaa cgacctgtgc gcgtccatgt ggttcaccta cctgctgctc 300
tacctgcact cggtgcgcgc ctacagctcc cgcggcgcgg ggctgctgct gctgctgggc 360
caggtggccg acgggctgtg cacaccgctc gtgggctacg aggccgaccg cgccgccagc 420
tgctgcgccc gctacggccc gcgcaaggcc tggcacctgg tcggcaccgt ctgcgtcctg 480
ctgtccttcc ccttcatctt cagcccctgc ctgggctgtg gggcggccac gcccgagtgg 540
gctgccctcc tctactacgg cccgttcatc gtgatcttcc agtttggctg ggcctccaca 600
cagatctccc acctcagcct catcccggag ctcgtcacca acgaccatga gaaggtggag 660
ctcacggcac tcaggtatgc gttcaccgtg gtggccaaca tcaccgtcta cggcgccgcc 720
tggctcctgc tgcacctgca gggctcgtcg cgggtggagc ccacccaaga catcagcatc 780
agcgaccagc tggggggcca ggacgtgccc gtgttccgga acctgtccct gctggtggtg 840
ggtgteggcg ccgtgttctc actgctattc cacctgggca cccgggagag gcgccggccg 900
catgcggagg agccaggcga gcacaccccc ctgttggccc ctgccacggc ccagcccctgl960
ctgctctgga agcactggct ccgggagccg gctttctacc aggtgggcat actgtacatg 1020
accaccaggc tcatcgtgaa cctgtcccag acctacatgg ccatgtacct cacctactcg 1080
ctccacctgc ccaagaagtt catcgcgacc attcccctgg tgatgtacct cagcggcttc 1140
ttgtcctcct tcctcatgaa gcccatcaac aagtgcattg ggaggaacat gacctacttc 1200
tcaggcctcc tggtgatcct ggcctttgcc gcctgggtgg cgctggcgga gggactgggt 1260
gtggccgtgt acgcagcggc tgtgctgctg ggtgctggct gtgccaccat cctcgtcacc 1320
tcgctggcca tgacggccga cctcatcggt ccccacacga acagcggagc gttcgtgtac 1380
ggctccatga gcttcttgga taaggtggcc aatgggctgg cagtcatggc catccagagc 1440
ctgcaccctt gcccctcaga gctctgctgc agggcctgcg tgagctttta ccactgggcg 1500
atggtggctg tgacgggcgg cgtgggcgtg gccgctgccc tgtgtctctg tagcctcctg 1560
ctgtggccga cccgcctgcg acgctgggac cgtgatgccc ggccctgact cctgacagcc 1620
tcctgcacct gtgcaaggga actgtgggga cgcacgagga tgccccccag ggccttgggg 1680
aaaagccccc actgcccctc actcttctct ggacccccac cctccatcct cacccagctc 1740
ccgggggtgg ggtcgggtga gggcagcagg gatgcccgcc agggacttgc aaggaccccc 1800
tgggttttga gggtgtccca ttctcaactc taatccatcc cagccctctg gaggatttgg 1860
ggtgcccctc tcggcaggga acaggaagta ggaatcccag aagggtctgg gggaacccta 1920
accctgagct cagtccagtt cacccctcac ctccagcctg ggggtctcca gacactgcca 1980
gggccccctc aggacggctg gagcctggag gagacagcca cggggtggtg ggctgggcct 2040
ggaccccacc gtggtgggca gcagggctgc ccggcaggct tggtggactc tgctggcagc 2100
aaataaagag atgacggcag cctggctcct gtctgcctgc gggggggctc tgggcagggg 2160
tagcctgggc atctcagccc tgccctggtt gtgggcggcc agcgagccca gtgtctgcct 2220
ctgtcccgag cctctggtcc cctgggacta ggttagtgcc ccctcatctg ggtgcagaga 2280
cagtgggtgc atcctggtag catgccttta tcggggagtg ggtgtgaggg aaggcgggga 2340
ccgctggcag gtggaggggc agtatggttc caggacccac tcccggtagt tctgggtggt 2400
gccgggcggg cgctggggtg ccgacaggga gggcacgtag tctgatgccc tccacagtgg 2460
ctccaccccg taccggttcc tgttgagcac tgtaggtggg actcgggtca ccatgtgccc 2520
ccacctcctc gccgttggcc agcaaggggc tcctggatcg ccccgggcag tttcaccctg 2580
gcctaggtgg ccttgtcccc ctggcctccc aaggacccac cctgcaccta gcctcaccgt 2640
64/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
attccttgcc ccggattggc ctgtctttcc acagcgcgct cccccaccgg gtgctggggg 2700
cctggtactg ggcagggacg atggggtcat gccaggcggt ctcccgcagg tgctgggtgt 2760
aggctgcggt ggggcggggg ctggcggtca ttcctgtccc cctctggcag gcccgctgcc 2820
