Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TRANSPORTERS AND ION CHANNELS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of
transporters and ion
channels and to the use of these sequences in the diagnosis, prevention, and
treatment of taransport,
neurological, muscular, immunological, and cell proliferative disorders, as
well as disorders of iron
metabolism, and in the assessment of the effects of exogenous compounds on the
expression of
nucleic acid and amino acid sequences of transporters and ion channels.
BACKGROUND OF THE INVENTION
Eukaryotic cells are surrounded and subdivided into functionally distinct
organelles by
hydrophobic lipid bilayer membranes which are highly impermeable to most polar
molecules. Cells and
organelles require transport proteins to import and export essential nutrients
and metal ions including
K+, NH4+, P;, 504, sugars, and vitamins, as well as various metabolic waste
products. Transport
proteins also play roles in antibiotic resistance, toxin secretion, ion
balance, synaptic neurotransmission,
kidney function, intestinal absorption, tumor growth, and other diverse cell
functions (Griffith, J. and C.
Sansom (1998) The Transporter Facts Book, Academic Press, San Diego CA, pp. 3-
29). Transport
can occur by a passive concentration-dependent mechanism, or can be linked to
an energy source
such as ATP hydrolysis or au ion gradient. Pxoteins that function in transport
include carrier proteins,
which bind to a specific solute and undergo a conformational change that
translocates the bound solute
across the membrane, and channel proteins, which form hydrophilic pores that
allow specific solutes to
diffuse through the membrane down an electrochemical solute gradient.
Carrier proteins which transport a single solute from one side of the membrane
to the other
are called uniporters. In contrast, coupled transporters link the transfer of
one solute with
simultaneous or sequential transfer of a second solute, either in the same
direction (symport) or in the
opposite direction (antiport). For example, intestinal and kidney epithelium
contains a variety of
symporter systems driven by the sodium gradient that exists across the plasma
membrane. Sodium
moves into the cell down its electrochemical gradient and brings the solute
into the cell with it. The
3o sodium gradient that provides the driving force for solute uptake is
maintained by the ubiquitous
Na+/K+ ATPase system. Sodium-coupled transporters include the mammalian
glucose transporter
(SGLT1), iodide transporter (NIS), and multivitamin transporter (SMVT). All
three transporters have
twelve putative txansmembrane segments, extracellular glycosylation sites, and
cytoplasmically-
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oriented N- and C-termini. NIS plays a crucial role in the evaluation,
diagnosis, and treatment of
various thyroid pathologies because it is the molecular basis for radioiodide
thyroid-imaging techniques
and for specific targeting of radioisotopes to the thyroid gland (Levy, O. et
al. (1997) Proc. Natl.
Acad. Sci. USA 94:5568-5573). SMVT is expressed in the intestinal mucosa,
kidney, and placenta,
and is implicated in the transport of the water-soluble vitamins, e.g., biotin
and pantothenate (Prasad,
P.D. et al. (1998) J. Biol. Chem. 273:7501-7506).
One of the largest families of transporters is the major facilitator
superfamily (MFS), also
called the uniporter-symporter-antiporter family. MFS transporters are single
polypeptide carriers that
transport small solutes in response to ion gradients. Members of the MFS are
found in all classes of
living organisms, and include transporters for sugars, oligosaccharides,
phosphates, nitrates,
nucleosides, monocarboxylates, and drugs. MFS transporters found in eukaryotes
all have a structure
comprising 12 transmembrane segments (Pao, S.S. et al. (1998) Microbiol.
Molec. Biol. Rev. 62:1-34).
The largest family of MFS transporters is the sugar transporter family, which
includes the seven
glucose transporters (GLUT1-GLUT7) found in humans that are required for the
transport of glucose
and other hexose sugars. These glucose transport proteins have unique tissue
distributions and
physiological functions. GLUT1 provides many cell types with their basal
glucose requirernerlts and
transports glucose across epithelial and endothelial barrier tissues;, GLUT2
facilitates glucose uptake
or efflux from the liver; GLUT3 regulates glucose supply to neurons; GLUT4 is
responsible for insulin-
. regulated glucose disposal; and GLUTS regulates fructose uptake into
skeletal muscle. Defects in
glucose transporters are involved in a recently identified neurological
syndrome causing infantile
seizures and developmental delay, as wall as glycogen storage disease, Fanconi-
Bickel syndrome, and
non-insulin-dependent diabetes mellitus (Mueckler, M. (1994) Eur. J. Biochem.
219:713-725; Longo,
N. and L.J. Elsas (1998) Adv. Pediatr. 45:293-313).
Monocarboxylate anion transporters are proton-coupled symporters with a broad
substrate
specificity that includes L-lactate, pyruvate, and the ketone bodies acetate,
acetoacetate, and
beta-hydroxybutyrate. At least seven isoforms have been identified to date.
The isoforms are
predicted to have twelve transme~Cnbrane (TM) helical domains with a Iarge
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 monocarboxyla'tes. Other cells possess H+-linked
monocarboxylate
transporters with differing substrate and inhibitor selectivities. In
particular, cardiac muscle and tumor
cells have transporters that differ in their K"~ values for certain
substrates, including stereoselectivity
2
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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
transporters for organic cations and organic anions in organs including the
kidney, intestine and liver.
Organic anion transporters are selective for hydrophobic, charged molecules
with electron-attracting
side groups. Organic cation transporters, such as the ammonium transporter,
mediate the secretion of
a variety of drugs and endogenous metabolites, and contribute to the
maintenance of intercellular pH
(Poole, R.C. and A.P. Halestrap (1993) Am. J. Physiol. 264:C761-C782; Price,
N.T. et al. (1998)
Biochem. J. 329:321-328; and Martinelle, K. and I. Haggstrom (1993) J.
Biotechnol. 30:339-350).
ATP-binding cassette (ABC) transporters are members of a superfamily of
membrane
proteins that transport substances ranging from small molecules such as ions,
sugars, amino acids,
peptides, and phospholipids, to lipopeptides, large proteins, and complex
hydrophobic drugs. ABC
transporters consist of four modules: two nucleotide-binding domains (NBD),
which hydrolyze ATP to
supply the energy required for transport, and two membrane-spanning domains
(MSD), each
containing six putative transmembrane segments. These four modules may be
encoded by a single
gene.as;is the case for the cystic fibrosis transmembrane regulator (CFTR), or
by separate genes.
When.encoded by separate genes, each gene product contains a single NBD and
MSD. These '.'half '
molecules" forth homo- and heterodimers, such as Tap1 and Tap2, the
endoplasmic reticulum-Based
major histocompatibility (MHC) peptide transport system. Several genetic
diseases are attributed to
defects in ABC transporters, such as the following diseases and their
corresponding proteins: cystic
fibrosis (CFTR, an ion channel), adrenoleukodystrophy (adrenoleukodystrophy
protein, ALDP),
Zellweger syndrome (peroxisonlal membrane protein-70, PMP70), and
hyperinsulinemic hypoglycemia
(sulfonylurea receptor, SUR). Overexpression of the multidrug resistance (MDR)
protein, anothex
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.
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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
signi~.cant fraction of fatty
acid transport appears to occur via a high affinity, low capacity protein-
mediated transport process.
Fatty acid transport protein (FATP), an integral membrane protein with four
transmembrane
segments, is expressed in tissues exhibiting high levels of plasma membrane
fatty acid flux, such as
muscle, heart, and adipose. Expression of FATP is upregulated in 3T3-L1 cells
during adipose
conversion, and expression in COS7 fibroblasts elevates uptake of long-chain
fatty acids (Hui, T.Y. et
al. (1998) J. Biol. Chem. 273:27420-27429).
Mitochondrial carrier proteins are transmembrane-spanning proteins which
transport ions and
charged metabolites between the cytosol and the mitochondrial matrix. Examples
include the ADP,
ATP carrier protein; the 2-oxoglutarate/malate carrier; the phosphate carrier
protein; the pyruvate
carrier; the dicarboxylate carrier which transports malate, succinate,
famerete, 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.);:-:;_: :;..
BiochemistryW'.H. Freeman and Company, New York NY, p. 551; PROSITE
PDOC00'189. !,, ,~~ ~ .
Mitochondrial energy transfer proteins signature; Online Mendelian Inheritance
in Man (C)MINI)~.
*275000 Graves Disease).
This class of transporters also includes the mitochondrial uncoupling
proteins, which create
proton leaks across the inner xnitochondrial 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).
Urea trausporters (UT, UrT) play a central role in urea excretion and water
balance by
allowing the accumulation and concentration of urea in the kidney medulla
(Hediger, M.A. et al.
(1996) Kidney Int. 49:1615-1623). Urea is a major solute found in urine and is
the principal means by
which mammals dispose of nitrogen-based waste products. Urea transporter
proteins have been
identified in erythropoietic cells (UT-B) and in the kidney medula (UT-A).
Several isoforms of the
renal urea transporter (UT-A) have been cloned (i.e., UT-A1, UT-A2, UT-A3, and
UT-A4). The
expression of UT-A2 may be upregulated in response to uremia. UT-A3 may be
expressed in the
4
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testis. Urea transporters rnay also be expressed in the brain (I~arakashian,
A. et al. (1999) J. Am.
Soc. Nephrol. 1999 10:230-237; Couriaud, C. et al. (1996) Biochim Biophys
Acta. 1996 1309:197-19).
At least two distinct classes of urea transporters are present in humans:
constitutively-expressed
transporters, and vasopressin-regulated transporters (Olives, B. et al. (1996)
FEBS Lett.
386:156-160).
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).
Iron plays an essential role in oxygen transport and redox reactions,
particularly cell
respiration~:liowever,: iron is also toxic when present in excess. In humans,
unregulated ironabsorption;
.leads to cirrhosis, endocrine. failure, arthritis and cardiomyopathy, as well
as hepatocellular.carcinoW a ~.;;
(GriffithsW:rJ;H. et al. (1999) Mol. Med. Today: 5:431-438). Ferritin is a
ubiquitous iron-binding u'~='~ ";
protein that i's~involved in iron storage and detoxification in microbes,
plants, and animals. Mammalian
ferritin consists of 24 subunits of two types, H (for heart, or heavy) and L
(for light or liver). These
subunits assemble into a spherical structure which can accommodate up to 4,000
iron atoms as
ferrihydrite, FeOOH (Aisen, P. et al. (1999) Curr. Opin. Chem. Biol. 3:200-
206).
The nuclear pore complex (NPC) is a large multiprotein complex spanning the
nuclear
envelope which mediates the transport of proteins and RNA molecules between
the nucleus and the
cytoplasm, thus contributing to the regulation of gene expression. The NPC
allows passive diffusion
of ions, small molecules, and macromolecules under about 60kD, while larger
macromolecules are
transported by facilitated, energy-dependent pathways. Nuclear localization
signals (NLS), consisting
of short stretches of amino acids enriched in basic residues, are found on
proteins that are targeted to
the nucleus, such as the glucocorticoid receptor. The NLS is recognized by the
NLS receptor,
importin, which then interacts with the monomeric GTP-binding protein Ran.
This NLS
protein/receptor/Ran complex navigates the nuclear pore with the help of the
homodimeric protein
nuclear transport factor 2 (NTF2) (Nakielny, S. and Dreyfuss, G. (1997) Curr.
Opin. Cell Biol. 9:420-
429; Gorlich, D. (1997) Curr. Opin. Cell Biol. 9:412-419). Four O-linked
glycoproteins, p62, p58, p54,
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and p45, exist as a stable "p62 complex" that foams a ring localized on both
nucleoplasmic and
cytoplasmic surfaces of the NPC. The p62, p58, and p54 proteins all interact
directly with the
cytosolic trausport factors p97 and NTF2, suggesting that the p62 complex is
an important ligand
binding site near the central gated channel of the NPC (Hu, T. et al. (1996)
J. Cell Biol. 134:589-601).
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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 ton against the ion's concentration gradient. Gated ion channels
allow passive flow of au
ion down the ion's electrochemical gradient under restricted conditions.
Together; these types of ion
channels generate, maintain, and utilize an electrochemical gradient that is
used in 1) electrical impulse
conduction down the axon of a nerve cell, 2) transport of molecules into cells
against concentration
gradients, 3) initiation of muscle contraction, and 4) endocrine cell
secretion.
Ion Transporters
Ion transporters generate and maintain the resting electrical potential of a
cell. Utilizing the
energy derived from ATP hydrolysis, they transport ions against the ion's
concentration gradient.
These transmembrane ATPases are divided into three families. The
phosphorylated (P) class ion
transporters, including Na+-K+ ATPase, Ca2+-ATPase, and H+-ATPase, are
activated by a
phosphorylation event. P-class ion transporters are responsible for
maintaining resting potential
distributions such that cytosolic concentrations of Na+ and Ca2+ are low and
cytosolic concentration of ,
K+ is high. The.vacuolar (V) class of ion transporters includes H+ pumps on
intracellular organelles,
such as lysosomes and Golgi. V-class ion~transporters 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 trausmembrane domains
and
several large cytoplasmic regions that may play a role in ion binding
(Scarborough, G.A. (1999) Curr.
Opin. Cell Biol. 11:517-522). The V-ATPases are composed of two functional
domains: the Vl
domain, a peripheral complex responsible for ATP hydrolysis; and the Vo
domain, an integral complex
responsible for proton trauslocation across the membrane. The F-ATPases are
structurally and
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 trausmembrane 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.
7
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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 licks transfer
of Ca2+ out of the cell
with transport of Na* into the cell (antiport).
Gated Ion Channels
Gated ion channels control ion flow by regulating the opening and closing of
pores. The ability
1o to control ion flux through various gating mechanisms allows ion channels
to mediate such diverse
signaling and homeostatic functions as neuronal and endocrine signaling,
muscle contraction,
fertilization, and regulation of ion and pH balance. Gated ion channels are
categorized according to
the manner of regulating the gating function. Mechanically-gated channels open
their pores in
response to mechanical stress; voltage-gated channels (e.g., Na+, K+, Caz+,
and Cl-channels) open
their pores in response to changes in membrane potential; and ligand-gated
channels (e.g.,
acetylcholine-, serotonin-, and glutamate-gated cation~channels, and GABA- and
glycine-gated
chloride channels) open their poxes 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 modulatedbyassociation with auxiliary channel proteins
andlor 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 Caz+ and Na+ (Suzuki, M. et al.
(1999) J. Biol. Chem.
274:6330-6335).
The pore-forming subunits of the voltage-gated cation channels form a
superfamily of ion
channel proteins. 'The characteristic domain of these channel proteins
comprises six transmembrane
domains (S1-S6), a pore-forming region (P) located between SS and S6, and
intracellular amino and
carboxy termini. In the Na+ and Ca2+ subfamilies, this domain is repeated four
times, while in the K+
channel subfamily, each channel is formed from a tetramer of either identical
or dissimilar subunits.
The P region contains information specifying the ion selectivity for the
channel. In the case of K+
channels, a GYG tripeptide is involved in this selectivity (Ishii, T.M. et al.
(1997) Proc. Natl. Aced.
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Sci. USA 94:11651-11656).
Voltage-gated Na+ and K+ channels are necessary for the function of
electarically excitable
cells, such as nerve and muscle cells. Action potentials, which lead to
neurotransmitter release and
muscle contraction, arise from large, transient changes in the permeability of
the membrane to Na+
and K+ ions. Depolarization of the membrane beyond the threshold level opens
voltage-gated Na+
channels. Sodium ions flow into the cell, further depolarizing the membrane
and opening more
voltage-gated Na+ channels, which propagates the depolarization down the
length of the cell.
Depolarization also opens voltage-gated potassium channels. Consequently,
potassium ions flow
outward, which leads to repolarization of the membrane. Voltage-gated channels
utilize charged
residues in the fourth transmembrane segment (S4) to sense voltage change. The
open state lasts
only about 1 millisecond, at which time the channel spontaneously converts
into an inactive state that
cannot be opened irrespective of the membrane potential. Inactivation is
mediated by the channel's
N-terminus, which acts as a plug that closes the pore. The transition from an
inactive to a closed state .
requires a return to resting potential.
Voltage-gated Nay channels are heterotrimeric complexes composed of a 260 kDa
pore-
forming a subunit that associates with two smaller auxiliary subunits, (31 and
j32. The J32 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 extxacellular 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
xecently characterized
H+-gated cation channels or acid-sensing ion channels (ASIC). ASIC subunits
are expressed in the
brain and form heteromultimeric Nay-permeable channels. These channels require
acid pH
fluctuations for activation. ASIC subunits show homology to the degenerins, a
family of mechanically-
gated channels originally isolated from C. elegans. Mutations in the
degenerins cause
neurodegeneration. ASIC subunits may also have a role in neuronal function, or
in pain perception,
since tissue acidosis causes pain (Waldmann, R. and M. Lazdunski (1998) Curr.
Opin. Neurobiol.
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8:418-424; Eglen, R,M. et al. (1999) Trends Pharmacol. Sci. 20:337-342).
K+ channels are located in all cell types, and may be regulated by voltage,
ATP concentration,
or second messengers such as Ca2+ and CAMP. In non-excitable tissue, K+
channels are involved in
protein synthesis, control of endocrine secretions, and the maintenance of
osmotic equilibrium across
membranes. In neurons and other excitable cells, in addition to regulating
action potentials and
repolarizing membranes, K+ channels are responsible for setting resting
membrane potential. The
cytosol contains non-diffusible anions and, to balance this net negative
charge, the cell contains a Na+-
K+ pump and ion channels that provide the redistribution of Na+, K+, and Cl-.