cagggcgggg gggggggcac tcacccgatg gcatgctgca ctcacggtgt tggtagcagc 2880
tgtgccagcg gttgtaggcc tcgcggaagg ggctggccgg ggcccgtggc aggttgtacc 2940
aggcttccca ggcgtccgag ttggtcaggc ctgtgtacca cagctggccg gctgcgtccc 3000
gtcccatggg cgtgtacttc cagcgtgtgg cctgcctgat ggctggtggc cagcgggaac 3060
cctccaggga caggtagtca tcgctgaggg tgggcagggg gcagagactg agcccatgtc 3120
tacagcgagt gctttgaccc ctttgcgatg tctgccaggg tggatgatgt agaggcctgg 3180
cccacggcgt ggggtctccc tccctcgcca cttggagtct gtccttcagc cctgtacccc 3240
tcaccccaga gtgggtgctt gaggagagag gctgactccc cctctcccca cacatcgcac 3300
cccaagcacc caagtcagca ctaaaccttt ctgttctcag cttttcttgc ctggagaaga 3360
gggaggggag aggacaaggg ccctggctac tcctggattc ctacagtcct tgtccagcct 3420
ccaagaccca caagtccctt cctctgggaa gcccccctgg cctggaggtg caccaggaag 3480
aagtggtctg gggctggcac taagccatgg cccagggaag actgggggac ccactaggcc 3540
aggtgtgtgg ctcacgcttg taaacccagc actttgggag gctgaggcag gtggatcact 3600
tgaggtcagg agttcgagac cagcctggcc agcatggtaa aaccccatct ctactaaaaa 3660
tacgaaaatt aagccaggca tggtgtgggg gcggggggca cctgtaatcc cagctactca 3720
ggaggctgag gcaggagaat cgcttgaacc caggaagtgg agtttgcagt aagctgagat 3780
cgtgcccttt gactccagcc ctgggaaaag agtgagactc cgtcctcaaa aaccaaaggg 3840
ccaggagact caaagaatgt cttatgcttt gaaccttgct ccttggaata atgtcccagg 3900
gaagtcatcc cagaaaacaa 3920
<210> 45
<211> 1361
<212> ANA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Tncyte ID No: 2395967CB1
<400> 45
ctggaagcat gtcggagttt tggttaattt ctgcccctgg cgataaggaa aatttgcaag 60
ctctggagag gatgaatact gtaacctcca agtccaacct gtcttataat accaaattcg 120
ctattcctga cttcaaggtg gggaccttgg attccctggt tggcctctct gatgagttgg 180
ggaaactcga cacctttgct gaaagcctca taaggagaat ggctcagagc gtggtggaag 240
tcatggagga ctcaaagggg aaggtccagg agcacctcct ggcaaacgga gttgacttaa 300
catcctttgt gacccacttt gaatgggaca tggccaaata tcctgtcaag cagccgctcg 360
tgagtgtggt ggacacaata gccaagcaac tggcgcagat cgagatggac ctgaagtccc 420
gaacggccgc ctacaacact ctgaagacaa acctggagaa cctggaaaag aaatccatgg 480
ggaacctctt cacccggaca ctgagtgata ttgtgagcaa agaggacttc gtgctggatt 540
ctgaatatct cgtcacactt ctggtcatcg tccccaaacc aaactactca caatggcaaa 600
aaacctacga atctctctca gacatggtag tccctcgatc aaccaaactc attactgagg 660
acaaggaagg gggccttttc actgtgactc tgtttcgaaa agtgattgaa gatttcaaaa 720
ccaaggccaa agaaaacaag ttcactgttc gtgaatttta ctatgatgag aaggaaattg 780
aaagggaaag ggaggagatg gccagattgc tgtctgataa gaagcaacag tatggccccc 840
tgctgcgctg gctcaaggtg aacttcagtg aagccttcat tgcctggatc cacatcaagg 900
ccctgagagt gtttgtggag tccgtgctca ggtatggact accagtgaac ttccaggcag 960
tgctcctgca gccgcataag aagtcatcca ccaagcgttt aagagaggtt ctaaactctg 1020
tcttccgaca tctggatgaa gtagccgcta caagtatact ggatgcatct gtggagatcc 1080
cgggactgca actcaataac caagactatt ttccttatgt ctacttccat attgacctta 1140
gtcttcttga ctagaaaggc cagctggcac ctctgtctca tgttcgtgca gattattaca 1200
gacacctctt tcctttagcc agagaatggt tcaaatgtct tacagaacta agatcttttt 1260
cagagaaatt gctcacaaaa.gttagtgaca gttgtattta tttttttaag ttacaataaa 1320
atgctctcaa gtcctttgaa tgttccaaca aattcaaaaa a 1361
65/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
<210> 46
<211> 1867
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte~.ID No: 3586648CB1
<400> 46
cagaattagc cggtatagga atgaacgagc atgaagattt gaaattgctc cgattggaag 60
gaagcccagg ttaggtttgg gcacctccaa acgcacccgt tttaaagcca cctggactga 120
ggcgtcgagc tttcagctcc accaaacgct cacctggcct ggcagcgagc ggcggaagag 180
cccgggagcc cctcacagag cgcaccgagc cgggcggaga gctgagccgc aggcacccgc 240
gtctccagga tgataggcga cattgcaaca aatctctaca cccagcagct cagggggctc 300
caagcagagc agcaagttcg aggatccggg cgtggagccg agtgaggccg cagcccagcg 360
ggcctcgggc gaaaaatctt ggaaaatgta taccagtcat gaagatattg ggtatgattt 420
tgaagatggc cccaaagaca aaaagacact gaagccccac ccaaacattg atggcggatg 480
ggcttggatg atggtgctct cctctttctt tgtgcacatc ctcatcatgg gctcccagat 540
ggccctgggt gtcctcaacg tggaatggct ggaagaattc caccagagcc gcggcctgac 600
cgcctgggtc agctccctca gcatgggcat caccttgata gtgggccctt tcatcggctt 660
gttcattaac acctgtgggt gccgccagac tgcgatcatt ggagggctcg tcaactccct 720
gggctgggtg ttgagtgcct atgctgcaaa cgtgcattat ctcttcatta cttttggagt 780
cgcagctggc ctgggcagcg ggatggccta cctgccagcg gtggtcatgg tgggcaggta 840
tttccagaag agacgcgccc tcgcccaggg cctcagcacc acggggaccg gattcggtac 900
gttcctaatg actgtgctgc tgaagtacct gtgcgcagag tacggctgga ggaatgccat 960
gttgatccaa ggtgccgttt ccctaaacct gtgtgtttgt ggggcgctca tgaggcccct 1020
ctctcctggt aaaaacccaa acgacccagg agagaaagat gtgcgtggcc tgccagcgca 1080
ctccacagaa tctgtgaagt caactggaca gcagggaaga acagaagaga aggatggtgg 1140
gctcgggaac gaggagaccc tctgcgacct gcaagcccag gagtgccccg atcaggccgg 1200
gcacaggaag aacatgtgtg ccctccggat tctgaagact gtcagctggc tcaccatgag 1260
agtcaggaag ggcttcgagg actggtattc gggctacttt gggacagcct ctctatttac 1320
aaatcgaatg tttgtagcct ttattttctg ggctttgttt gcatacagca gctttgtcat 1380
ccccttcatt cacctcccag aaatcgtcaa tttgtataac ttatcggagc aaaacgacgt 1440
tttccctctg acgtcaatta tagcaatagt tcacatcttt ggaaaagtga tcctgggcgt 1500
catagccgac ttgccttgca ttagtgtttg gaatgtcttc ctgttggcca acttcaccct 1560
tgtcctcagt atttttattc tgccgttgat gcacacgtac gctggcctgg cggtcatctg 1620
tgcgctgata gggttttcca gtggttattt ctccctaatg cccgtagtga ctgaagactt 1680
ggttggcatt gaacacctgg ccaatgccta cggcatcatc atctgtgcta atggcatctc 1740
tgcattgctg ggaccacctt ttgcaggtaa actctctgag gttttaagag ctcagagtgc 1800
atgtacatat ggtgcgttat gttataaagt cccagattaa gaaacaaaaa aaaaaaaaaa 1860
agatcgg 1867
<210> 47
<211> 2211
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473396CB1
<400> 47
atgcagaata ttaccaaaga atttggaaca ttcaaggcaa atgacaacat caatttacaa 60
gtaaaggcag gagagattca tgcgttgctt ggagaaaacg gtgctggcaa atctacattg 120
atgaacgtgc tttccggatt attagagccg acatcaggga aaattttgat gcgtgggaaa 180
66/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
gaagtacaga tcacaagccc gacaaaagcc aatcaattag ggattgggat ggtccatcag 240
cactttatgc ttgttgatgc ctttactgta acagaaaaca tcgtgttggg aagcgaacct 300
agtcgtgcag ggatgcttga ccataaaaaa gcgcgaaaag agatccaaaa agtttctgaa 360
caatatggat tatcagtcaa cccggatgct tatgttcgtg atatttcagt tgggatggaa 420
caacgggtag aaattttaaa aacactttac cgaggagcag atgtactgat ttttgatgag 480
ccgacagctg tattgacccc tcaggaaatt gatgaattaa tcgtgatcat gaaggaatta 540