The pump actively
transports Na+ out of the cell and K~ into the cell in a 3:2 ratio. Ion
channels in the plasma membxane
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
IS transmembrane/1 pore domain structure. Four subunits combine as homo- or
heterotetramers to form
functional K channels. These pore-forming subunits also associate with various
cytoplasmic (3
subunits that alter channel inactivation.kinetics. The Shaker-like channel
family includes the voltage-
gated K+ channels as well as the delayed rectifier type channels such as the
human ether-a-go-go
related gene (HERG) associated with long QT, a cardiac dysrythmia'syndrome
(Curran, M.E. (1998)
2o Curr. Opin. Biotechnol. 9:565-572; Kaczorowski, G.J. and M.L. Garcia (1999)
Curr. Opin. Chem.
Biol. 3:448-458).
A second superfamily of K+ channels is composed of the inward rectifying
channels (Kir).
Kir channels have the property of preferentially conducting K+ currents in the
inwaxd direction. These
proteins consist of a single potassium selective pore domain and two
transmembrane domains, which
25 correspond to the fifth and sixth transmembrane domains of voltage-gated K+
channels. Kir subunits
also associate as tetramers. The Kir family includes ROMK1, mutations in which
lead to Banter
syndrome, a renal tubular disorder. Kir channels are also involved in
regulation of cardiac pacemaker
activity, seizures and epilepsy, and insulin regulation (Doupnik, C.A. et al.
(1995) Curr. Opin.
Neurobiol. 5:268-277; Curran, supra).
30 The recently recognized TWIK K+ channel family includes the mammalian TWIK-
1, TREK-1
and TASK proteins. Members of this family possess an overall structure with
four transmembrane
domains and two P domains. These proteins are probably involved in controlling
the resting potential
in a large set of cell types (Duprat, F. et al. (1997) EMBO J 16:5464-5471).
I0
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The voltage-gated Ca2+ channels have been classified into several subtypes
based upon their
electrophysiological and pharmacological characteristics. L-type Ca~* channels
are predominantly
expressed in heart and skeletal muscle where they play an essential role in
excitation-contraction
coupling. T-type channels are important for cardiac pacemaker activity, while
N-type and P/Q-type
channels are involved in the control of neurotransmitter release in the
central and peripheral nervous
system. The L-type and N-type voltage-gated Ca 2+ channels have been purified
and, though their
functions differ dramatically, they have similar subunit compositions. The
channels are composed of
three subunits. The al subunit forms the membrane pore and voltage sensor,
while the a28 and (3
subunits modulate the voltage-dependence, gating properties, and the current
amplitude of the channel.
These subunits are encoded by at least six al, one az~, and four ~3 genes. A
fourth subunit, y, has
been identified in skeletal muscle (Walker, D. et al. (1998) J. Biol. Chem.
273:2361-2367; McCleskey,
E.W. (1994) Curr. Opin. Neurobiol. 4:304-312).
The high-voltage-activated Ca(2+) channels that have been characterized
biochemically
include complexes of a pore-forming alphal subunit of approximately 190-250
kDa; a transmembrane
complex of alpha2 and delta subunits; an intracellular beta subunit; and in
some cases a
transmembrane gamma subunit. A variety of alphal subunits, alpha2delta
complexes, beta subunits,
and gamma subunits are known. The Cav1 family of alphal subunits conduct L-
type Ca(2+) currents,
which initiate muscle contraction, endocrine secretion, and gene
transcription, and are regulated
primarily by second messenger-activated protein phosphorylation~pathways. The
Cad family of
alphal subunits conduct N-type, P/Q-type, and R-type Ca(2+) currents, which
initiate rapid synaptic
transmission and are regulated primarily by direct interaction with G proteins
and SNARE proteins and
secondarily by protein phosphorylation. The Cav3 family of alphal subunits
conduct T-type Ca(2+)
currents, which are activated and inactivated more rapidly and at more
negative membrane potentials
than other Ca(2+) current types. The distinct structures and patterns of
regulation of these three
families of Ca(2+) channels provide an array of Ca(2+) entry pathways in
response to changes in
membrane potential and a range of possibilities for regulation of Ca(2+) entry
by second messenger
pathways and interacting proteins (Catterall, W.A. (2000) Annu. Rev. Cell Dev.
Biol. 16:521-555).
The alpha-2 subunit of the voltage-gated Ca2+-chancel may include one or more
Cache
domains. An extracellular Cache domain may be fused to an intracellular
catalytic domain, such as
3o the histidine kinase, PP2C phosphatase, GGDEF (a predicted diguanylate
cyclase), HD-GYP (a
predicted phosphodiesterase) or adenylyl cyclase domain, or to a noncatalytic
domain, like the
methyl-accepting, DNA binding winged helix-turn-helix, GAF, PAS or HAMP
(domain found in
istidine kiuases, denylyl cyclases, ethyl-binding proteins and phosphatases).
Small molecules are bound
11
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via the Cache domain and this signal is converted into diverse outputs
depending on the intracellular
domains (Anantharaman, V, and Aravind, L.(2000) Trends Biochem. Sci. 25:535-
537).
The transient receptor family (Trp) of calcium ion channels are thought to
mediate
capacitative calcium entry (CCE). CCE is the Ca2+ influx into cells to
resupply Ca2+ stores depleted
by the action of inositol triphosphate (IP3) and other agents iu response to
numerous hormones and
growth factors. Trp and Trp-like were first cloned from Drosophila and have
similarity to voltage
gated Ca2+ channels in the S3 through S6 regions. This suggests that Trp
and/or related proteins may
foam 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
r . an Na+, I~~/Cl- cotxansporter, accumulating in the cell above its
electrochemical equilibrium. .
. ~ concentration. Secretion of Cl- from the apical surface, in response to
hormonal stimulation, leads to
flow of Na * and water into the secretory lumen. The cystic fibrosis
transmembrane conductance
regulator (CFTR) is a chloride channel encoded by the gene for cystic
fibrosis, a common fatal genetic
disorder in humans. CFTR is a member of the ABC transporter family, and is
composed of two
domains each consisting of six transmembrane domains followed by a nucleotide
binding site. Loss of
CFIR function decreases transepithelial water secretion and, as a result, the
layers of mucus that coat
the respiratory tree, pancreatic ducts, and intestine are dehydrated and
difficult to clear. The resulting
blockage of these sites leads to pancreatic insufficiency, "meconium ileus",
and devastating "chronic
obstructive pulmonary disease" (Al-Awqati, ~. 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) C~rr. Opin. Neurobiol. 6:303-310).
12
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Ligand-gated channels open their pores when an extracellular or intracellular
mediator binds to
the channel. Neurotransmitter-gated channels are channels that open when a
neurotransmitter binds
to their extracellular domain. These channels exist in the postsynaptic
membrane of nerve or muscle
cells. There are two types of neurotransmitter-gated channels. Sodium channels
open in response to
excitatory neurotransmitters, such as acetylcholine, glutamate, and serotonin.
This opening causes an
influx of Na+ and produces the initial localized depolarization that activates
the voltage-gated channels
and starts the action potential. Chloride channels open in response to
inhibitory neurotransmitters,
such as y-aminobutyric acid (GABA) and glycine, leading to hyperpolarization
of the membrane and
the subsequent generation of an action potential. Neurotransmitter-gated ion
channels have four
transmembrane domains and probably function as pentamers (Jentsch, s_upra).
Amino acids in the
second transmembrane domain appear to be important in determining channel
permeation and
selectivity (Sather, W.A. et al. (1994) Curr. Opin. Neurobiol. 4:313-323).
Ligand-gated channels can be regulated by intracellular second messengers. For
example,
calcium-activated K+ channels are gated by internal calcium ions. In nerve
cells, an influx of calcium
during depolarization opens K+ channels to modulate the magnitude of the
action potential (Ishi et al.,
., supra). The large conductance (BK) channel has been purified from brain and
its subunit~composition
determined. The et 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, supra;
Vergara, C. et al. (1998)
C~rr. Opin. Neurobiol. 8:321-329).
Cyclic nucleotide-gated (CNG) channels are gated by cytosolic cyclic
nucleotides. The best
examples of these are the CAMP-gated Na+ channels involved in olfaction and
the cGMP-gated
canon channels involved in vision. Both systems involve ligand-mediated
activation of a G-protein
coupled receptor which then alters the level of cyclic nucleotide within the
cell. CNG channels also
represent a major pathway for Ca2+ entry into neurons; and play roles in
neuronal development and
plasticity. CNG channels are tetramers contain'tug at least two types of
subunits, an a subunit which
can form functional homomeric channels, and a (3 subunit, which modulates the
channel properties.
All CNG subunits have six transmembrane domains and a pore forming region
between the fifth and
sixth transmembrane domains, similar to voltage-gated K+ channels. A large C-
terminal domain
contains a cyclic nucleotide binding domain, while the N-terminal domain
confers variation among
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channel subtypes (Zufall, F. et al. (1997) Curr. Opin. Neurobiol. 7:404-412).
The activity of other types of ion channel proteins may also be modulated by a
variety of
intracellular signalling proteins. Many channels have sites for
phosphorylation by one or more protein
kinases including protein kinase A, protein kinase C, tyrosine kinase, and
casein kinase II, all of which
regulate ion channel activity in cells. Kir channels are activated by the
binding of the G(3~y subunits of
heterotrimeric G-proteins (Reimann, F. and F.M. Ashcroft (1999) Curr. Opin.
Cell.,Biol. 11:503-508).
Other proteins are involved in the localization of ion channels to specific
sites in the cell membrane.
Such proteins include the PDZ domain proteins known as MAGUKs (membrane-
associated guanylate
kinases) which regulate the clustering of ion channels at neuronal synapses
(Craven, S.E. and D.S.
1o Bredt (1998) Cell 93:495-498).
Disease Correlation
The etiology of numerous human diseases and disorders can be attributed to
defects in the
transport of molecules across membranes. Defects in the trafficking of
membrane-bound transporters
and ion channels are associated with several disorders, e.g., cystic fibrosis,
glucose-galactose
malabsorption syndrome, hypercholesterolemia, von Gierke disease, and certain
forms of diabetes
mellitus. Single-gene defect diseases resulting in an inability to transport
small molecules across
membranes include, e.g., cystinuria, iminoglycinuria, Hartup disease, and
Fanconi disease.(van't Hoff, .,.
W.G.~ (1996) Exp. Nephrol. 4:253-262; Talente, G.M. et al. (1994) Ann. Intern.
Med. 120:218-226; - .
and Chillon, M. et al. (1995) New Engl. J. Med. 332:1475-1480).
Human diseases caused by mutations in ion channel genes include disorders of
skeletal
muscle, cardiac muscle, and the central nervous system. Mutations in the pore-
forming subunits of
sodium and chloride channels cause myotonia, a muscle disorder in which
relaxation after voluntary
contraction is delayed. Sodium channel myotonias have been treated with
channel blockers.
Mutations in muscle sodium and calcium channels cause forms of periodic
paralysis, while mutations in
the sarcoplasmic calcium release channel, T-tubule calcium channel, and muscle
sodium channel
cause malignant hyperthermia. Cardiac arrythmia disorders such as the long QT
syndromes and
idiopathic ventricular fibrillation are caused by mutations in potassium and
sodium channels (Cooper,
E.C. and L.Y. Jan (1998) Proc. Natl. Acad. Sci. USA 96:4759-4766). All four
known human
idiopathic epilepsy genes code for ion channel proteins (Berkovic, S.F. and
LE. Scheffer (1999) Curr.
Opin. Neurology 12:177-182). Other neurological disorders such as ataxias,
hemiplegic migraine and
hereditary deafness can also result from mutations in ion channel genes (Jen,
J. (1999) Curr. Opin.
Neurobiol. 9:274-280; Cooper, su ra).
Ion channels have been the target for many drug therapies. Neurotransmitter-
gated channels
14
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have been targeted in therapies for treatment of insomnia, anxiety,
depression, and schizophrenia.
Voltage-gated chancels 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
immunomodulation. T-cell activation depends upon calcium signaling, and a
diverse set of T-cell
specific ion channels has been characterized that affect this signaling
process. Channel blocking
agents can inhibit secretion of lymphokines, cell proliferation, and killing
of target cells. A peptide
antagonist of the T-cell potassium channel Kvl.3 was found to suppress delayed-
type hypersensitivity
and allogenic responses in pigs, validating the idea of channel blockers as
safe and efficacious
immunosuppressants (Cahalan, M.D. and K.G. Chandy (1997) Curr. Opin.
Biotechnol. 8:749-756).
In addition, several SLC26 gene family (solute carrier family 26) ion
trausporters have been
associated with human disease. Defects in the sulfate transporter encoded by
the DTDST gene . . .
cause diastrophic dysplasia, atelosteogenesis type It, or achondrogenesis type
IB. Defects in the
chloride transporter encoded by the CLD (formerly known as DRA) gene causes
congenital chloride
diarrhea. Defects in the iodide transporter encoded by the PDS gene is
associated with Pendred
syndrome (PS) and nonsyndromic deafness type DFNB4. A fourth member of the
family transports
anions such as sulfate, oxalate, and bicarbonate. A fifth member functions as
a motor protein of the
cochlear outer hair cells. A sixth member, SLC26A6, has recently been
identified as a sulfate
transporter (Waldegger, S. et al. (2001) Genomics 72:43-50 and references
within).
Expression profiling
Array technology can provide a simple way to explore the expression of a
single polymorphic
gene or the expression profile of a large number of related or unrelated
genes. When the expression
of a single gene is examined, arrays are employed to detect the expression of
a specific gene or its
variants. When an expression profile is examined, arrays provide a platform
for identifying genes that
are tissue specific, are affected by a scbstance being tested in a toxicology
assay, are part of a
signaling cascade, carry out housekeeping functions, or are specifically
related to a particular genetic
predisposition, condition, disease, or disorder.
The potential application of gene expression profiling is particularly
relevant to improving
CA 02447662 2003-11-18
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diagnosis, prognosis, and treatment of disease that affect the immune
response. Jurkat is an acute T
cell leukemia cell line that grows actively in the absence of external
stimuli. Jurkat has been
extensively used to study signaling in human T cells.
PMA is a broad activator of the protein kinase C-dependent pathways. Ionomycin
is a
calcium ionophore that permits entry of calcium into the cell, hence
increasing the cytosolic calcium
concentration. The combination of PMA and ionomycin activates two of the major
signaling pathways
used by mammalian cells to interact with their environment. In T cells, the
combination of PMA and
ionomycin mimics the type of secondary signaling events elicited during
optimal B cell activation.
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, muscular, immunological, and cell
proliferative disorders, as
well as disorders of iron metabolism, 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," and "TRICH-9." In one aspect,
the invention
provides an isolated polypeptide selected from the group consisting of a) a
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID NO:1-9, 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:1-9, c) a
biologically active fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID N0:1-9, and
d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-9. In one alternative, the invention provides an
isolated polypeptide
comprising the amino acid sequence of SEQ ID NO:1-9.
The invention further provides an isolated polynucleotide encoding a
polypeptide selected from
the group consisting of a) a polypeptide comprising an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-9, 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-
9, e) a biologically active fragment of a polypeptide having an amino acid
sequence selected from the
group consisting of SEQ ID N0:1-9, and d) an irnmunogenic fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID N0:1-9. In
one alternative, the
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polynucleotide encodes a polypeptide selected from the group consisting of SEQ
m N0:1-9. In
another alternative, the polynucleotide is selected from the group consisting
of SEQ ID N0:10-18.
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 m N0:1-9, 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 m N0:1-9, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ m N0:1-9, and d) an immunogenic fragment of a polypeptide
having an amino acid
sequence selected from the group consisting of SEQ m N0:1-9. In one
alternative, the invention
provides a cell transformed with the recombinant polynucleotide. In another
alternative, the invention
provides a transgenic organism comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide selected from
the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ m N0:1-9, 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 m N0:1-9, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ m N0:1-9, and d) an immunogenic fragment of a polypeptide
having an amino acid
sequence selected from the group consisting of SEQ m N0:1-9. 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 N0:1-9, 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 m NO:I-9, c) a biologically active fragment of a
polypeptide having an amino
acid sequence selected from the group consisting of SEQ m N0:1-9, and d) an
immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
3o m N0:1-9.
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
m N0:10-I8, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least
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90% identical to a polynucleotide sequence selected from the group consisting
of SEQ m N0:10-18,
c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide complementary to
the polynucleotide of b), and e) an RNA equivalent of a)-d). In one
alternative, the polynucleotide
comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide selected from
the group consisting of
a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of SEQ
ID N0:10-18, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ )D N0:10-18,
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 ll~
N0:10-18, b) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ ~ NO:10-18, c) a
polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
complementary to the
polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises
a) amplifying said
target polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b)
detecting the presence or absence of said amplified target polynucleotide or
fragment thereof, and,
optionally, if present, the amount thereof.