gtcaaagaag gcaagtcaat cattttgatt acgcataagt tagatgaaat caaagcagta 600
gctgaccgtt gtacagttat ccgccgtgga aaaggaatcg gtacagtcaa cgttaaagac 660
gttacctcac agcaattagc tgatatgatg gtcggaagag cggtttcatt caaaacgatg 720
aaaaaagaag cgaagcctca agaagtcgtt ttgtctattg aaaatctagt ggtaaaagaa 780
aatcgtggat tagaagccgt gaaaaacctg aacttagagg ttcgtgctgg cgaagtactt 840
ggtatcgctg gaatcgatgg aaacgggcag tcggagttga tccaagcttt gactggtttg 900
cgaaaggcag aaagcggaca tatcaagcta aaaggggaag acatcaccaa taaaaaacct 960
cgaaagatca ctgaacatgg tgtaggacat gtgccagaag accgtcataa atacgggttg 1020
gtcctagata tgacattgtc tgaaaacatt gccctgcaaa ~cgtatcatca aaaaccttac 1080
agtaaaaacg gtatgctgaa ttattcagtg ataaatgaac atgccagaga attgatcgaa 1140
gaatatgatg ttcgaacaac gaatgaactt gttcctgcaa aagctttatc aggcggaaat 1200
cagcaaaaag caatcatcgc tcggatagtc gaccgagatc ctgatctgtt gatcgttgca 1260
aatccaactc gtgggctgga tgtaggagaa tttgtagcag tcacaggtgt gtctggttct 1320
ggaaagagta cattggtcaa tagtatctta aagaaatcgt tagcgcaaaa attaaataag 1380
aattctgcta agccaggtaa attcaagaca atttccggct acgaaagtat cgaaaagatc 1440
atcgatatcg atcaaagccc aatcggccgg acgccgagaa gtaatccagc gacttataca 1500
agtgtatttg atgatatccg tgggttattt gctcaaacga acgaggcaaa aatgcggggt 1560
tataagaaag ggcgttttag tttcaacgta aaaggcggtc gttgtgaagc ttgtcgcggg 1620
gatggaatta ttaagatcga aatgcacttt ttgcctgatg tctatgttcc ttgtgaagta 1680
tgtcatggca aacgatataa ctctgaaaca ttagaagtgc attacaaagg aaaaagcatt 1740
gctgatattt tggaaatgac agtagaagat gctgtagaat tcttcaagca cattccaaag 1800
attcatcgca aactgcaaac gattgttgat gttggcttag gttatgtgac tatggggcaa 1860
ccagcaacga cattgtccgg tggtgaggca caacggatga aacttgccag tgaattgcac 1920
aaaatctcta atggaaagaa tttctatata ctagatgaac caacgacagg acttcatagc 1980
gatgacatcg cccgcttgtt gcatgtatta caaagattag tagatgctgg taacacagtt 2040
ttagtgattg aacacaatct agatgtaatc aaaacagcag attatatcat tgatttagga 2100
ccagaaggtg gagaaggtgg aggaacgatc cttacgactg gaacaccaga agaaatcatt 2160
aacgtaaaag aaagttatac aggtcactat ttgaaaaaaa taatggtata a 2211
<210> 48
<211> 1446
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7476283CB1
<400> 48
tggctgggag aattgagcta gtgcagcaca cgtaaaaaag cgattccgat gggtcctttg 60
aaagcttttc tcttctcccc ttttcttctg cggagtcaaa gtagaggggt gaggttggtc 120
ttcttgttac tgaccctgca tttgggaaac tgtgttgata aggcagatga tgaagatgat 180
gaggatttaa aggtgaacaa aacctgggtc ttggccccaa aaattcatga aggagatatc 240
acacaaattc tgaattcatt gcttcaaggc tatgacaata aacttcgtcc agatatagga 300
gtgaggccca cagtaattga aactgatgtt tatgtaaaca gcattggacc agttgatcca 360
attaatatgg aatatacaat agatataatt tttgcccaaa cctggtttga cagtcgttta 420
aaattcaata gtaccatgaa agtgcttatg cttaacagta atatggttgg aaaaatttgg 480
attcctgaca ctttcttcag aaactcaaga aaatctgatg ctcactggat aacaactcct 540
aatcgtctgc ttcgaatttg gaatgatgga cgagttctgt atactctaag attgacaatt 600
aatgcagaat gttatcttca gcttcataac tttcccatgg atgaacattc ctgtccactg 660
67/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
gaattttcaa gcgatggata ccctaaaaat gaaattgagt ataagtggaa aaagccctcc 720
gtagaagtgg ctgatcctaa atactggaga ttatatcagt ttgcatttgt agggttacgg 780
aactcaactg aaatcactca cacgatctct ggggattatg ttatcatgac aatttttttt 840