The invention further provides a composition comprising an effective amount of
a polypeptide
selected from the group consisting of a) a polypeptide comprising an amino
acid sequence selected
from the group consisting of SEQ )D NO:1-9, 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 m NO:1-9, c) a biologically active fragment of a polypeptide having an
amino acid sequence
selected from the group consisting of SEQ m N0:1-9, and d) an immunogenic
fragment of a
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polypeptide having an amino acid sequence selected from the group consisting
of SEQ m NO:1-9, and
a pharmaceutically acceptable excipient. In one embodiment, the composition
comprises an amino
acid sequence selected from the group consisting of SEQ m N0:1-9. The
invention additionally
provides a method of treating a disease or condition associated with decreased
expression of
functional TRICH, comprising administering to a patient in need of such
treatment the composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ m N0:1-9, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90°Io identical to an
amino acid sequence selected
from the group consisting of SEQ a7 N0:1-9, c) a biologically active fragment
of a polypeptide having
an amino acid sequence selected from the group consisting of SEQ m N0:1-9, and
d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ m N0:1-9. 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 T1RICH, 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 m N0:1-9, 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 a7 NO:1-9, c) a
biologically active fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ m N0:1-9, and
d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ m NO:1-9. °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
adnvnistering to a patient in need of such treatment the composition.
The invention further provides a method of screening fox a compound that
specifically binds to
a polypeptide selected from the group consisting of a) a polypeptide
comprising an amino acid
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sequence selected from the group consisting of SEQ l~ N0:1-9, 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 m N0:1-9, c) a biologically active fragment
of a polypeptide having
au amino acid sequence selected from the group consisting of SEQ m N0:1-9, and
d) an
irnmunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ m N0:1-9. 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 au amino
acid sequence selected from the group consisting of SEQ m N0:1-9, 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 ~ N0:1-9, c) a biologically active fragment
of a polypeptide having
an amino acid sequence selected from the group consisting of SEQ ll7 N0:1-9,
and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ m NO:1-9. The method comprises a) combining. the polypeptide
with at least one
test compound under conditions permissive for the activity of the polypeptide,
b) assessing the activity
of the polypeptide in the presence of the test compound, and c) comparing the
.activity of the
polypeptide in the presence of the test compound with the activity of the
polypeptide in the absence of
the test compound, wherein a change in the activity of the polypeptide in the
presence of the test
compound is indicative of a compound that modulates the activity of the
polypeptide.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
polynucleotide sequence selected from the group consisting of SEQ m N0:10-18,
the method
comprising a) exposing a sample comprising the target polynucleotide to a
compound, b) detecting
altered expression of the target polynucleotide, and c) comparing the
expression of the target
polynucleotide in the presence of varying amounts of the compound and in the
absence of the
compound.
The invention further provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound; b)
hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide selected from the group consisting
of i) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
ll~ NO:10-18, ii) a
CA 02447662 2003-11-18
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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 ID N0:10-18,
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 occuxs under conditions
whereby a specific
hybridization complex is formed between said probe and a target polynucleotide
in the biological
sample, said target polynucleotide selected from the group consisting of i) a
polynucleotide comprising
a polynucleotide sequence selected from the group consisting of SEQ ID N0:10-
18, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ ID NO:10-18,
iii) a polynucleotide
complementary to the polynucleotide of i), iv) a polynucleotide complementary
to the polynucleotide of
ii), and v) an RNA equivalent of i)-iv). Alternatively, the target
polynucleotide comprises a fragment
of a polynucleotide sequence selected from the group consisting of i)-v)
above; c) quantifying the
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 scores for the
matches between each
polypeptide and its homolog(s) are also shown.
Table 3 shows structural features of polypeptide sequences of the invention,
including
predicted motifs and domains, along with the methods, algorithms, and
searchable databases used for
analysis of the polypeptides.
Table 4 lists the cDNA and/or genomic DNA fragments which were used to
assemble
polynucleotide sequences of the invention, along with selected fragments of
the polynucleotide
sequences.
Table 5 shows the representative cDNA library for polynucleotides of the
invention.
Table 6 provides an appendix which describes the tissues and vectors used for
construction of
the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
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polypeptides of the invention, along with applicable descriptions, references,
and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which will
be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include pluxal 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
"TRICK' 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
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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
oIigonucleotide probe of
the polynucleotide encoding TRICH, and improper or unexpected hybridization to
allelic variants, with
a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding TRICH.
The encoded protein may also be "altered," and may contain deletions,
insertions, or substitutions of
amino acid residues which produce a silent change and xesult in a functionally
equivalent TRICH.
Deliberate amino acid substitutions may be made on the basis of similarity in
polarity, charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues,
as long as the biological
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 polymerase chain reaction (PCR)
technologies well known
in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of TRICH. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of TRICH either by
directly interacting with TRICH or by acting on components of the biological
pathway in which
TRICH participates.
The term "antibody" refers to intact i_m_m__unoglobulin molecules as well as
to fragments
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thereof, such as Fab, Flab' )2, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind TRICH polypeptides can be prepared using intact
polypeptides or using fragments
containing small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide used
to immunize au animal (e.g., a mouse, a rat, or a rabbit) can be derived from
the translation of RNA,
or synthesized chemically, and can be conjugated to a carrier protein if
desired. Commonly used
carriers that are chemically coupled to peptides include bovine serum albumin,
thyroglobulin, and
keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize
the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures on
the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen used
to elicit the immune response) for binding to an antibody.
The term "aptamer" refers to a nucleic acid or oligonucleotide molecule that
binds to a
specific molecular target. Aptamers are derived from an iw 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-licked 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" xefers 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 "spiegeliner" refers to an aptamer which includes L-DNA, L-RNA, or
other left
handed nucleotide derivatives or nucleotide-like molecules. Aptamers
containing left-handed
nucleotides are resistant to degradation by naturally occurring enzymes, which
normally act on
substrates containing right handed nucleotides.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
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(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
phosphorotluoates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2 =methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
designation "positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic TRICH, or
of any oligopeptide thereof,
to induce a specific immune response in appropriate animals or cells and to
bind with specific
antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising a
given amino acid sequence" refer broadly to any composition containing the
given polynucleotide or
amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding TRICH or fragments
of TRICH may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., 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 moxe overlapping cDNA, EST, or genomic DNA fragments
using a computer
program for fragment assembly, such as the GELVIEW fragment assembly system
(GCG, Madison
WI) or Phrap (University of Washington, Seattle WA). Some sequences have been
both extended
and assembled to produce the consensus sequence.
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"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of the
protein is conserved and not significantly changed by such substitutions. The
table below shows amino
acids which may be substituted for an original amino acid in a protein and
which are regarded as
conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser . Cys, Thr
Thr Ser, Val
Trp ' Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide can include, for example,
replacement of hydrogen by an
alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a
polypeptide which retains
at least one biological or immunological function of the natural molecule. A
derivative polypeptide is
one modified by glycosylation, pegylation, or any similar process that retains
at least one biological or
immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
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"Differential expression" refers to increased or upregulated; or decreased,
downregulated, or
absent gene or protein expression, determined by comparing at least two
different samples. Such
comparisons may be carried out between, for example, a treated and an
untreated sample, or a
diseased and a normal sample.
"Exon shuffling" refers to the recombination of different coding regions
(exons). Since an
exon may represent a structural or functional domain of the encoded protein,
new proteins may be
assembled through the novel reassortment of stable substructures, thus
allowing acceleration of the
evolution of new protein functions.
A "fragment" is a unique portion of TRICH or the polynucleotide encoding TRICH
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example, a
fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or fox other purposes,
may be at least 5, 10, 15,
16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 2S% or 50%) of a polypeptide
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ ID N0:10-18 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ m N0:10-18, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ m N0:10-18 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish SEQ
D7 NO:10-18 from related polynucleotide sequences. The precise length of a
fragment of SEQ >D
N0:10-18 and the region of SEQ 1D N0:10-18 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 m N0:1-9 is encoded by a fragment of SEQ m N0:10-18. A
fragment
of SEQ >I7 NO:1-9 comprises a region of unique amino acid sequence that
specifically identifies SEQ
JD N0:1-9. Fox example, a fragment of SEQ m N0:1-9 is useful as an immunogenic
peptide for the
development of antibodies that specifically recognize SEQ m N0:1-9. The
precise length of a
fragment of SEQ m NO:1-9 and the region of SEQ ll~ NO:1-9 to which the
fragment corresponds
are routinely determinable by one of ordinary skill in the art based on the
intended purpose for the
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fragment.
A "full length" polynucleotide sequence is one containing at least a
translation initiation codon
(e.g., methionine) followed by an open reading frame and a translation
termination codon. A "full
length" polynucleotide sequence encodes a "full length" polypeptide sequence.
"Homology' refers to sequence similarity or, interchangeably, sequence
identity, between two
or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide
sequences, refer to
the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps in
the sequences being compared in order to optimize alignment between two
sequences, and therefore
achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Iliggins, 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/BLASTI. 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:
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Matrix: BLOSUM62
Reward for' match: 1
Penalty for' mismatch: -2
Open Gap: 5 arid Extension Gap: 2 penalties
Gap x drop-off.' S0
Expect: 10
Word Size: ~11
r' filter': OIZ
Percent identity may be measured over the length of an. entire defined
sequence, for example,
as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length supported
by the sequences shown herein, in the tables, figures, or Sequence Listing,
may be used to describe a
length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity" 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 pxogram (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
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comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version
2Ø12 (April-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Opeft Gap: 11 and Extension Gap: 1 penalties
Gap x drop-off. 50
Expect: 10
Word Size: 3
Filter: ort
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ ID number, or may be measured over
a shorter length,
for example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
2o The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the
stringency of the hybridization process, with more stringent conditions
allowing less non-specific
binding, i.e., binding between pairs of nucleic acid strands that are not
perfectly matched. Pernussive
conditions for annealing of nucleic acid sequences are routinely determinable
by one of ordinary skill in
the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
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1% (w/v) SDS, and about 100 ~,g/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (T"~ for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tin and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook, J. et al. (1989)
Molecular Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring Harbor
Press, 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 ,ug/ml. Organic
solvent, such as
formamide at a concentration of about 35-SO% v/v, may~also be used under
particular circumstances,
such as for P,NA: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 similaritybetween the
nucleotides. Such similarity is
strongly indicative of a similar role for the nucleotides and their encoded
polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"hnmune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of TRICH
which is
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capable of eliciting an immune response when introduced into a living
organism, for example, a
mammal. The term "immunogenic fragment" also includes any polypeptide or
oligopeptide fragment
of TRICH which is useful in any of the antibody production methods disclosed
herein or known in the
art.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides,
polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of TRICH. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other biological,
functional, or immunological properties of TRICH.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
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 Iifespan in the cell.
"Post-translational modification" of an TRICH may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in the
art. These processes may occur synthetically or biochemically. Biochemical
modifications will vary
by cell type depending on the enzymatic milieu of TRICH.
"Probe" refers to nucleic acid sequences encoding TRICH, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents,
and enzymes. "Primers"
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CA 02447662 2003-11-18
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are short nucleic acids, usually DNA oligonucleotides, which may be annealed
to a target
polynucleotide by complementary base-pairing. The primer may then be extended
along the target
DNA strand by a DNA polymerase enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence, e.g., by the polymerase chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In. order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
20 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
Bioloev, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al. (1990) PCR
15 Protocols, A Guide to Methods and Applications, Academic Press, San Diego
CA. PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
20 purpose. For example, OLIGO 4.06 software is useful fox the selection of
PCR primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to 5,000
nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection
programs have incorporated additional features for expanded capabilities. For
example, the PrimOU
primer selection program (available to the public from the Genome Center at
University of Texas
25 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 pxogram (available to the public from the Whitehead Institute/MIT
Center for Genome
Research, Cambridge MA) allows the user to input a "mispriming library," in
which sequences to
avoid as primer binding sites are user-specified. Primer3 is useful, in
particular, for the selection of
30 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
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selection of primers that hybridize to either the most conserved or least
conserved regions of aligned
nucleic acid sequences. Hence, this program is useful for identification of
both unique and conserved
oligonucleotides and polynucleotide fragments. The oligonucleotides and
polynucleotide fragments
identified by any of the above selection methods are useful in hybridization
technologies, for example,
as PCR or sequencing primers, microarray elements, or specific probes to
identify fully or partially
complementary polynucleotides in a sample of nucleic acids. Methods of
oligonucleotide selection are
not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supf-a. 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
(UTRs). Regulatory elements interact with host or viral proteins which control
transcription,
translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
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,
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in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" anal "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular structure
of the protein, e.g., the antigenic determinant or epitope, recognized by the
binding molecule. For
example, if an antibody is specific for epitope "A," the presence of a
polypeptide comprising the
epitope A, or the presence of free unlabeled A, in a reaction containing free
labeled A and the
antibody will reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 75% free, and most preferably at least 90% free from other
components with
which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides
by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" or "expression profile" refers to the collective pattern
of gene expression
by a particular cell type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into
a recipient
cell. Transformation may occur under natural or artificial conditions
according to various methods
well known in the art, and may rely on any known method for the insertion of
foreign nucleic acid
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,
CA 02447662 2003-11-18
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by way of deliberate genetic manipulation, such as by microinjection or by
infection with a
recombinant virus. In one alternative, the nucleic acid can be introduced by
infection with a
recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002)
Science 295:868-872). The
term genetic manipulation does not include classical cross breeding, or in
vitro fertilization, but rather is
directed to the introduction of a recombinant DNA molecule. The transgenic
organisms contemplated
in accordance with the present invention include bacteria, cyanobacteria,
fungi, plants and animals.
The isolated DNA of the present invention can be introduced into the host by
methods known in the
art, for example infection, transfection, transformation or transconjugation.
Techniques for
transferring the DNA of the present invention into such organisms are widely
known and provided in
references such as Sambrook et al. (1989), 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
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least
92%, at least 93%, at least
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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,
prevention, and treatment of transport, neurological, muscular, immunological,
and cell proliferative
disorders, as well as disorders of iron metabolism.
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to a
single Incyte project identification number (Incyte Project ID). Each
polypeptide sequence is denoted
by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:)
and an Incyte
polypeptide sequence number (Incyte Polypeptide ID) as shown. Each
polynucleotide sequence is
denoted by both a polynucleotide sequence identification number
(Polynucleotide SEQ 11? NO:) and an
Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as
shown.
Table 2 shows sequences with homology to the polypeptides of the invention as
identified by
BLAST analysis against the GenBank protein (genpept) database. Columns 1 and 2
show the
polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the
invention. Column 3
shows the GenBank identification number (GenBank lD NO:) of the nearest
GenBankhomolog.
Column 4 shows the probability scores for the matches between each polypeptide
and its homolog(s).
Column 5 shows the annotation of the GenBank homolog(s) along with relevant
citations where
applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the
invention. Columns 1 and
2 show the polypeptide sequence identification number (SEQ 117 NO:) and the
corresponding Incyte
polypeptide sequence number (Iucyte 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,
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SEQ ~ N0:3 is 50% identical, from residue A14 to residue 8236, to Caulobacter
crescentus
MotA/TolQ/ExbB proton channel family protein (GenBank m g13424917) as
determined by the Basic
Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability
score is 6.2e-53,
which indicates the probability of obtaining the observed polypeptide sequence
alignment by chance.
SEQ m N0:3 also contains a MotA/TolQ/ExbB proton channel family domain as
determined by
searching for statistically significant matches in the hidden Markov model
(I~~IM) based PFAM
database of conserved protein family domains. (See Table 3.) Data from further
BLAST analyses
pxovide further corroborative evidence that SEQ ll~ N0:3 is a pxoton channel.
In an alternative
example, SEQ m N0:4 is 99% identical, from residue G88 to residue 8947, to
human calcium channel
to alpha-2-delta3 subunit (GenBank B7 g7105926) 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 m N0:4
also contains a
cache domain as determined by searching for statistically significant matches
in the hidden Markov
model (I~VVIM)-based PFAM database of conserved protein family domains. (See
Table 3.) Data
from BLM'S amd MOTIFS analyses provide further corroborative evidence that SEQ
m N0:4 is a
calcium channel alpha-2-delta3 subunit. In an alternative example, SEQ m NO:S
is 81% identical,
from residue E8 to residue E461, to the murine urea transporter UTA-3 (GenBank
D7 g11177180) as
determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.)
The BLAST
probability score is 4.0e-207, which indicates the probability of obtaining
the observed polypeptide
sequence alignment by chance. In an alternative example, SEQ m N0:6 is 40%
identical, from
residue E43 to residue L443, to the human solute carrier family 26 member 6
protein (SLC26A6), an
anion transporter (GenBank m g13344999), as determined by BLAST analysis with
a probability
score of 4.0e-93. SEQ m NO:6 also contains a sulfate transporter domain as
determined by
searching for statistically significant matches in the hidden Markov model
(I~VIM)-based PFAM
database of conserved protein family domains. (See Table 3.) Data from BLIMPS
analysis provide
further corroborative evidence that SEQ ll~ N0:6 is a sulfate transporter. In
an alternative example,
SEQ ID N0:7 is 96% identical, from residue M1 to residue E323, to human GT
mitochondrial solute
carrier protein (GenBank m g386960) as determined by the Basic Local Alignment
Search Tool
(BLAST). (See Table 2.) The BLAST probability score is 6.2e-167, which
indicates the probability
of obtaining the observed polypeptide sequence alignment by chance. SEQ m N0:7
also contains
mitochondrial carrier protein domains as determined by searching for
statistically significant matches in
the hidden Markov model (I~~IM) based PFAM database of conserved protein
family domains. (See
Table 3.) Data from BLIMPS, MOTIFS, and PROF1LESCAN analyses provide further
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corroborative evidence that SEQ ID N0:7 is a mitochondrial carrier protein.
SEQ ll~ N0:1-2 and
SEQ U~ N0:8-9 were analyzed and annotated in a similar manner. The algorithms
and parameters
for the analysis of SEQ ID N0:1-9 are described in Table 7.
As shown in Table 4, the full length polynucleotide sequences of the pxesent
invention were
assembled using cDNA sequences or coding (exon) sequences derived from genomic
DNA, or any
combination of these two types of sequences. Column 1 lists the polynucleotide
sequence
identification number (Polynucleotide SEQ ID NO:), the corresponding Incyte
polynucleotide
consensus sequence number (Incyte ID) for each polynucleotide of the
invention, and the length of
each polynucleotide sequence iu basepairs. Column 2 shows the nucleotide start
(5') and stop (3')
positions of the cDNA and/or genomic sequences used to assemble the full
length polynucleotide .
sequences of the invention, and of fragments of the polynucleotide sequences
which are useful, for
example, in hybridization or amplification technologies that identify SEQ 117
N0:10-18 or that
distinguish between SEQ ID NO:10-18 and related polynucleotide sequences.
The-polynucleotide fragments described in Column 2 of Table 4 may refer
specifically, for
example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from
pooled cDNA
libraries. Alternatively, the polynucleotide fragments described in column 2
may refer to GenBank
cDNAs or. ESTs vcrhich contributed to the assembly of the full length
polynucleotide sequences. In
addition, the polynucleotide fragments described in column 2 may identify
sequences derived from the
ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences
including the w
designation "ENST"). Alternatively, the polynucleotide fragments described in
column 2 may be
derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those
sequences
including the designation "NM" or "NT") or the NCBI RefSeq Protein Sequence
Records (i.e., those
sequences including the designation "NP"). Alternatively, the polynucleotide
fragments described in
column 2 may refer to assemblages of both eDNA and Genscan-predicted exons
brought together by
an "exon stitching" algorithm. For example, a polynucleotide sequence
identified as
FL_XXXXXX~ NI 1V2 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 analysis (See Example V).
Alternatively, the
polynucleotide fragments in column 2 may refer to assemblages of exons brought
together by an
"exon-stretching" algorithm. For example, a polynucleotide sequence identified
as
FL~.'XXXXX_g<4AAAA~BBBBB_1 N is a "stretched" sequence, with X~'~~XXX being
the Iucyte
project identification number, gAAAAA being the GenBank identification number
of the human
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genomic sequence to which the "exon-stretching" algorithm was applied, gBBBBB
being the GenBank
identification number or NCBI RefSeq identification number of the nearest
GenBank protein homolog,
and N referring to specific exons (See Example V). In instances where a RefSeq
sequence was used
as a protein homolog for the "exon-stretching" algorithm, a RefSeq identifier
(denoted by "NM,"
"NP," or "NT") may be used in place of the GenBank identifier (i. e., gBBBBB).
Alternatively, a prefix identifies component sequences that were hand-edited,
predicted from
genomic DNA sequences, or derived from a combination of sequence analysis
methods. The
following Table lists examples of component sequence prefixes and
corresponding sequence analysis
methods associated with the prefixes (see Example IV and Example V).
1o 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 . Fall length transcript and exon prediction
from mapping of EST
sequences to the genome. Genomic location
and EST composition
data are combined to predict the exons and
resulting transcript.
In some cases, Incyte cDNA coverage redundant with the sequence coverage shown
in
Table 4 was obtained to confirm the final consensus polynucleotide sequence,
but the relevant Tncyte
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 confrtm 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 ~0%, 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
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from the group consisting of SEQ ID N0:10-18, which encodes TRICH. The
polynucleotide
sequences of SEQ 117 N0:10-18, as presented in the Sequence Listing, embrace
the equivalent RNA
sequences, wherein occurrences of the nitrogenous base thymine are replaced
with uracil, and the
sugar backbone is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding
TRICH. In
particular, such a variant polynucleotide sequence will have at least about
70%, or alternatively at least
about 85%, or even at least about 95% polynucleotide sequence identity to the
polynucleotide
sequence encoding TRICH. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID N0:10-
18 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 N0:10-18. Any one of the polynucleotide variants described above can encode
an amino acid
sequence which contains at least one functional or structural characteristic
of TRICH.
In addition, or in the alternative, a polynucleotide variant of the invention
is a splice variant of a
polynucleotide sequence encoding TRICH. A splice variant may have portions
which have significant
sequence identity to the polynucleotide sequence encoding TRICH, but will
generally have a greater or
lesser number of polynucleotides due to additions or deletions of blocks of
sequence arising from
alternate splicing of exons during mRNA processing. A splice variant may have
less than about 70%,
or alternatively less than about 60%, or alternatively less than about 50%
polynucleotide sequence
identity to the polynucleotide sequence encoding TRICH over its entire length;
however, portions of
the splice variant will have at least about 70%, or alternatively at least
about 85%, or alternatively at
least about 95%, or alternatively 100% polynucleotide sequence identity to
portions of the
polynucleotide sequence encoding TRICH. Any one of the splice variants
described above can
encode an amino acid sequence which contains at least one functional or
structural characteristic of
TRICH.
It will be appreciated by those skilled in the art that as a result of the
degeneracy of the
genetic code, a multitude of polynucleotide sequences encoding TRICH, some
bearing minimal
similarity to the polynucleotide sequences of any known and naturally
occurring gene, may be
produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
sequence that could be made by selecting combinations based on possible codon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring TRICH, and all such variations
are to be considered as
being specifically disclosed.
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Although nucleotide sequences which encode TRICH and its variants are
generally capable of
hybridizing to the nucleotide sequence of the naturally occurring TRICH under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding TRICH or
its derivatives possessing a substantially different codon usage, e.g.,
inclusion of non-naturally
occurring codons. Codons may be selected to increase the rate at which
expression of the peptide
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
NO:10-18 and fragments thereof undex various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; Kim_m__el, A.R. (1987)
Methods Enzymol. 152:507-
511.) Hybridization conditions, including annealing and wash conditions, are
described in "Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(Applied
Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ),
or combinations
of polymerases and proofreading exonucleases such as those found in the
ELONGASE amplification
system (Invitrogen, Carlsbad CA). 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 (Amersham Biosciences),
or other
systems known in the art. The resulting sequences are analyzed using a variety
of algorithms which
are well known in the art. (See, e.g., Ausubel, F.M. (1997) Short Protocols in
Molecular Biolo~y, John
Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biology and
Biotechnolo~y,
42
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
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-stranded sequence into a
region of unknown
sequence before performing PCR. Other methods which may be used to retrieve
unknown sequences
are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids
Res. 19:3055-3060).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries
(Clontech, Palo
Alto CA) to walk genomic DNA. This procedure avoids the need to screen
libraries and is useful in
finding intron/exon junctions. For all PCR-based methods, primers may be
designed using
commercially available software, such as OLIGO 4.06 primer analysis software
(National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30
nucleotides in length,
to have a GC content of about 50%o or more, and to anneal to the template at
temperatures of about
68°C to 72°C.
When screening for full length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence
into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems); and the
entire
43
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof which
encode TRICH may be cloned in recombinant DNA molecules that direct expression
of TRICH, or
fragments or functional equivalents thereof, in appropriate host cells. Due to
the inherent degeneracy
of the genetic code, other DNA sequences which encode substantially the same
or a functionally
equivalent amino acid sequence may be produced and used to express TRICH.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter TRICH-encoding sequences for a variety of
purposes including, but
not limited to, modification of the cloning, processing, and/or expression of
the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction
sites, alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc.; Santa Clara CA; described in U.S. Patent
No.
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or improve
the biological properties of TRICH, such as its biological or enzymatic
activity or its ability to bind to
other molecules or compounds. DNA shuffling is a process by which a library of
gene variants is
produced using PCR-mediated recombination of gene fragments. The library is
then subjected to
selection or screening procedures that identify those gene variants with the
desired properties. These
preferred variants may then be pooled and further subjected to recursive
rounds of DNA shuffling and
selection/screening. Thus, genetic diversity is created through "artificial"
breeding and rapid molecular
evolution. For example, fragments of a single gene containing random point
mutations may be
recombined, screened, and then reshuffled until the desired properties are
optimized. Alternatively,
fragments of a given gene may be recombined with fragments of homologous genes
in the same gene
family, either from the same or different species, thereby maximizing the
genetic diversity of multiple
naturally occurring genes in a directed and controllable manner.
In another embodiment, sequences encoding TRICH may be synthesized, in whole
or in part,
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
al. (1980) Nucleic Acids
Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.) Alternatively,
44.
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
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 Robexge, 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., Cliiez, 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 trauslational control of the
inserted coding sequence in
a suitable host. These elements include regulatory sequences, such as
enhancers, constitutive and
inducible promoters, and 5' and 3' untranslated regions in the vector and in.
polynucleotide sequences
encoding TRICH. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
TRICH. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding TRICH and its initiation codon and upstream regulatory
sequences are inserted
into the appropriate expression vector, no additional transcriptional or
translational control signals may
be needed. However, in cases where only coding sequence, or a fragment
thereof, is inserted,
exogenous translational control signals including an in-frame ATG initiation
codon should be provided
by the vector. Exogenous translational elements and initiation codons may be
of various origins, both
natural and synthetic. The efficiency of expression may be enhanced by the
inclusion of enhancers
appropriate for the particular host cell system used. (See, e.g., Scharf, D.
et al. (1994) Results Probl.
Cell Differ. 20:125-162.)
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding TRICH and appxopriate transcriptional
and translational control
elements. These methods include if2 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)
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
Current Protocols in Molecular Biolo~y, John Wiley ~z Sons, New York NY, ch.
9, 13, and 16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding TRICH. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems taransformed 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, supf~a; 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. linrmunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature
389:239-242.) The
invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding TRICH. For
example, routine cloning,
subcloning, and propagation of polynucleotide sequences encoding TRICH can be
achieved using a
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORT1
plasmid (Invitrogen). Ligation of sequences encoding TRICH into the vector's
multiple cloning site
disrupts the lacZ gene, allowing a colorimetric screening procedure for
identification of transformed
bacteria containing recombinant molecules. In addition, these vectors may be
useful for in vitro
transcription, dideoxy sequencing, single strand rescue with helper phage, and
creation of nested
deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M. Schuster
(1989) J. Biol. Chem.
264:5503-5509.) When large quantities of TRICH are needed, e.g. for the
production of antibodies,
vectors which direct high level expression of TRICH may be used. For example,
vectors containing
the strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of TRICH. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
46
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
promoters, may be used in the yeast Saccharomyces cer~evisiae or Pichia
pastor~is. 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, su ra;
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 TR1CH may be driven by viral promoters, e.g., the 35S and 19S
promoters of CaMV used
alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small subunit of
RUBTSCO or heat shock
promoters rnay 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 Techuolo~y
(1992) McGraw Hill,
New York NY, pp. 191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an~ adenovirus is used as an expression vector, sequences encoding TRICH
may be ugated into
an adenovirus transcription/translation complex consisting of the late
promoter and tizpartite leader
sequence. .Insertion in a non-essential E1 or E3 region of the viral genome
maybe used to obtain
infective virus which expresses TRTCH in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalan 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 (uposomes,
polycationic amino polymers,
or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J. et al.
(1997) Nat. Genet. 15:345-
355.)
For long term production of recombinant proteins in mammalan systems, stable
expression of
TRICH in cell lines is preferred. For example, sequences encoding TRICH can be
transformed into
cell ones using expression vectors which may contain viral origins of
replication and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media before
being switched to selective media. The purpose of the selectable marker is to
confer resistance to a
47
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
selective agent, and its presence allows growth and recovery of cells which
successfully express the
introduced sequences. Resistant clones of stably transformed cells may be
propagated using tissue
culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk and Apr. cells, respectively.
(See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic, or
herbicide resistance can be used as the basis for selection: For example, dhfr-
confers resistance to
methotrexate; taeo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltrausferase,
respectively. (See, e.g.,
Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. (1981)
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hisD, which
alter cellular requirements for metabolites. (See, e.g., Hat~tmau, S.C. and
R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., authocyanins, green
fluorescent proteins
(GFP; Clontech), J3 glucuronidase and its substrate !3-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system. ,
(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 anal expression of the gene may need to be
confirmed. For example, if
the sequence encoding TRICH is inserted within a marker gene sequence,
transformed cells
containing sequences encoding TRICH can be identified by the absence of marker
gene function.
Alternatively, a marker gene can be placed in tandem with a sequence encoding
TRICH under the
control of a single promoter. Expression of the marker gene in response to
induction or selection
usually indicates expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding TRICH
and that express
TRICH may be identified by a variety of procedures known to those of skill in
the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations,
PCR
amplification, and protein bioassay or immunoassay techniques which include
membrane, solution, or
chip based technologies for the detection and/or quantification of nucleic
acid or protein sequences.
T_m_m__unologlcal 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
48
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
fluorescence activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on TRICH is
preferred, but a
competitive binding assay may be employed. These and other assays are well
known in the art. (See,
e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN,
Sect. IV; Coligau, J.E. et al. (1997) Current Protocols in hxununolo~y, Greene
Pub. Associates and
Wiley-Interscience, New York NY; and Pound, J.D. (1998) Tmmunochemical
Protocols, Humana
Press, Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled hybridization
or PCR probes for detecting sequences related to polynucleotides encoding
TRICH ixtclude
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 i~t vitro by addition of an
appropriate RNA polymerase
such as T7, T3, or SP6 and labeled nucleotides. These procedures may be
conducted using a variety
of commercially available kits, such as those provided by Amersham
Biosciences, Promega (Madison
WI), and US Biochemical. Suitable reporter molecules or labels which may be
used for ease of
detection include radionuclides, enzymes, fluorescent, chemiluminescent, or
chromogenic agents, as
well as substrates, cofactors, inhibitors, magnetic patfiicles, and the like.
2o Host cells transformed with nucleotide sequences encoding TRICH may be
cultured under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the sequence
and/or the vector used. As will be understood by those of skill in the art,
expression vectors containing
polynucleotides which encode TRICH may be designed to contain signal sequences
which direct
secretion of TRICH through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of
the polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which cleaves a
"prepro" or "pro" form of the
protein may also be used to specify protein targeting, folding, and/or
activity. Different host cells
which have specific cellular machinery and characteristic mechanisms for post-
translational activities
(e.g., CHO, HeLa, MDCI~, HEI~293, and WI38) are available from the American
Type Culture
Collection (ATCC, Manassas VA) and may be chosen to ensure the correct
modification and
49
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding TRICH may be ligated to a heterologous sequence resulting
in translation of a
fusion protein in any of the aforementioned host systems. For example, a
chimeric TR1CH 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
to hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of
their cognate fusion
proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin,
and metal-chelate resins,
respectively. FLAG, c-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,
supra, ch. 10). A variety of
commercially available kits may also be used to facilitate expression and
purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled TRICH may
be achieved
in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These
systems couple transcription and translation of protein-coding sequences
operably associated with the
T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
precursor, for example, 35S-methionine.
TRICH of the present invention or fragments thereof may be used to screen for
compounds
that specifically bind to TRICH. At least one and up to a plurality of test
compounds may be screened
for specific binding to TRICH. Examples of test compounds include antibodies,
oligonucleotides,
proteins (e.g., ligands or receptors), or small molecules. In one embodiment,
the compound thus
identified is closely related to the natural ligand of TRICH, e.g., a ligand
or fragment thereof, a natural
substrate, a structural or functional mimetic, or a natural binding partner.
(See, e.g., Coligan, J.E. et al.
(1991) Current Protocols in Immunology 1(2):Chapter 5.) In another embodiment,
the compound thus
identified is a natural ligand of a receptor TRICH. (See, e.g., Howard, A.D.
et al. (2001) Trends
Pharmacol. Sci.22:132-140; Wise, A. et al. (2002) Drug Discovery Today 7:235-
246.)
In other embodiments, the compound can be closely related to the natural
receptor to which
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
TRICH binds, at least a fragment of the receptor, or a fragment of the
receptor including all or a
portion of the ligand binding site or binding pocket. For example, the
compound may be a receptor for
TRICH which is capable of propagating a signal, or a decoy receptor for TRICH
which is not capable
of propagating a signal (Ashkenazi, A. and V.M. Divit (1999) Curr. Opin. Cell
Biol. 11:255-260;
Mantovani, A. et al. (2001) Trends Tmmunol. 22:328-336). The compound can be
rationally designed
using known techniques. Examples of such techniques include those used to
construct the compound
etanercept (ENBREL; Tm_m__unex Corp., Seattle WA), which is efficacious for
treating rheumatoid
arthritis in humans. Etanercept is an engineered p75 tumor necrosis factor
(TNF) receptor dimer
linked to the Fc portion of human IgGl (Taylor, P.C. et al. (2001) Curr. Opin.
Trrmmunol. 13:611-616).