gacctgagca gaagaatggg atatttcact attcagacct acattccatg cattctgaca 900
gttgttcttt cttgggtgtc tttttggatc aataaagatg cagtgcctgc aagaacatcg 960
ttgggtatca ctacagttct gactatgaca accctgagta caattgccag gaagtcttta 1020
cctaaggttt cttatgtgac tgcgatggat ctctttgttt ctgtttgttt catttttgtt 1080
tttgcagcct tgatggaata tggaaccttg cattatttta ccagcaacca aaaaggaaag 1140
actgctacta aagacagaaa gctaaaaaat aaagcctcga tgactcctgg tctccatcct 1200
ggatccactc tgattccaat gaataatatt tctgtgccgc aagaagatga ttatgggtat 1260
cagtgtttgg agggcaaaga ttgtgccagc ttcttctgtt gctttgaaga ctgcagaaca 1320
ggatcttgga gggaaggaag gatacacata cgcattgcca aaattgactc ttattctaga 1380
atatttttcc caaccgcttt tgccctgttc aacttggttt attgggttgg ctatctttac 1440
ttataa 1446
<210> 49
<211> 1332
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte 2D No: 7477105C81
<400> 49
ttcggctcga gggaccccag gccgggccgg gccgagaggc tgccatgggc tccgtgggga 60
gccagcgcct tgaggagccc agcgtggcag gcacaccaga cccgggcgta gtgatgagct 120
tcaccttcga cagtcaccag ctggaggagg cggcggaggc ggctcagggc cagggcctta 180
gggccagggg cgtcccagct ttcacggata ctacattgga cgagccagtg cccgatgacc 240
gttatcacgc catctacttt gcgatgctgc tggctggcgt gggcttcctg ctgccataca 300
acagcttcat cacggacgtg gactacctgc atcacaagta cccagggacc tccatcgtgt 360
ttgacatgag cctcacctac atcttggtgg cactggcagc tgtcctcctg aacaacgtcc 420
tggtggagag actgaccctg cacaccagga tcaccgcagg ctacctctta gccttgggcc 480
ctctcctttt tatcagcatc tgcgacgtgt ggctgcagct cttctctcgg gaccaggcct 540
acgccatcaa cctggccgct gtgggcaccg tggccttcgg ctgcacagtg cagcaatcca 600
gcttctacgg gcaccgcctg gcccagcctc caccagggac ccctcctcat gaactctgga 660
gccctgagag gagaggggca gccccccacc ttgtcaccct cagggcttcc ccttctgtcc 720
tcattcttag agactgcttc tcccaaacat aacgcgttag ccatgaagga gtcggagccc 780
tgggtccgaa tggacccgcc tgcggtctgc atcagcctct gggaaaccac agcagtgatg 840
ccagctgggc acgtcaggac ctccccacac acccacacga tgccacaggt cagggggctg 900
tgcctgacta gggagccctc ccattgcctt cctggcccgg gatagaagag gggaggtaag 960
tctgggggct acgaagccgg gcccccacac cctggctgaa gtcagcttga cctaggtctt 1020
gaccctcatc cagcaaggga ctcgacagac ccaagggtcc ctggaacgta gggaggggct 1080
gggggtcact ccagcccggg cctcccagaa caccaggccc gtgtgggtgg caccctgagg 1140
tcaggggatc ctaagggtgt ccttccagag acggtgtttc cagggggagg accgcccccg 1200
cttccagatc cccggccccg gctgtgactg ccctgtttca cccctgctgt gtcccatccc 1260
ccgtctgtcc actaactgta ccgcaccggc cattaaaaga tgaaggcaga ccgctggaaa 1320
aaaaaaaaaa as 1332
<210> 50
<211> 2298
<212> DNA
<213> Homo sapiens
<220>
<221> misc feature
68/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
<223> Incyte ID No: 7482079CB1
<400> 50
atgctcaaac agagtgagag gagacggtcc tggagctaca ggccctggaa cacgacggag 60
aatgagggca gccaacaccg caggagcatt tgctccctgg gtgcccgttc cggctcccag 120
gccagcatcc acggctggac agagggcaac tataactact acatcgagga agacgaagac 180
ggcgaggagg aggaccagtg gaaggacgac ctggcagaag aggaccagca ggcaggggag 240
gtcaccaccg ccaagcccga gggccccagc gaccctccgg ccctgctgtc cacgctgaat 300
gtgaacgtgg gtggccacag ctaccagctg gactactgcg agctggccgg cttccccaag 360
acgcgcctag gtcgcctggc cacctccacc agccgcagcc gccagctaag cctgtgcgac 420