In one embodiment, screening for compounds which specifically bind to,
stimulate, or inhibit
TRICH involves producing appropriate cells which express T1ZICH, either as a
secreted protein or on
the cell membrane. Preferred cells include cells from mammals, yeast,
Drosophila, or E. coli. Cells
expressing TRICH or cell membrane fractions which contain TRICH are then
contacted with a test
compound and binding, stimulation, or inhibition of activity of either TRICH
or the compound is
analyzed.
An assay may simply test binding of a test compound to the polypeptide,
wherein binding is
detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable
label. For example, the
assay may comprise the steps of combining at least one test compound with
TRICH, either in solution
or affixed to a solid support, and detecting the binding of TRICH to the
compound. Alternatively, the
assay may detect or measure binding of a test compound in the presence of a
labeled competitor.
Additionally, the assay may be carried out using cell-free preparations,
chemical libraries, or natural
product mixtures, and the test compounds) may be free in solution or affixed
to a solid support.
An assay can be used to assess the ability of a compound to bind to its
natural ligand and/or to
inhibit the binding of its natural ligand to its natural receptors. Examples
of such assays include radio
labeling assays such as those described in U.S. Patent No. 5,914,236 and U.S.
Patent No. 6,372,724.
In a related embodiment, one or more amino acid substitutions can be
introduced into a polypeptide
compound (such as a receptor) to improve or alter its ability to bind to its
natural ligands. (See, e.g.,
Matthews, D.J. and J.A. WelIs. (1994) Chem. Biol. 1:25-30.) In another related
embodiment, one or
more amino acid substitutions can be introduced into a polypeptide compound
(such as a ligand) to
improve or alter its ability to bind to its natural receptors. (See, e.g.,
Cunningham, B.C. and J.A. Wells
(1991) Proc. Natl. Acad. Sci. USA 88:3407-3411; Lowman, H.B. et al. (1991) J.
Biol. Chem.
266:10982-10988.)
TRICH of the present invention or fragments thereof may be used to screen for
compounds
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that modulate the activity of TRICH. Such compounds may include agonists,
antagonists, or partial or
inverse agonists. In one embodiment, an assay is performed under conditions
permissive for TRICH
activity, wherein TRICH is combined with at least one test compound, and the
activity of TRICH in
the presence of a test compound is compared with the activity of TRICH in the
absence of the test
compound. A change in the activity of TRICH in the presence of the test
compound is indicative of a
compound that modulates the activity of TRICH. Alternatively, a test compound
is combined with an
in vitro or cell-free system comprising TRICH under conditions suitable for
TRICH activity, and the
assay is performed. In either of these assays, a test compound which modulates
the activity of
TRICH may do so indirectly and need not come in direct contact with the test
compound. At least
one and up to a plurality of test compounds may be screened.
In another embodiment, polynucleotides encoding TRICH or their mammalian
homologs may
be "knocked out" in an animal model system using homologous recombination in
embryonic stem (ES)
cells. Such techniques are well known in the art and are useful for the
generation of animal models of
human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No.
5,767,337.) 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 phosphotrausferase gene (neo; Capeccbi,
M.R. (1989) Science
244:1288-1292). The vector integrates .into the corresponding region of the
host genome by
homologous recombination. Alternatively, homologous recombination takes place
using the Cre-loxP
system to knockout a gene of interest in a tissue- or developmental stage-
specific manner (Marth, J.D.
(1996) Cliu. Iuvest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids
Res. 25:4323-4330).
Transformed ES cells are identified and micxoinjected 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. Trausgenic animals thus generated may be tested with potential
therapeutic or toxic agents.
Polynucleotides encoding TRICH may also be manipulated in vitro in ES cells
derived from
human blastocysts. Human ES cells have the potential to differentiate into at
least eight separate cell
lineages including endoderm, mesoderm, and ectodermal cell types. These cell
lineages differentiate
into, for example, neural cells, hematopoietic lineages, and cardiomyocytes
(Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding TRTCH can also be used to create "knockin" humanized
animals
(pigs) or transgenic animals (mice or rats) to model human disease. With
knockin technology, a region
of a polynucleotide encoding TRICH is injected into animal ES cells, and the
injected sequence
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integrates into the animal cell genome. Transformed cells are injected into
blastulae, and the blastulae
are implanted as described above. Transgenic progeny or inbred Hues are
studied and treated with
potential pharmaceutical agents to obtain information on treatment of a human
disease. Alternatively,
a mammal inbred to overexpress TRICH, e.g., by secreting TRICH in its milk,
may also serve as a
convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu.
Rev. 4:55-74).
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, examples of
tissues expressing
TRICH can be found in Table 6 and can also be found in Example XI. Therefore,
TRICH appears to
play a role in transport, neurological, muscular, immunological, and cell
proliferative disorders, as well
as disorders of iron metabolism. In the treatment of disorders associated with
increased TRICH
expression or activity, it is desirable to decrease the expression or activity
of TRICH. In the treatment
of disorders associated with decreased TRICH expression or activity, it is
desirable to increase the
expression or activity of TRICH.
Therefore, in one embodiment, TRICH or a fragment or derivative thereof may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of TRICH. Examples of such disorders include, but are not limited to,
a transport disorder
such as akinesia, amyotrophic lateral sclerosis, ataxia telaugiectasia, 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 dyskiuesia, 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 trausport, 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
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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 centaral nervous system
disease, prion diseases including
kuru, Creutzfeldt-Jakob disease, and Gerstmann-Stxaussler-Scheinker syndrome,
fatal familial
insomnia, nutritional and metabolic diseases of the nervous system,
neurofibromatosis, tuberous
1o sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal
syndrome, mental retardation
and other developmental disorders of the central nervous system including Down
syndrome, cerebral
palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial
nerve disorders, spinal cord
diseases, muscular dystrophy and other neuromuscular disorders, peripheral
nervous system disorders,
dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic
myopathies, myasthenia
gravis, periodic paralysis, mental disorders including mood, anxiety, and
schizophrenic disorders,
seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic
neuropathy, tardive
dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's
disorder, progressive
supranuclear palsy, corticobasal degeneration, and familial frontotemporal
dementia; a muscle .disorder
such as cardiomyopathy, myocarditis, Duchenne's muscular dystrophy, Beckex'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 syndxome, 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
spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune
hemolytic anemia, autoimmune
thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy
(APECED), bronchitis,
cholecystitis, contact dermatitis, Crohu'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,
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myocardial or pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis, polymyositis, psoriasis,
Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome,
systemic anaphylaxis,
systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura,
ulcerative colitis, uveitis,
Werner syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral,
bacterial, fungal, parasitic, protozoal, and hehninthic infections, and
trauma; and a cell proliferative
disorder such as actinic keratosis, arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis, mixed
connective tissue disease (MCTD), myeloflbrosis, 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; and a disorder of iron metabolism
such as
hypotransferrinaemia, acaeruloplasminaemia, adult, juvenile, and neonatal
haemochromatosis.
In another embodiment, a vector capable of expressing TRICH or a fragment or
derivative
thereof may be administered to a subject to treat or prevent a disorder
associated with decreased
expression or activity of TRICH including, but.not limited to, those described
above.
In a further embodiment, a composition comprising a substantially purified
TRICH in
conjunction with a suitable pharmaceutical carrier may be administered to a
subject to treat or prevent
a disorder associated with decreased expression or activity of TRICH
including, but. not limited to,
those provided above.
In still another embodiment, an agonist which modulates the activity of TRICH
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of TRICH including, but not limited toy those listed above.
In a further embodiment, an antagonist of TRICH may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of TRICH.
Examples of such
disorders include, but are not limited to, transport, neurological, muscular,
immunological, and cell
proliferative disorders, as well as disorders of iron metabolism described
above. In one aspect, an
antibody which specifically binds TRICH may be used directly as an antagonist
or indirectly as a
targeting or delivery mechanism for bringing a pharmaceutical agent to cells
or tissues which express
3o TRICH.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding TRICH may be administexed to a subject to treat ox prevent a disorder
associated with
increased expression or activity of TRICH including, but not limited to, those
described above.
CA 02447662 2003-11-18
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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. Single chain
antibodies (e.g., from
15. camels or llamas) may be potent enzyme inhibitors and may have advantages
in the design of peptide
mimetics, and in the development of immuno-adsorbents and biosensors
(Muyldermans, S. (2001) J. .
Biotechnol. 74:277-302).
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, camels,
dromedaries, llamas, humans, and others may be immunized by injection with
TRICH or with any
fragment or oligopeptide thereof which has immunogenic properties. Depending
on the host species,
various adjuvants may be used to increase immunological response. Such
adjuvants include, but are
not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface
active substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH,
and dinitrophenol. Among
adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium
parvum are especially
preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
TRICH have an amino acid sequence consisting of at least about 5 amino acids,
and generally will
consist of at least about 10 amino acids. It is also preferable that these
oligopeptides, peptides, or
fragments are identical to a portion of the amino acid sequence of the natural
protein. Short stretches
of TRICH amino acids may be fused with those of another protein, such as I~LH,
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
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to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-
hybridoma
technique. (See, e.g., Kohlex, G. et al. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.
Tmmunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; 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 immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in
the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.
USA 86:3833-3837; Winter,
G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for TRICH may also be
generated.
For example, such fragments include, but are not limited to, F(ab')2 fragments
produced.by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab')2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
TRICH and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive
to two non-interfering TRICH epitopes is generally used, but a competitive
binding assay may also be
3o employed (Pound, supja).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay techniques
may be used to assess the affinity of antibodies for TRTCH. Affinity is
expressed as an association
constant, Ka, which is defined as the molar concentration of TRICH-antibody
complex divided by the
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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 axe preferred for use in immunoassays in
which the TRICH-
antibody complex must withstand rigorous manipulations. Low-affinity antibody
preparations with Ka
ranging from about 106 to 10' L/mole are preferred for use in
immunopurification and similar
procedures which ultimately require dissociation of TRICH, preferably in
active form, from the
antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL
Press, Washington DC;
Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies,
John Wiley & Sons,
New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to determine
the quality and suitability of such preparations for certain downstream
applications. For example, a
polyclonal antibody preparation containing at least 1-2 mg specific
antibody/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 qualityand usage in various applications, are generally available.
(See, e.g., Catty, supra,
and Coligan et al. supra.)
2o In another embodiment of the invention, the polynucleotides encoding TRICH,
or any
fragment or complement thereof, may be used for therapeutic purposes. In one
aspect, modifications
of gene expression can be achieved by designing complementary sequences or
antisense molecules
(DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory
regions of the gene
encoding TRICH. Such technology is well known in the art, and antisense
oligonucleotides or larger
fragments can be designed from various locations along the coding or control
regions of sequences
encoding TRICH. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics,
Humana Press Inc.,
Totawa NJ.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. (1998) J. Allergy Clip. Tm_m__unol. 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
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vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. (1990) Blood
76:271; Ausubel, supf-a; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi,, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et
al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al. (1997)
Nucleic Acids Res.
25(14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding TRICH may be
used for
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease
characterized by X-
linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672),
severe combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypexcholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-
242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites (e.g.,
against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore, D. (1988)
2o 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 falciparwm and
Trypanosoma cruzi). In
the case where a genetic deficiency in TRICH expression or regulation causes
disease, the expression
of TRICH from an appropriate population of transduced cells may alleviate the
clinical manifestations
caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by
deficiencies in
TRICH are treated by constructing mammalian expression vectors encoding TRICH
and introducing
these vectors by mechanical means into TRICH-deficient cells. Mechanical
transfer technologies for
use with cells in vivo or ex vitro include (i) direct DNA microinjection into
individual cells, (ii) ballistic
gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-
mediated gene transfer, and
(v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Anna.
Rev. Biochem.
62:191-217; Ivies, Z. (1997) Cell 91:501-510; Boulay, J-L. and H. Recipon
(1998) Curr. Opin.
Biotechnol. 9:445-450).
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Expression vectors that may be effective for the expression of TRICH include,
but are not
limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors
(Iuvitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La
Jolla CA),
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 (TIC), or (3-actin genes),
(ii) an inducible promoter
(e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992)
Proc. Natl. Acad. Sci.
USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi,
F.M.V. and H.M. Blau
(1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen));
the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIIVD;
Invitrogen); the
FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible
promoter (Rossi, F.M.V.
and H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native
promoter of the endogenous
gene encoding TRICH from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of these
standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to TRICH expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding TRICH under the control of an independent promoter or
the retrovirus long
terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and
(iii) a Rev-responsive
element (RRE) along with additional retrovirus cis-acting RNA sequences and
coding sequences
required for efficient vector propagation. Retrovirus vectors (e.g., PFB and
PFBNEO) are
commercially available (Stratagene) and are based on published data (Riviere,
I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The
vector is propagated in
an appropriate vector producing cell Line (VPCL) that expresses an envelope
gene with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; 2ufferey, R. et
al. (1998) J. Virol. 72:9873-9880). U.S. Patent No. 5,910,434 to Rigg ("Method
for obtaining
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retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant") discloses
a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by reference.
Propagation of retrovirus vectors, transduction of a population of cells
(e.g., CD4+ T-cells), and the
return of transduced cells to a patient are procedures well known to persons
skilled in the art of gene
therapy and have been well documented (Ranga, U. et al. (1997) J. Virol.
71:7020-7029; Bauer, G. et
al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-4716;
Ranga, U. et al. (1998)
Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding TRICH to cells which have one or more genetic
abnormalities with respect
to the expression of TRICH. The construction and packaging of adenovirus-based
vectors are well
known to those with ordinary skill in the art. Replication defective
adenovirus vectors have proven to
be versatile for importing genes encoding immunoregulatory proteins into
intact islets in the pancreas
(Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful
adenoviral vectors are
described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors for
gene therapy"),
hereby incorporated by reference. For adenoviral vectors, see also Antinozzi,
P.A. et al. (1999)
Annu. Rev. Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature
18:389:239-242, both
incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding TRICH to target cells which have one or more genetic
abnormalities with
respect to the expression of TRICH. The use of herpes simplex virus (HSV)
based vectors may be
especially valuable for introducing TRICH to cells of the central nervous
system, for which HSV has
a tropism. The construction and packaging of herpes-based vectors are well
known to those with
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
type 1 based vector has
been used to deliver a reporter gene to the eyes of primates (Liu, X. et al.
(1999) Exp. Eye Res.
169:385-39S). The construction of a HSV-1 virus vector has also been disclosed
in detail in U.S.
Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is hereby
incorporated by reference. U.S. Patent No. 5,804,413 teaches the use of
recombinant HSV d92
which consists of a genome containing at least one exogenous gene to be
transferred to a cell under
the control of the appropriate promoter for purposes including human gene
therapy. Also taught by
this patent are the construction and use of recombinant HSV strains deleted
for ICP4, ICP27 and
ICP22. For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-
532 and Xu, H. et al.
(1994) Dev. Biol. 163:152-161, hereby incorporated by reference. The
manipulation of cloned
herpesvirus sequences, the generation of recombinant virus following the
transfection of multiple
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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 Itnmunolo~ic Approaches, Futura Publishing, Mt. Kisco NY, pp.
163-177.) A
complementary sequence or antisense molecule may also be designed to block
translation of mRNA
by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
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molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding TRICH.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared
by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA
sequences encoding TRICH. Such DNA sequences may be incorporated into a wide
variety of
vectors with suitable RNA polymerase promoters such as T7 or SP6.
Alternatively, these cDNA
constructs that synthesize complementary RNA, constitutively or inducibly, can
be introduced into cell
lines, cells, or tissues. '
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-, tbio-, and similarly modified
forms of adenine, cytidine,
guanine, thymine, and uridine which are not as easily recognized by endogenous
endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding TRICH. Compounds
which may be effective in altering expression of a specific polynucleotide may
include, but are not
limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming
oligonucleotides,
transcription factors and other polypeptide transcriptional regulators, and
non-macromolecular
chemical entities which are capable of interacting with specific
polynucleotide sequences. Effective
compounds may alter polynucleotide expression by acting as either inhibitors
or promoters of
polynucleotide expression. 'Thus, in the treatment of disorders associated
with increased TRICH
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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 i~2 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 Schizosacchar-omyces 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.
28:815) 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 iyi vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposorne injections, or by polycationic amino
polymers may be achieved
64.
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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 Remin tg on's
Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may
consist of TRICH,
antibodies to TRICH, and mimetics, agonists, antagonists, or inhibitors of
TRICH.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, intra-
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 drugs),
aerosol delivery of fast-
acting formulations is well-known in the art. In the case of macromolecules
(e.g. larger peptides and
proteins), recent developments in the field of pulmonary delivery via the
alveolar region of the lung
have enabled the practical delivery of drugs such as insulin to blood
circulation (see, e.g., Patton, J.S.
et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage of
administration without
needle injection, and obviates the need for potentially toxic penetration
enhancers.
Compositions suitable for use in the invention include compositions wherein
the active
ingredients are contained in an effective amount to achieve the intended
purpose. The determination
of an effective dose is well within the capability of those skilled in the
art.