gactacgagg agcagacaga cgaatacttc ttcgaccgcg acccggccgt cttccagctg 480
gtctacaatt tctacctgtc cggggtgctg ctggtgctcg acgggctgtg tccgcgccgc 540
ttcctggagg agctgggcta ctggggcgtg cggctcaagt acacgccacg ctgctgccgc 600
atctgcttcg aggagcggcg cgacgagctg agcgaacggc tcaagatcca gcacgagctg 660
cgcgcgcagg cgcaggtcga ggaggcggag gaactcttcc gcgacatgcg cttctacggc 720
ccgcagcggc gccgcctctg gaacctcatg gagaagccat tctcctcggt ggccgccaag 780
gccatcgggg tggcctccag caccttcgtg ctcgtctccg tggtggcgct ggcgctcaac 840
accgtggagg agatgcagca gcactcgggg cagggcgagg gcggcccaga cctgcggccc 900
atcctggagc acgtggagat gctgtgcatg ggcttcttca cgctcgagta cctgctgcgc 960
ctagcctcca cgcccgacct gaggcgcttc gcgcgcagcg ccctcaacct ggtggacctg 1020
gtggccatcc tgccgctcta ccttcagctg ctgctcgagt gcttcacggg cgagggccac 1080
caacgcggcc agacggtggg cagcgtgggt aaggtgggtc aggtgttgcg cgtcatgcgc 1140
ctcatgcgca tcttccgcat cctcaagctg gcgcgccact ccaccggact gcgtgccttc 1200
ggcttcacgc tgcgccagtg ctaccagcag gtgggctgcc tgctgctctt catcgccatg 1260
ggcatcttca ctttctctgc ggctgtctac tctgtggagc acgatgtgcc cagcaccaac 1320
ttcactacca tcccccactc ctggtggtgg gccgcggtga gtacctttgc cctgggcttt 1380
cccatcctct tccccagccc agtgagctgc tcctccctcc cctggttatc agccaccagg 1440
ctttggcttc tgatcctcgt cttccccccc acccccaatc gccgcataca gctaacaaaa 1500
cggcgatgga tgtcaaaagt ggtggaaaga gaactcagca gatcagtaaa ctccagcagc 1560,
cacatgtcga tggctgtggc aaagaacaag agagagaatg caagccccat catgcaaaca 1620
cttcataagt ttcttttcat ggcatttgct cagcccattg gccagagtaa gtcacatggc 1680
caagctgcaa gtcaaagggc agggcaggtg agcatctcca ccgtgggcta cggagacatg 1740
tacccagaga cccacctggg caggtttttt gccttcctct gcattgcttt tgggatcatt 1800
ctcaacggga tgcccatttc catcctctac aacaagtttt ctgattacta cagcaagctg 1860
aaggcttatg agtataccac catacgcagg gagaggggag aggtgaactt catgcagaga 1920
gccagaaaga agatagctga gtgtttgctt ggaagcaacc cacagctcac cccaagacaa 1980
gagaattagt attttatagg acatgtggct ggtagattcc atgaacttca aggcttcatt 2040
gctctttttt taatcattat gattggcagc aaaaggaaat gtgaagcaga catacacaaa 2100
ggccatttcg ttcacaaagt actgcctcta gaaatactca ttttggccca aactcagaat 2160
gtctcatagt tgctctgtgt tgtgtgaaac atctgacctt ctcaatgacg ttgatattga 2220
aaacctgagg ggagcaacag cttagatttt tcttgtagct tctcgtggca tctagctcaa 2280
taaatatttt tggacttg 2298
<210> 51
<211> 2250
<212> DNA
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 55145506CB1
<400> 51
agaaacagat ctctcggatc aataagcatg aatgacgaag actacagcac catctatgac 60
acaatccaaa atgagaggac gtatgaggtt ccagaccagc cagaagaaaa tgaaagtccc 120
cattatgatg atgtccatga gtacttaagg ccagaaaatg atttatatgc cactcagctg 180
69/71
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
aatacccatg agtatgattt tgtgtcagtc tataccatta agggtgaaga gaccagcttg 240
gcctctgtcc agtcagaaga cagaggctac ctcctgcctg atgagatata ctctgaactc 300
caggaggctc atccaggtga gccccaggag gacaggggca tctcaatgga agggttatat 360
tcatcagccc aggaccagca actctgcgca gcagaactcc aggagaatgg gagtgtgatg 420
aaggaagatc tgccttctcc ttcaagcttc accattcagc acagtaaggc cttctctacc 480
accaagtatt cctgctattc tgatgctgaa ggtttggaag aaaaggaggg agctcacatg 540
aaccctgaga tttacctctt tgtgaaggct ggaatcgatg gagaaagcat cggcaactgt 600
cctttctctc agcgcctctt catgatcctc tggctgaaag gagtcgtgtt caatgtcacc 660
actgtggatc tgaaaagaaa gccagctgac ctgcacaacc tagcccccgg cacgcacccg 720
cccttcctga ccttcaacgg ggacgtgaag acagacgtca ataagatcga ggagttcctg 780
gaggagacct tgacccctga aaagtacccc aaactggctg caaaacaccg ggaatccaac 840
acagcgggca tcgacatctt ttccaagttt tctgcctaca tcaaaaatac caagcagcag 900
aacaatgctg ctcttgaaag aggcctaacc aaggctctaa agaaattgga tgactacctg 960
aacacccctc taccagagga gattgacgcc aacacttgtg gggaagacaa ggggtcccgg 1020
cgcaagttcc tggatgggga tgagctgacc ctggctgact gcaatctgtt gcccaagctc 1080
catgtggtca agacccacct tctcacttcc tccagcaact tcctaaggaa caagtaccac 1140
tgaaagggat gatataattc cagctcagtc acactgtgtc agagtgatac aatgcaaaga 1200
tcaggagacc cgagttccgg tcctgtattt gctgccaact agcagcatga gctgaggcac 1260
atcatttaat ctttttggaa ttcatttttc tcatgcctag aagaacagaa gtggattgta 1320
ttccttcttg ccttcttttc ctttcttctt tccctccttc tttccttttc tcttgctcaa 1380
acatgtattc actaccactc aaaaaccatt tgttgaacaa agcaaacaaa tgaatctccc 1440
aagccttggg cttcatcctg tgatttcctc aattcccacc tgccttaaat tactcagtga 1500
agccctgtcc ttggagaaaa ttcagtgggt ggttaaccca gagaagctgg agatcaaaaa 1560
gaagatggcc aatgaaagaa caaaggccag cccttggccc ctatctcttt ggatttctgc 1620
tgatccagct tatcagatcc cagaaacctg gcaaacctct aaagttcaca aagagcgaag 1680
gggaagccaa gtcaggcctc cagtttggct tcggatgcca aaacttaatc tgggctgtgg 1740
gagctaactg ttttcatatg aaagagcaaa ttcagaacat gagcatggaa gtccctgcga 1800
acgtcagatc tccgtgtgca tccttacccc cttgctgctt tcatgctcac tctcctcttg 1860
cgtggctcgc tttcaggttt atctccatcc ctggaagcag agttgctctg gcccaggctc 1920
tccatgagag tttggcttga acattcattg tctggccccc tcctagttct catctcccaa 1980
agtcaagcca atgtgtgaag aaatgaccag ctcagcagcc aaggcccagg gtgcacaggt 2040
cttcgttggg agaggcatct gcaggccttt ccttgcccac tgggatcctt gcctagcata 2100
gtgacgatgt tcagccctgg agacaaacaa gaaggggaac accaacatca atagaagtat 2160
atatttacaa attgcatttc tgctgtattg aaactaacat tctgcccttt aaaatcctga 2220
aaataaaatt tcagtatgaa atgaaaaaaa 2250
<210> 52
<211> 3430
<212> DNA
<213> Homo sapiens
<220>
<221> misc feature
<223> Incite ID No: 5950519CB1
<400> 52
gagctgaccc tgcggggtcc cgggggggga gggggagccg cgaagccccc actgaggccg 60
ccgctgccgg gcctcccctc ccccccgggc gggcgccatg cgggggagcc cgggcgacgc 120
ggagcggcgg cagcgctggg gtcgcctgtt cgaggagctg gacagtaaca aggatggccg 180
cgtggacgtg cacgagttgc gccaggggct ggccaggctg ggcgggggca acccagaccc 240
cggcgcccaa cagggtatct cctctgaggg tgatgctgac ccagatggcg ggctcgacct 300
ggaggaattt tcccgctatc tgcaggagcg ggaacagcgt ctgctgctca tgtttcacag 360
tcttgaccgg aaccaggatg gtcacattga tgtctctgag atccaacaga gtttccgagc 420
tctgggcatt tccatctcgc tggagcaggc tgagaaaatt ttgcacagca tggaccgaga 480
cggcacaatg accattgact ggcaagaatg gcgcgaccac ttcctgttgc attcgctgga 540
aaatgtggag gacgtgctgt atttctggaa gcattccacg gtcctggaca ttggcgagtg 600
70171
CA 02422497 2003-03-14
WO 02/22684 PCT/USO1/28938
cctgacagtg ccggacgagt tctcaaagca agagaagctg acgggcatgt ggtggaaaca 660
gctggtggcc ggcgcagtgg caggtgccgt gtcacggaca ggcacggccc ctctggaccg 720
cctcaaggtc ttcatgcagg tccatgcctc aaagaccaac cggctgaaca tccttggggg 780
gcttcgaagc atggtccttg agggaggcat ccgctccctg tggcgcggca atggtattaa 840