Specialized forms of compositions may be prepared for direct intracellular
delivery of
macromolecules comprising TRICH or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of the
macromolecule. Alternatively, TRICH or a fragment thereof may be joined to a
short cationic N-
terminal portion from the H1V 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°Io 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,ug to 100,000 ,ug, 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
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treated with TRICH or agonists, antagonists, or inhibitors of TRICH.
Antibodies useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic
assays for TRICH include methods which utilize the antibody and a label to
detect TRICH in human
body fluids or in extracts of cells or tissues. The antibodies may be used
with or without modification,
and may be labeled by covalent or non-covalent attachment of a reporter
molecule. A wide variety of
reporter molecules, several of which are described above, are known in the art
and may be used.
A variety of protocols for measuring TRICH, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
TRICH expression. Normal
or standard values for TRICH expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibodies to TRICH under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of TRICH
expressed in
subject, control, and disease samples frombiopsied 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 lD
N0:10-18 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
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production of mRNA probes. Such vectors are known in the art, are commercially
available, and may
be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as 32P or 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,
bradyarlytlnnia, tachyarrythmia, hypertension, Long QT syndrome, myocarditis,
cardiomyopathy,
nemaline myopathy, centronuclear myopathy, lipid myopathy, mitochondria)
myopathy, thyrotoxic
rnyopathy, 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, eystinuria, 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
extxapyramidal 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
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syndrome, mental retardation and other developmental disorders of the central
nervous system
including Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic
nervous system
disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy
and other neuromuscular
disorders, peripheral nervous system disorders, dermatomyositis and
polymyositis, inherited, metabolic,
endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental
disorders including
mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD),
akathesia, amnesia,
catatonia, diabetic neuropathy, tardive dyskinesia; dystonias, paranoid
psychoses, postherpetic
neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal
degeneration, and familial
frontotemporal dementia; a muscle disorder such as cardiomyopathy,
myocarditis, Duchenne's
muscular dystrophy, Becker's muscular dystrophy, myotonic dystrophy, central
core disease, nemaline
myopathy, centronuclear myopathy, lipid myopathy, mitochondrial myopathy,
infectious myositis,
polymyositis, dermatomyositis, inclusion body myositis, thyrotoxic myopathy,
ethanol myopathy, angina,
anaphylactic shock, arrhythmias, asthma, cardiovascular shock, Cushing's
syndrome, hypertension,
hypoglycemia, myocardial infarction, migraine, pheochromocytoma, and
myopathies including
encephalopathy, epilepsy, Kearns-Sayre syndrome, lactic acidosis, myoclonic
disorder,
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 spondylitis,
amyloidosis, anemia, asthma,
atherosclerosis, autoirnmune hemolytic anemia, autoimmune thyroiditis,
autoimmune
polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis,
cholecystitis, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema, episodic
lymphopen2a with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum,
atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis,
hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's
syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial,
fungal, parasitic, protozoal, and helminthic infections, and trauma; and a
cell proliferative disorder such
as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis,
hepatitis, mixed connective
tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria,
polycythemia vera,
psoriasis, primary thrombocythemia, and cancers including adenocarcinoma,
leukemia, lymphoma,
melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of
the adrenal gland,
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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; and a disorder of iron metabolism such as
hypotransferrinaemia,
acaeruloplasminaemia, adult, juvenile, and neonatal haemochromatosis. The
polynucleotide sequences
encoding TRICH may be used in Southern or northern analysis, dot blot, or
other membrane-based
technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-
like assays; and in
microarrays utilizing fluids or tissues from patients to detect altered TRICH
expxession. 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
ef~.cacy of a particular therapeutic treatment regimen in animal studies, in
clinical trials, or to monitor
the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with
expression of
TRICH, a normal or standard profile for expression is established. This may be
accomplished by
combining body fluids or cell extracts taken from normal subjects, either
animal or human, with a
sequence, or a fragment thereof, encoding TRICH, under conditions suitable for
hybridization or
amplification. Standard hybridization may be quantified by comparing the
values obtained from normal
subjects with values from an experiment in which a known amount of a
substantially purified
polynucleotide is used. Standard values obtained in this manner may be
compared with values
obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard
values is used to establish the presence of a disorder.
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
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overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the development
of the disease, or may provide a means for detecting the disease prior to the
appearance of actual
clinical symptoms. A more definitive diagnosis of this type may allow health
professionals to employ
preventative measures or aggressive treatment earlier thereby preventing the
development or further
progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding TRICH
may involve the use of PCR. These oligomers may be chemically synthesized,
generated
enzymatically, or produced in vitro. Oligomers will preferably contain a
fragment of a polynucleotide
encoding TRICH, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
TRICH, and will be employed under optimized conditions for identification of a
specific gene or
condition. Oligomers may also be employed under less stringent conditions for
detection or
quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding TRICH may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are
substitutions, insertions and deletions that are a frequent cause of inherited
or acquired genetic disease
in humans. Methods of SNP detection include, but are not limited to, single-
stranded conformation
polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP,
oligonucleotide primers
derived from the polynucleotide sequences encoding TRICH are used to amplify
DNA using the
polymerase chain reaction (PCR). The DNA may be derived, for example, from
diseased or normal
tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause
differences in the
secondary and tertiary structures of PCR products in single-stranded form, and
these differences are
detectable using gel electrophoresis in non-denaturing gels. In fSCCP, the
oligonucleotide primers are
fluorescently labeled, which allows detection of the amplimers in high-
throughput equipment such as
DNA sequencing machines. Additionally, sequence database analysis methods,
termed in silico SNP
(isSNP), are capable of identifying polymorphisms by comparing the sequence of
individual
overlapping DNA fragments which assemble into a common consensus sequence.
These computer-
based methods filter out sequence variations due to laboratory preparation of
DNA and sequencing
errors using statistical models and automated analyses of DNA sequence
chromatograms. In the
alternative, SNPs may be detected and characterized by mass spectrometry
using, for example, the
high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
SNPs may be used to study the genetic basis of human disease. For example, at
least 16
common SNPs have been associated with non-insulin-dependent diabetes mellitus.
SNPs are also
useful for examining differences in disease outcomes in monogenic disorders,
such as cystic fibxosis,
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sickle cell anemia, or chronic granulomatous disease. For example, variants in
the mannose-binding
lectin, MBL2, have been shown to be correlated with deleterious pulmonary
outcomes in cystic
fibrosis. SNPs also have utility in pharmacogenomics, the identification of
genetic variants that
influence a patient's response to a drug, such as life-threatening toxicity.
For example, a variation in
N-acetyl txansferase is associated with a high incidence of peripheral
neuropathy in response to the
anti-tuberculosis drug isoniazid, while a variation in the coxe promoter of
the ALOXS gene results in
diminished clinical response to treatment with an anti-asthma drug that
targets the 5-lipoxygenase
pathway. Analysis of the distribution of SNPs in different populations is
useful for investigating
genetic drift, mutation, recombination, and selection, as well as for tracing
the origins of populations
and their migrations. (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512;
Kwok, P.-Y, and Z. Gu
(1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) C~rr. Opin.
Neurobiol. 11:637-641.)
Methods which may also be used to quantify the expression of TRICH include
radiolabeling or
biotinylatiug nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Tmrnunol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives rapid
quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described below. The microarray may also be
used to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, to monitor
progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used
to develop a pharmacogenomic profile of a patient in order to select the most
appropriate and effective
treatment regimen for that patient. For example, therapeutic agents which are
highly effective and
display the fewest side effects may be selected for a patient based on his/her
pharmacogenomic
profile.
In another embodiment, TRICH, fragments of TRICH, or antibodies specific for
TRICH may
be used as elements on a microarray. The microarray may be used to monitor or
measure protein-
protein interactions, drug-target interactions, and gene expression profiles,
as described above.
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A particular embodiment relates to the use of the polynucleotides of the
present invention to
generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern of
gene expression by a particular tissue or cell type. Global gene expression
patterns are analyzed by
quantifying the number of expressed genes and their relative abundance under
given conditions and at
a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent No.
5,840,484, expressly incorporated by reference herein.) Thus a transcript
image may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines, biopsies,
or other biological samples. The transcript image may thus reflect gene
expression in vivo, as in the
case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the
present invention
may also .be used in conjunction with in vitro model systems and preclinical
evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and toxicity
(Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L.
Anderson (2000)
Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein).
If a test compound has a
signature similar to that of a compound with known toxicity, it is likely to
share those toxic properties.
These fingerprints or signatures are most useful and refined when they contain
expression information
from a large number of genes and gene families. Ideally, a genome-wide
measurement of expression
provides the highest quality signature. Even genes whose expression is not
altered by any tested
compounds are important as well, as the levels of expression of these genes
are used to normalize the
rest of the expression data. The normalization procedure is useful for
comparison of expression data
after treatment with different compounds. While the assignment of gene
function to elements of a
toxicant signature aids in interpretation of toxicity mechanisms, knowledge of
gene function is not
necessary for the statistical matching of signatures which leads to prediction
of toxicity. (See, for
example, Press Release 00-02 from the National Institute of Environmental
Health Sciences, released
February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm.)
Therefore, it is
important and desirable in toxicological screening using toxicant signatures
to include all expressed
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gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological sample
containing nucleic acids with the test compound. Nucleic acids that are
expressed in the treated
biological sample are hybridized with one or more probes specific to the
polynucleotides of the present
invention, so that transcript levels corresponding to the polynucleotides of
the present invention may be
quantified. The transcript levels in the treated biological sample are
compared with levels in an
untreated biological sample. Differences in the transcript levels between the
two samples are
indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a proteome
can be subjected individually to further analysis. Proteome expression
patterns, or profiles, are
analyzed by quantifying the number of expressed proteins and their relative
abundance under given
conditions and at a given time. A profile of a cell's proteome may thus be
generated by separating
and analyzing the polypeptides of a particular tissue or cell type. In one
embodiment, the separation is
achieved using two-dimensional gel electrophoresis, in which proteins from a
sample are separated by
isoelectric focusing in the first dimension, and then according to molecular
weight by sodium dodecyl .
sulfate slab gel electrophoresis in the second dimension (Steiner and
Anderson, su ra). The proteins
are visualized in the gel as discrete and uniquely positioned spots, typically
by staining the gel with an
agent such as Coomassie Blue or silver or fluorescent stains. The optical
density of each protein spot
is generally proportional to the level of the protein in the sample. The
optical densities of equivalently
positioned protein spots from different samples, for example, from biological
samples either treated or
untreated with a test compound or therapeutic agent, are compared to identify
any changes in protein
spot density related to the treatment. The proteins in the spots are partially
sequenced using, for
example, standard methods employing chemical or enzymatic cleavage followed by
mass
spectrometry. The identity of the protein in a spot may be determined by
comparing its partial
sequence, preferably of at.least 5 contiguous amino acid residues, to the
polypeptide sequences of the
present invention. In some cases, further sequence data may be obtained for
definitive protein
identification.
A proteomic profile may also be generated using antibodies specific for TRICH
to quantify
the levels of TRICH expression. In one embodiment, the antibodies are used as
elements on a
microarray, and protein expression levels are quantified by exposing the
microarray to the sample and
detecting the levels of protein bound to each array element (Lueking, A. et
al. (1999) Anal. Biochem.
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270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection
may be performed by
a variety of methods known in the art, for example, by reacting the proteins
in the sample with a thiol-
or amino-reactive fluorescent compound and detecting the amount of
fluorescence bound at each
array element.
Toxicant signatures at the proteome level axe 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 pxohling may be more reliable and
informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
each protein is compared to the amount of the corresponding protein in an
untreated biological sample.
A difference in the amount of protein between the two samples is indicative of
a toxic response to the
test compound in the treated sample. Individual proteins are identified by
sequencing the amino acid
residues of the individual proteins and comparing these partial sequences to
the polypeptides of the
present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are incubated
with antibodies specific to the polypeptides of the present invention. The
amount of protein recognized
by the antibodies is quantified. The amount of protein in the treated
biological sample is compared
with the amount in an untreated biological sample. A difference in the amount
of protein between the
two samples is indicative of a toxic response to the test compound in the
treated sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad.
Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application
W095/251116; Shalom D. et
al. (1995) PCT application WO95/35505; Heller, R.A. et a1. (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
microarxays are well known and thoroughly described in DNA Microarrays: A
Practical Appxoach,
M. Schena, ed. (1999) Oxford University Press, London, hereby expressly
incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding TRICH
may be
CA 02447662 2003-11-18
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used to generate hybridization probes useful in mapping the naturally
occurring genomic sequence.
Either coding or noncoding sequences may be used, and in some instances,
noncoding sequences may
be preferable over coding sequences. For example, conservation of a coding
sequence among
members of a multi-gene family may potentially cause undesired cross
hybridization during
chromosomal mapping. The sequences may be mapped to a particular chromosome,
to a specific
region of a chromosome, or to artificial chromosome constructions, e.g., human
artificial chromosomes
(HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes
(BACs), bacterial P1
constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop
genetic linkage maps, for example, which correlate the inheritance of a
disease state with the
inheritance of a particular chromosome region or restriction fragment length
polymorphism (RFLP).
(See, for example, 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-UJrich, et al. (1995) in Meyers, s-upra, pp. 965-
968.) Examples of genetic
map data can be found in various scientific journals or at the Online
Mendelian Inheritance in Man
(OM1M) 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.
2o In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
may reveal associated markers even if the exact chromosomal locus is not
known. This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely
localized by genetic linkage to a particular genomic region, e.g., ataxia-
telangiectasia to 11q22-23, any
sequences mapping to that area may represent associated or regulatory genes
for further investigation.
(See, e.g., Gatti, R.A. et a1. (1988) Nature 336:577-580.) The nucleotide
sequence of the instant
invention may also be used to detect differences in the chromosomal location
due to translocation,
inversion, etc., among normal, carrier, or affected individuals.
In another embodiment of the invention, TRICH, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used for screening libraries of compounds in any
of a variety of drug
screening techniques. The fragment employed in such screening may be free in
solution, affixed to a
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solid support, borne on a cell surface, or located intracellularly. The
formation of binding complexes
between TRICH and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. (1984) PCT
application WO84/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with TRICH,
or fragments thereof,
and washed. Bound TRICH is then detected by methods well known in the art.
Purified TRICH can
also be coated directly onto plates for use in the aforementioned drug
screening techniques.
Alternatively, non-neutralizing antibodies can be used to capture the peptide
and immobilize it on a
solid support.
In another embodiment, one may use competitive drug screening assays in which
neutralizing
antibodies capable of binding TRICH specifically compete with a test compound
for binding TRICH.
Tn this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with TRICH.
In additional embodiments, the nucleotide sequences which encode TRICH may be
used in
any molecular biology techniques that have yet to be developed, provided the
new techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following embodiments are, therefore,
to be construed as merely illustrative, and not limitative of the remainder of
the disclosure in any way
whatsoever.
The disclosures of all patents, applications and publications, mentioned above
and below,
including U.S. Ser. No. 60/296,881, U.S. Ser. No. 60/305,105, U.S. Ser No.
60/293,722, and U.S. Ser
No. 60/304,593, are expressly incorporated by reference herein.
EXAMPLES
I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the LIFESBQ GOLD
database (Incyte Genomics, Palo Alto CA). Some tissues were homogenized and
lysed in guanidinium
isothiocyanate, while others were homogenized and lysed in phenol or in a
suitable mixture of
denaturants, such as TRIZOL (Invitrogen), a monophasic solution of phenol and
guanidine
isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or
extracted with
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chloroform. RNA was precipitated from the lysates with either isopropanol or
sodium acetate and
ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were~repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A)+ RNA was
isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX
latex particles
(QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
Alternatively,
RNA was isolated directly from tissue lysates using other RNA isolation kits,
e.g., the
POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
Iu some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
20 libraries. Otherwise, cDNA was synthesized and cDNA libraries were
constructed with the
UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Invitrogen),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, sera, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the°cDNA
was size-selected (300-
1000 bp) using SEPHACRYL 51000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Biosciences) or preparative agarose gel
electrophoresis. cDNAs were
ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCR1P'T plasmid (Stratagene), PSPORT1 plasmid (Invitrogen), PCDNA2.1
plasmid
(Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid
(Invitrogen),
PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE
(Incyte
Genomics), or pINCY (Incyte Genomics), or derivatives thereof. Recombinant
plasmids were
transformed into competent E. coli cells including XL1-Blue, XL1-BlueMRF, or
SOLR from
Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Invitrogen.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo
excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega); an
AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid,
QIA,WELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
R.E.A.L. PREP
96 plasmid purification kit from QIAGEN. Following precipitation, plasmids
were resuspended in 0.1
ml of distilled water and stored, with or without lyophilization, at
4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
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high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically using
PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence
scanner
(Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation such
as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200
thermal cyclex
(MJ Research) in conjunction with the HYDRA rnicrodispenser (Bobbins
Scientific) or the
MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Biosciences or supplied in ABI sequencing
kits such as the
ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied
Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carried out using the MEGABACE 1000 DNA sequencing system (Amersham
Biosciences);
the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction
with standard
ABI protocols and base calling software; or other sequence analysis systems
known in the art.
Reading frames within the cDNA sequences were identified using standard
methods (reviewed in
Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequences were selected for
extension using the
techniques disclosed in Example V1R.
The polynucleotide sequences derived from Incyte cDNAs were validated by
removing
vector, linker, and poly(A) sequences and by masking ambiguous bases, using
algorithms and
programs based on BLAST, dynamic programming, and dinucleotide nearest
neighbor analysis. 'The
Incyte cDNA sequences or translations thereof were then queried against a
selection of public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases, and
BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo
Sapiens, Rattus fiofvegicus, Mus musculus, Caenorhabditis elegans, Sacchaf-
omyces cer-evisiae,
Scl2iz~sacchar-omyces porvbe, and Carvdida albicaris (Incyte Genomics, Palo
Alto CA); hidden
Markov model (HMM)-based protein family databases such as PFAM,1NCY, and
TIGRFAM (Haft,
D.H. et al. (2001) Nucleic Acids Res. 29:41-43); and H1VIIVI-based protein
domain databases such as
SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864; Letunic,
I. et al. (2002)
Nucleic Acids Res. 30:242-244). (HMM is a probabilistic approach which
analyzes consensus
primary structures of gene families. See, for example, Eddy, S.R. (1996) C~rr.
Opin. Struct. Biol.
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6:361-365.) The queries were performed using programs based on BLAST, FASTA,
BLIZVVIPS, and
~R. The Incyte cDNA sequences were assembled to produce full length
polynucleotide
sequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences,
stretched
sequences, or Genscan-predicted coding sequences (see Examples IV and V) were
used to extend
Incyte cDNA assemblages to full length. Assembly was performed using programs
based on Phred,
Phrap, and Consed, and cDNA assemblages were screened for open reading frames
using programs
based on GeneMark, BLAST, and FASTA. The full length polynucleotide sequences
were translated
to derive the corresponding full length polypeptide sequences. Alternatively,
a polypeptide of the
invention may begin at any of the methionine residues of the full length
translated polypeptide. Full
20 length polypeptide sequences were subsequently analyzed by querying against
databases such as the
GenBank protein databases (genpept), SwissProt, the PROTEOME databases,
BLOCKS, PRINTS,
DOMO, PRODOM, Prosite, hidden Markov model (ITVIM)-based protein family
databases such as
PFAM, INCY, and TIGRFAM; and HNINI based protein domain databases such as
SMART. Full
length polynucleotide sequences are also analyzed using MACDNASIS PRO software
(Hitachi
Software Engineering, South San Francisco CA) and LASERGENE software
(DNASTAR).
Polynucleotide and polypeptide sequence alignments are generated using default
parameters specified ,
by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence
alignment program
(DNASTAR), which also calculates the percent identity between aligned
sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis
and assembly of
Incyte cDNA and full length sequences and provides applicable descriptions,
references, and threshold
parameters. The first column of Table 7 shows the tools, programs, and
algorithms used, the second
column provides brief descriptions thereof, the third column presents
appropriate references, all of
which are incorporated by reference herein in. their entirety, and the fourth
column presents, where
applicable, the scores, probability values, and other parameters used to
evaluate the strength of a
match between two sequences (the higher the score or the lower the probability
value, the greater the
identity between two sequences).
The programs described above for the assembly and analysis of full length
polynucleotide and
polypeptide sequences wexe also used to identify polynucleotide sequence
fragments from SEQ ID
N0:10-18. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
amplification technologies are described in Table 4, column 2.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative transporters and ion channels were initially identified by running
the Genscan gene
identification program against public genomic sequence databases (e.g., gbpri
and gbhtg). Genscan is
~0
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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) C~rr. Opin. Struct. Biol. 8:346-354). The program
concatenates predicted exons to
form an assembled cDNA sequence extending from a metllionine 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
aualyzed by quexying against PFAM models for trausporters and ion channels.
Potential transporters
and ion channels were also identified by homology to Incyte cDNA sequences
that had been
annotated as transporters and ion channels. These selected Genscan-predicted
sequences were then
compared by BLAST analysis to the genpept and gbpri public databases. Where
necessary, the
Genscan-predicted sequences were then edited by comparison to the top BLAST
hit from genpept to
correct errors in the sequence predicted by Genscan, such as extra or omitted
exons. BLAST
analysis was also used to find any Iucyte cDNA or public cDNA coverage of the
Genscan-predicted
sequences, thus providing evidence for transcription. When Incyte cDNA
coverage was available,
this information was used to correct or confirm the Genscan predicted
sequence. Full length
polynucleotide sequences were obtained by assembling Genscan-predicted coding
sequences with
Incyte cDNA sequences andlor public cDNA sequences using the assembly process
described in
Example III. Alternatively, full length polynucleotide sequences were derived
entirely from edited or
unedited Genscan-predicted coding sequences.
Y. Assembly of Genomic Sequence Data with cDNA Sequence Data
"Stitched" Sequences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification program described in Example IV. Partial cDNAs assembled as
described in Example
III were mapped to genomic DNA and parsed into clusters containing related
cDNAs and Genscan
exon predictions from one or more genomic sequences. Each cluster was analyzed
using an algorithm
based on graph theory and dynamic programming to integrate cDNA and genomic
information,
generating possible splice variants that were subsequently confirmed, edited,
or extended to create a
full length sequence. Sequence intervals in which the entire length of the
interval was present on
more than one sequence in the cluster were identified, and intervals thus
identified wexe 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
81
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thus identified were then "stitched" together by the stitching algorithm in
the order that they appear
along their parent sequences to generate the longest possible sequence, as
well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence
(cDNA to cDNA or
genomic sequence to genomic sequence) were given preference over linkages
which change parent
type (cDNA to genomic sequence). The resultant stitched sequences were
translated and compared
by BLAST analysis to the genpept and gbpri public databases. Incorrect exons
predicted by Genscan
were corrected by comparison to the top BLAST hit from genpept. Sequences were
further extended
with additional cDNA sequences, or by inspection of genomic DNA, when
necessary.
"Stretched" Sequences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example III were
queried against public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases
using the BLAST program. The nearest GenBank protein homolog was then compared
by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences
described in
Example IV. A chimeric protein was generated by using the resultant high-
scoring segment pairs
(HSPs) to map the translated sequences onto the GenBank protein homolog.
Insertions or deletions
may occur in the chimeric protein with respect to the original GenBank protein
homolog. The .
GenBank protein homolog, the chimexic protein, or both were used as probes to
search for homologous
genomic sequences from the public human genome databases. Partial DNA
sequences were
therefore "stretched" or extended by the addition of homologous genomic
sequences. The resultant
stretched sequences were examined to determine whether it contained a complete
gene.
VI. Chromosomal Mapping of TRICH Encoding Polynucleotides
The sequences which were used to assemble SEQ ID N0:10-18 were compared with
sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ ID NO:10-18 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 m 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-
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arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
chromosomal markers. On average, 1 cM is roughly equivalent to I megabase (Mb)
of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM distances
are based on genetic markers mapped by Genethon which provide boundaries for
radiation hybrid
markers whose sequences were included in each of the clusters. Human genome
maps and other
resources available to the public, such as the NCBI "GeneMap'99" World Wide
Web site
(http://www.ncbi.nlm.nih.gov/genemap~, can be employed to determine if
previously identified disease
genes map within. or in proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
from a particular cell type or tissue have been bound. (See, e.g., Sambrook,
supra, ch. 7; Ausubel
(1995) sera, 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 L1FESEQ (Iucyte 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 pxoduct score of 100 is produced only for I00%
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 fall length
sequences are
assembled, at least in part, with overlapping Incyte cDNA sequences (see
Example llI). 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;
unclassi~ed/mixed; or
urinary tract. The number of libraries in each category is counted and divided
by the total number of
libraries across all categories. Similarly, each human tissue is classified
into one of the following
disease/condition categories: cancer, cell line, developmental, inflammation,
neurological, trauma,
cardiovascular, pooled, and other, and the number of libraries in each
category is counted and divided
by the total number of libraries acxoss all categories. The resulting
percentages reflect the tissue- and
disease-specific expression of cDNA encoding TRICH.
VIII. Extension of TRICH Encoding Polynucleotides
Full length polynucleotide sequences were also produced by extension of an
appropriate
fragment of the full length molecule using oligonucleotide primers designed
from this fragment. One
primer was synthesized to initiate 5' extension of the known fragment, and the
other primer was
synthesized to initiate 3' extension of the known fragment. The initial
primers were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68°C to about 72°C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mgz+, (NH4)ZSO4,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE
enzyme
(Invitrogen), and Pfu DNA polymerase (Stratagene), with the following
parameters for primer pair
PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step
3: 60°C, 1 min; Step 4: 68°C, 2
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??m?n; 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 ~tl
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 ~tl of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ~1 to 10 ,u1 aliquot of the xeaction 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 religation into pUC 18 vector (Amersham
Biosciences). 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
Biosciences), treated with Pfu DNA polymerase (Stxatagene) to fill-in
restriction site overhangs, and
transfected into competent E. coli cells. Transformed cells were selected on
antibiotic-containing
media, and individual colonies were picked and cultured overnight at 37
°C in 384-well plates in LB/2x
carb liquid media.
The cells were lysed, and DNA was amplibed by PCR using Taq DNA polymerase
(Amersham Biosciences) and Pfu DNA polymerase (Stratagene) with the following
parameters: Step
1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1
min; Step 4: 72°C, 2 min; Step 5: steps 2, 3, and
4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at
4°C. DNA was quantified by
PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA
recoveries
were reamplified using the same conditions as described above. Samples were
diluted with 20%
dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer
sequencing primers
and the DYENAMIC DIRECT kit (Amersham Biosciences) or the ABI PRISM BIGDYE
Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the
above procedure or
are used to obtain 5' regulatory sequences using the above procedure along
with oligonucleotides
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designed for such extension, and an appropriate genomic library.
IX. Identification of Single Nucleotide Polymorphisms in TRICH Encoding
PoIynucleotides
Common DNA sequence variants known as single nucleotide polymorphisms (SNPs)
were
identified in SEQ ID N0:10-18 using the LIFESEQ database (Iucyte Genomics).
Sequences from the
same gene were clustered together and assembled as described in Example III,
allowing the
identification of all sequence variants in the gene. An algorithm consisting
of a series of filters was
used to distinguish SNPs from other sequence variants. Preliminary filters
removed the majority of
basecall errors by requiring a minimum Phred quality score of 15, and removed
sequence alignment
errors and errors resulting from improper trimming of vector sequences,
chimeras, and splice variants.
An automated procedure of advanced chromosome analysis analysed the original
chromatogram files
in the vicinity of the putative SNP. Clone error filters used statistically
generated algorithms to identify
errors introduced during laboratory processing, such as those caused by
reverse transcriptase,
polymerase, or somatic mutation. Clustering error filters used statistically
generated algorithms to
identify errors resulting from clustering of close homologs or pseudogenes, or
due to contamination by
non human sequences. A final set of filters removed duplicates and SNPs found
in immunoglobulins
or T-cell receptors.
Certain SNPs were selected for further characterization by mass spectrometry
using the high
throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at
the SNP sites in
four different human populations. The Caucasian population comprised 92
individuals (46 male, 46
female), including 83 from Utah, four French, three Venezualan, and two Amish
individuals. The
African population comprised 194 individuals (97 male, 97 female), all African
Americans. The
I3ispanic population comprised 324 individuals (162 male, 162 female), all
Mexican Hispanic. The
Asian population comprised 126 individuals (64 male, 62 female) with a
reported parental breakdown
of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian.
Allele
frequencies were first analyzed in the Caucasian population; in some cases
those SNPs which showed
no allelic variance in this population were not further tested in the other
three populations.
X. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:10-18 are employed to screen
cDNAs,
genomic DNAs, ox 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
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[Y 32P] adenosine- triphosphate (Amersham Biosciences), and T4 polynucleotide
kinase (DuPont NEN,
Boston MA). The labeled oligonucleotides are substantially purified using a
SEPHADEX G-25
superfine size exclusion dextran bead column (Amersham Biosciences). An
aliquot containing 10'
counts per minute of the labeled probe is used in a typical membrane-based
hybridization analysis of
human genomic DNA digested with one of the following endonucleases: Ase I,
BgllI, Eco RI, Pst I,
Xba I, or Pvu lI (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40 °C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%a
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
XI. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, supra.), mechanical
microspotting technologies, and derivatives thereof. The substrate in each of
the aforementioned
technologies should be uniform and solid with a non-porous surface (Schena
(1999), so ra).
Suggested substrates include silicon, silica, glass slides, glass clops, and
silicon wafers. Alternatively, a
procedure analogous to a dot or slot blot may also be used to arrange and link
elements to the surface
of a substrate using thermal, W, chemical, or mechanical bonding procedures. A
typical array may
be produced using available methods and machines well known to those of
ordinary skill in the art and
may contain any appropriate number of elements. (See, e.g., Schena, M. et al.
(1995) Science
270:467-470; 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 canbe
selected using software well known in the art such as LASERGENE software
(DNASTAR). The
array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element on
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the microarray may be assessed. Iu one embodiment, microarray preparation and
usage is described
in detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/pl oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 units/~.1 RNase inhibitor, 500 ~.M dATP, 500 p.M dGTP, 500
~.M dTTP, 40 ~.M
dCTP, 40 ACM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences). The reverse
transcription
reaction is performed in a 25 ml volume containing 200 ng poly(A)+ RNA with
GEMBR.IGHT 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 ~15X SSC/0.2% SDS.
Microarra~paration
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 ~tg.
Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Biosciences).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water, and
coated with 0.05% amiuopropyl silane (Sigma) in 95% ethanol. Coated slides are
cured in a 110°C
oven.
Array elements are applied to the coated glass substrate using a procedure
described in U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 ~,l of the array
element DNA, at an average
concentration of 100 ng/pl, is loaded into the open capillary printing element
by a high-speed robotic
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apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are W-crosslinked using a STRATALINI~R W-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 ~tg
each of Cy3 and
Cy5 labeled cDNA synthesis products in SX SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65° C for 5 minutes and is aliquoted onto the
microarray surface and covered with
an 1.8 cmz coverslip. The arrays are transferred to a waterproof chamber
having a cavity just slightly
larger than a microscope slide. The chamber is kept at 100% humidity
internally by the addition of 140
~,1 of SX SSC in a corner of the chamber. The chamber containing the arrays is
incubated for about
6.5 hours at 60° C. The arrays are washed for 10 min at 45° C in
a first wash buffer (1X SSC, 0.1%
SDS), three times for 10 minutes each at 45° C in a second wash buffer
(0.1X SSC), and dried.
Detection
Reportex-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 82477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that location
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to be correlated with a weight ratio of hybridizing species of 1:100,000. When
two samples from
different sources (e.g., representing test and control cells), each labeled
with a different fluorophore,
are hybridized to a single array for the purpose of identifying genes that are
differentially expressed,
the calibration is done by labeling samples of the calibrating cDNA with the
two fluorophores and
adding identical amounts of each to the hybridization mixture.
The output of the photomultiplier. tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping emission
spectra) between the fluorophores using each fluorophore's emission spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each spot
is centered in each element of the grid. The fluorescence signal within each
element is then integrated
to obtain a numerical value corresponding to the average intensity of the
signal. The software used
for signal analysis is the GEMTOOLS gene expression analysis program (Iucyte).
Array elements
that exhibited at least about a two-fold change in expression, a signal-to-
background ratio of at least
2.5, and an element spot size of at least 40°~o were identified
as~differentially expressed using the
GEMTOOLS program (Incyte Genomics).
2o Expression
SEQ ID NO:10 showed differential expression in association with Jurkat cell
lines treated with
PMA and ionomycin as compared to untreated Jurkat cell lines, as determined by
microarray analysis.
The expression of SEQ ID N0:10 was decreased by at least two fold in Jurkat
cells treated with at
least 100 nM PMA and at least 1 microgram/ml ionomycin for 1 hour, as compared
to controls.
Therefore, in. an embodiment, SEQ ID NO:10 can be used in diagnostic assays
for and/or monitoring
treatment of immune response disorders.
XII. Complementary Polynucleotides
Sequences complementary to the TRICH-encoding sequences, or any parts thereof,
are used
to detect, decrease, or inhibit expression of naturally occurring TRICH.
Although use of
oligonucleotides comprising from about 15 to 30 base pairs is described,
essentially the same
procedure is used with smaller or with larger sequence fragments. Appropriate
oligonucleotides are
designed using OLIGO 4.06 software (National Biosciences) and the coding
sequence of TRICH. To
inhibit transcription, a complementary oligonucleotide is designed from the
most unique 5' sequence
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and used to prevent promoter binding to the coding sequence. To inhibit
translation, a complementary
oligonucleotide is designed to prevent ribosomal binding to the TRICH-encoding
transcript.
XIII. Expression of TRICH
Expression and purification of TRICH is achieved using bacterial or virus
based expression
systems. For expression of TRICH in bacteria, cDNA is subcloned into an
appropriate vector
containing an antibiotic resistance gene and an inducible promoter that
directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the
trp-lac (tac) hybrid
promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21(DE3).
Antibiotic resistant bacteria express TRICH upon induction with isopropyl beta-
D-
thiogalactopyranoside (IPTG). Expression of TRICH in eukaryotic cells is
achieved by infecting
insect or mammalian cell lines with recombinant Auto~raphica californica
nuclear polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding TRICH by either homologous recombination or
bacterial-mediated
transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect Spotlo~tera 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 Biosciences).