tgtactcaag attgcccccg agtcagctat caagttcatg gcctatgaac agatcaagag 900
ggccatcctg gggcagcagg agacactgca tgtgcaggag cgcttcgtgg ctggctccct 960
ggctggtgcc acagcccaaa ccatcattta ccctatggag gtgctgaaga cgcggctgac 1020
cttgcgccgg acgggccagt ataaggggct gctggactgc gccaggcgta tcctggagag 1080
ggaggggccc cgtgccttct accgcggcta cctccccaac gtgctgggca tcatccccta 1140
tgcgggcatc gacctggccg tctacgagac tctgaagaac tggtggcttc agcagtacag 1200
ccacgactcg gcagacccag gcatcctcgt gctcctggcc tgcggtacca tatccagcac 1260
ctgcggccag atagccagtt acccgctggc cctggtccgg acccgcatgc aggcacaagc 1320
ctccatcgag ggtggccccc agctgtccat gctgggtctg ctacgtcaca tcctgtccca 1380
ggagggcatg cggggcctct accgggggat cgcccccaac ttcatgaagg ttattccagc 1440
tgtgagcatc tcctatgtgg tctacgagaa catgaagcag gccttggggg tcacgtccag 1500
gtgagggacc cggagcccgt ccccccaatc cctcaccccc cacacctcag ccactggaga 1560
ctgatgatcc aaccacagga tccctactct ttggccacga gatcccagta cccagatcct 1620
ggatcctaga ctcctatgcc ccaaccattg ggtcatggga tcccagcacc cagatcctgg 1680
atcctagact cctatgcccc aaccactggg tcatgcgatc cccacccttc agccactaga 1740
tcccagatcc ccctgtaacc ataactgtgg atcccttact tcagcaactc aagtctgcta 1800
ccctaaccac aagattcaag attatccaca ccccagccct taatccccat cccccaaatc 1860
actggatcct gcagccccac atcctaaggt ggatcccacg cttccctgtg ccccctactg 1920
gatcctggac ctctacgtct taaccactgg atcccacaca aatcagtgaa tggatcccaa 1980
caccccaacc acaggagcac ggattccctg tacctcaaca cccagaccct gcctccctca 2040
ggcaccagat ccagtgtcct agtgaaacgc tggatcctag atccccaacc ccagatcccc 2100
atgcctcgag ccctggatct ccaagctcag ctgctggatt ctggatgtca acaaacctca 2160
ccactggatc ctgacaacca caatgcctgg atcctggggc ccccatcact ggatcccaga 2220
tcccctcact ccacccactg gattcctgca ttggtttttg gttttttgtt tttttttaac 2280
ctcgacactg ggtctcagat ccttctgctg actgccagat ccctgcattt caagcactac 2340
gccttccacc cccaggcact ggatcccaga ttcccaagcc ttcacccacc agattctggc 2400
tcctaaaaca agtgcggggg ccccagtggc acagcaagtg gatcctggca actgcagctg 2460
ctggattcca gattctgggt ccccaatccc tctgcccagt ccctcaatgt tgaaacctca 2520
tctcttgaag gcagatcctg atattccaag gcactgaatc ccaagccctg aatccccggt 2580
ttctgatctg aatcttccag gcgccgggtc ccaaatgttc aggccccaag tctagatcct 2640
ggcagcccag tcacagagta tcccacacac actggtgccc agagccggct tctcatgaca 2700
tgaaattgca tggtcgaggg agtctgtggg gaaggaagcc caggtcctgg ctgcaacctg 2760
cacggatgct ggattccccc tcaccccacc tctgcatggc caccccctcc cagccctgtg 2820
gggaaactgt tccctggaac cactccactc cctgcatccc cacacttcac agcatcttcc 2880
atccccctcc caccttctag gcgaatagtc cccagagctg tgttcctcca aggggtccga 2940
ggaatcactc actcctggag gctggcaagg agacagtctg aggccaggga cacatgaagg 3000
gatgtcccca ccccagcact atcagggcct ccccaggctt ccagagttga aagccaggag 3060
aaaatcggca aagaccaccc ttccctaaac ccaagcaccc aatgatgcaa aaaacaaaaa 3120
caaaaaaaaa ccaccaaatc cccaaattca ttccagatct atttttctac cagagagagg 3180
agcaaagtcc tcctcccctg cgcccttaca ttctgcactt catagttgga ttctgagGtt 3240
aggatcatct ggagacccca tggagggact tggaaagggg aactgggatt tggggagggg 3300
ctggaggact tccgcacgct tccacctcct tcgacctcca ctgcgcccca cctccctgcc 3360
tgtgtgtgtt atttcaaagg aaaagaacaa aaggaataaa ttttctaagc tctttaaaaa 3420
aaaaaaaaaa 3430
71/71