Following purification, the GST moiety can be proteolytically cleaved from
TRICH at specifically
engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffmity
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 (QLAGEN).
Methods for protein expression and purification are discussed in Ausubel
(1995, su ra, eh. 10 and 16).
Purified TRICH obtained by these methods can be used directly in the assays
shown in Examples
XVII, XVIII, and XIK where applicable.
XIV. Functional Assays
9I
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
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 plasmid (Invitrogen, Carlsbad CA) and PCR3.1 plasmid
(Invitrogen), both of
which contain the cytomegalovirus promoter. 5-10 ,ug of recombinant vector are
transiently
transfected into a human cell line, for example, an endothelial or
hematopoietic cell line, using either
liposome formulations or electroporation. 1-2 /,cg of an additional plasmid
containing sequences
encoding a marker protein are co-transfected. Expression of a marker protein
provides a means to
distinguish transfected cells from nontrausfected 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 trausfected cells expressing GFP
or CD64-GFP and to
evaluate the apoptotic state of the cells and other cellular properties. FCM
detects and quantifies the
uptake of fluorescent molecules that diagnose events preceding or coincident
with cell death. These
events include changes in nuclear DNA content as measured by staining of DNA
with propidium
iodide; changes in cell size and granularity as measured by forward light
scatter and 90 degree side
light scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine
uptake; alterations in expression of cell surface and intracellular proteins
as measured by reactivity
with specific antibodies; and alterations in plasma membrane composition as
measured by the binding
of fluorescein-conjugated Annexin V protein to the cell surface. Methods in
flow cytometxy 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
immunoglobuliu G (IgG). Transfected cells are efficiently separated from
nontransfected cells using
magnetic beads coated with either human IgG or antibody against CD64 (DYNAL,
Lake Success
NY). mRNA can be purified from the cells using methods well known by those of
skill in the art.
Expression of mRNA encoding TRICH and other genes of interest can be analyzed
by northern
analysis or microarray techniques.
3o XV. Production of TRICH Specific Antibodies
TRICH substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used to
immunize animals (e.g., rabbits, mice, etc.) and to produce antibodies using
standard protocols.
92
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
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, su ra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
KLH (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) to
increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are
immunized with the
oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are
tested for
antipeptide and anti-TRICH activity by, for example, binding the peptide or
TRICH to a substrate,
blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting
with radio-iodinated goat
anti-rabbit IgG.
XVI. Purification of Naturally Occurring TRICH Using Specific Antibodies
Naturally occurring or recombinant TRICH is substantially purified by
i_m_m__unoafhnity
chromatography using antibodies specific for TRICH. An immunoaffinity column
is constructed by
covalently coupling anti-TRICH antibody to an activated chromatographic resin,
such as
CNBr-activated SEPHAROSE (Amersham Biosciences). After the coupling, the resin
is blocked and
washed according to the manufacturer's instructions.
Media containing TRICH are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of TRICH (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/TRICH binding (e.g., a buffer of pH 2 to pH 3, or a high
concentration of a chaotrope, such
as urea or thiocyanate ion), and TRICH is collected.
XVII. Identification of Molecules Which Interact with TRICH
Molecules that interact with TRICH may include transporter substrates,
agonists or
antagonists, modulatory proteins such as G(3~y proteins (Reimann, supra) or
proteins involved in TRICH
localization or clustering such as MAGUKs (Craven, supra). TRICH, or
biologically active fragments
thereof, are labeled with luI 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.
93
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
Alternatively, molecules interacting with TRICH are analyzed using the yeast
two-hybrid
system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or
using commercially
available kits based on the two-hybrid system, such as the MATCHMAKER system
(Clontech).
TRICH, or fragments thexeof, are expressed as fusion proteins with the DNA
binding domain of Gal4
or lexA, and potential interacting proteins are expressed as fusion proteins
with an activation domain.
Interactions between the TRICH fusion protein and the TRICH interacting
proteins (fusion proteins
with an activation domain) reconstitute a transactivation function that is
observed by expression of a
reporter gene. Yeast 2-hybrid systems are commercially available, and methods
fox use of the yeast
2-hybrid system with ion channel proteins are discussed in Niethammer, M. and
M. Sheng (1998,
Methods Enzymol. 293:104-122).
TRICH may also be used in the PATHCALLING process (C~traGen Corp., New Haven
CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K,
et al. (2000) U.S.
Patent No. 6,057,101).
Potential TRICH agonists or antagonists may be tested for activation or
inhibition of TRICH
ion channel activity using the assays described in section XVIIZ.
XVIII. Demonstration of TRICH Activity
Ion channel activity of TRIGH 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
trausformation under conditions appropriate for the cell line to allow
expression and accumulation of
TRICH and 13-galactosidase.
Transformed cells expressing 13-galactosidase are stained blue when a suitable
colorimetric
substrate is added to the culture media under conditions that are well known
i_n 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
!3-galactosidase sequences alone, are used as controls and tested in parallel.
Cells expressing TR1CH
will have higher 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
94
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
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 Iaevis 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-S ng of
mRNA is injected into
mature stage IV oocytes. Injected oocytes are incubated at 18 °C for 1-
5 days. Inside-out
macropatches are excised into an intracellular solution containing 116 mM K-
gluconate, 4 mM 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 CaClz), where
appropriate.
Electrode resistance is set at 2-S MSZ and electrodes are filled with the
intracellular solution lacking
mediator. Experiments are performed at room temperature from a holding
potential of 0 mV. Voltage
ramps (2.5 s) from -100 to 100 mV are acquired at a sampling frequency of 500
Hz. Current
measured is proportional to the activity of TRICH in the assay.
For example, the activity of TRICH-3 is measured as proton conductance and the
activity of
TRICH-4 is measured as calcium conductance.
Transport activity of TRICH is assayed by measuring uptake of labeled
substrates into
Xenopus laevis oocytes. Oocytes at stages V and VI are injected with TRICH
mRNA (10 ng per
oocyte) and incubated for 3 days at 18°C in OR2 medium (82.5mM NaCl,
2.5 mM KCI, 1mM CaCl2,
1mM MgCla, 1mM NaaHP04, 5 mM Hepes, 3.8 mM NaOH , 50~.g/ml gentamycin, pH 7.8)
to allow
expression of TRICH. Oocytes are then transferred to standard uptake medium
(100mM NaCl, 2
mM KCl, 1mM CaCl2, 1mM MgCl2, 10 mM Hepes/Tris pH 7.5). Uptake of various
substrates (e.g.,
amizto 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. Test substrates include, but are not limited
to, melibiose or other
carbohydrates for TRICH-1, uxea for TRICH-5, and sulphate for TRICH-6.
ATPase activity associated with TRICH can be measured by hydrolysis of
radiolabeled ATP-
[y-32P], separation of the hydrolysis products by chromatographic methods, and
quantitation of the
recovered 32P using a scintillation counter. The reaction mixture contains ATP-
['y-32P] and varying
amounts of TRICH in a suitable buffer incubated at 37 °C fox a suitable
period of time. The reaction
is terminated by acid precipitation with trichloroacetic acid and then
neutralized with base, and an
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
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 propoxtional to the ATPase activity of
TRICH in the assay.
Alternatively, iron uptake activity of TRTCH is assayed in 100 mM HEPES/NaOH
buffer (pH
7.0) with a Fe2+fI'RICH molar ratio of 1000:1 at room temperature. Iron
incorporation is monitored by
measuring the absorbance at 310 mn using a W spectrophotometer (Masuda, T. et
al. (2001) J. Biol.
Chem. 276:19575-19579).
XIX. Identification of TRICH Agonists and Antagonists
TRICH is expressed in a eukaryotic cell line such as CHO (Chinese Hamster
Ovary) or HEK
(Human Embryonic Kidney) 293. Ion channel activity of the transformed cells is
measured in the
presence and absence of candidate agonists or antagonists. Ion channel
activity is assayed using
patch clamp methods well known. in the art or as described in Example XVII.
Alternatively, ion
chancel activity is assayed using fluorescent techniques that measure ion flux
across the cell
membrane (Velicelebi, G. et al. (1999) Meth. Enzymol. 294:20-47; West, M.R.
and C.R. Molloy
(1996) Anal. Biochem. 241:51-58). These assays may be adapted for high-
throughput screening using
microplates. Changes in internal ion concentration are measured using
fluorescent dyes such as the
Ca2+ indicator Fluo-4 AM (available from Molecular Probes) in combination with
the FLIPR
fluorimetric plate reading system (Molecular Devices). In a more generic
version of this assay,
changes in membrane potential caused by ionic flux across the plasma membrane
are measured using
oxonyl dyes such as DiBAC4 (Molecular Probes). DiBAC4 equilibrates between the
extracellular
solution and cellular sites according to the cellular membrane potential. The
dye's fluorescence
intensity is 20-fold greater when bound to hydrophobic intracellular sites,
allowing detection of
DiBAC4 entry into the cell (Gonzalez, J.E. and P.A. Negulescu (1998) Curr.
Opin. Biotechnol. 9:624-
631). Candidate agonists or antagonists may be selected from known ion channel
agonists or
antagonists, peptide libraries, or combinatorial chemical libraries.
Various modifications and variations of the described methods and systems of
the invention
will be apparent to those skilled in. the art without departing from the scope
and spirit of the invention.
Although the invention has been described in connection with certain
embodiments, it should be
understood that the invention as claimed should not be unduly limited to such
specific embodiments.
Indeed, various modifications of the described modes 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.
96
CA 02447662 2003-11-18
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N ~,> U~yN G N ~ ~~ ~" ~ OU1 ~ ~M
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o°°, ~ ~ o°n c\°n
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~ M '4j .n-a ~ ~''~ ''y ~ G4 O~ ~ N ~ N a~ a.~ N
V N U ~' ~' co ~ ,~ ~ s. ~ v O ~ N ~ N
'C ,_,
~ 0 0 0 ~ ~ '~ °,~~° ~~', ~ ~ b ~ GA x ~j ~ W °~
~ '~W oo ~ a ~ ~ o ~ ~ ~ o ~ o
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r--i ~ ° M o, o Z ° ',~ ,~ ~ ov 3 ~ ~ ~ p~''" '~ ~ ~ o ri ~ ~ ~.
c~ ov
x~N~~ ~Me~~w~ w~x~~~~~ c~Nz°.Ua~N~
N U ~ r3 N ~ bA rUn U 'O
S"" ~ ,.~~~ N ~ :a~.~ cYd
~ C/~ U U ~ '~ :C ~~ ~ ~ N 'Cy ~ ° y '~"'
O" ~ N N U 4j
ii r5'a ~ N O ~ ~ ~ ~ U ~ .~ ~ U a.~
o U ,~ a, ~ a~ ~ o ~. a~ ~ Z ~ w ~ ~ A,
~b ° ~ °° ~ ~ ~ 3 0.~ o ~ ~ ~ on ~
i . a ~ a~ a ø, o ~ ~ o o. ~. .c o
~ c'~'. ,~ R'" ~ p' N '' y ~n ~ .i~ ~ ~ ~y~" i o w W'-'
27 0 ~ w >~
o ~ O ,~ .C vW -~~ ~ ~ '~C ~ U c~ ~ ~ N ~ a:.
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°on ~ ~ ~ °on ~ ~ a~'~ ~ o ~ H ~ ~ ~ p., -fl ~ ,o ø' bn
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~ rn a~ ~ .~ d ~ ~ a. v~ d .l~ ~
U
N c~ v~ U
W P, P., .n'~, U ~ E-~i
210
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
b
0
Ei
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U
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111
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
<110> INCYTE GENOMICS, INC.
RAUMANN, Brigette E.
Griffin, Jennifer A.
HAFALIA, April J.A.
BATRA, Sajeev
YAO, Monique G.
FORSYTHE, Ian J,
RAMKUMAR, Jayalaxmi
DUGGAN, Brendan M.
BAUGHN, Mariah R.
AZTMZAI, Yalda
WARREN, Bridget A.
LAL, PREETI G.
GIETZEN, Kimberly J.
WALIA, Narinder K.
BECHA, Shanya D.
TANG, Y. Tom
YUE, Henry
CHIN, Anna M.
<120> TRANSPORTERS AND ION CHANNELS
<130> PF-0980 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/293,722; 60/296,881; 60/304,593; 60/305,105
<151> 2001-05-25; 2001-06-08; 2001-07-10; 2001-07-12
<160> 18
<170> PERL Program
<210> 1
<211> 473
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1561248CD1
<400> 1
Met Gly Pro Gly Pro Pro Ala Ala 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
1/19
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
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
125 130 135
Val Ile Phe Gln Phe Gly Trp Ala Ser Thr Gln Ile Ser His Leu
140 145 150
Ser Leu Ile Pro Glu Leu Val Thr Asn Asp His Glu Lys Val Glu
155 160 165
Leu Thr Ala Leu Arg Tyr Ala Phe Thr Val Val Ala Asn Ile Thr
170 275 180
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 Ala Gln Pro Leu Leu Leu Trp Lys His
260 265 270
Trp Leu Arg Glu Pro Ala 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
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 Ala Val
410 415 420
Met Ala Tle Gln Ser Leu His Pro Cys Pro Ser Glu Leu Cys Cys
425 430 435
Arg Ala Cys Val Ser Phe Tyr His Trp Ala Met Val Ala Val Thr
440 445 450
Gly GIy Val GIy Val Ala Ala Ala Leu Cys Leu Cys Ser Leu Leu
455 460 465
Leu Trp Pro Thr Arg Leu Arg Arg
470
<210> 2
<211> 201
<212> PRT
<213> Homo Sapiens
2/19
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
<220>
<221> misc_feature
<223> Incyte ID No: 4539525CD1
<400> 2
Met Gln Ala Gly Asp Arg Leu Val Ala Val Ala Gly Glu Ser Val
1 5 10 15
Glu Gly Leu Gly His Glu Glu Thr Val Ser Arg Ile Gln Gly Gln
20 25 30
Gly Ser Cys Val Ser Leu Thr Val Val Asp Pro Glu Ala Asp Arg
35 40 45
Phe Phe Ser Met Val Arg Leu Ser Pro Leu Leu Phe Leu GIu Asn
50 55 60
Thr Glu Ala Pra AIa Ser Pro Gln Gly Ser Ser Ser Ala Ser Leu
65 70 75
Val Glu Thr Glu Asp Pro Ser Leu Glu Asp Thr Ser Val Pro Ser
80 85 90
Val Pro Leu Gly Ser Arg Gln Cys Phe Leu Tyr Pro Gly Pxo Gly
95 100 105
Gly Ser Tyr Gly Phe Arg Leu Ser Cys Val Ala Ser Gly Pro Arg
110 115 120
Leu Phe Ile Ser Gln Val Thr Pro Gly Gly Ser Ala Ala Arg Ala
125 130 135
Gly Leu Gln Val Gly Asp Val Ile Leu Glu Val Asn Gly Tyr Pro
140 145 150
Val Gly Gly Gln Asn Asp Leu Glu Arg Leu Gln Gli1_Leu Pro Glu
155 160 165
Ala Glu Pro Pro Leu Cys Leu Lys Leu Ala Ala Arg~Ser Leu Arg
170 175 1 ~ 180
Gly Leu Glu Ala Trp Ile Pro Pro Gly Ala Ala Glu Asp Trp Ala
185 190 ~' 195
Leu Ala Ser Asp Leu Leu
200
<210> 3
<211> 237
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 72210802CD1
<400> 3
Met Asn Pro Ala Asp Val AIa GIn Ser Thr Leu Pro Leu Ala Ser
1 5 10 15
Ser Asp Val Ser Leu Ile Ala Leu Phe Trp Gln Ala His Trp VaI
20 25 30
Val Lys Cys Val Met Leu Gly Leu Leu Ser Cys Ser Val Trp Val
35 40 45
Trp Ala Ile Ala Ile Asp Lys Ile Leu Leu Tyr Ala Arg Thr Lys
50 55 60
Arg Ala Met Asp Lys Phe Glu Gln Ala Phe Trp Ser Gly Gln Ser
65 70 75
Ile Glu Glu Leu Tyr Arg Ala Leu Ser Ala Lys Pro Thr Gln Ser
80 g5 90
3l19
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
Met Ala Ala Cys Phe Val Ala Ala Met Arg Glu Trp Lys Arg Ser
95 ~ 100 105
Phe Glu Ser Gln Ser Arg Ser Phe Ala Gly Leu Gln Ala Arg Ile
110 115 120
Asp Lys Val Met Asn Val Ser Ile Ala Arg Glu Val G1u Arg Leu
125 130 135
Glu Arg Arg Leu Leu Val Leu Ala Thr Val Gly Ser Ala Gly Pro
140 145 150
Phe Val Gly Leu Phe Gly Thr Val Trp Gly Tle Met Ser Ser Phe
155 160 165
Gln Ser IIe Ala Ala Ser Lys Asn Thr Ser Leu Ala Val VaI Ala
170 175 180
Pro Gly Ile Ala Glu Ala Leu Phe Ala Thr Ala Ile Gly Leu Ile
185 190 195
Ala Ala Ile Pro Ala Thr Ile Phe Tyr Asn Lys Phe Thr Ser Glu
200 205 210
Val Asn Arg Gln Ala Ala Arg Leu Glu Gly Phe Ala Asp Glu Phe
215 220 225
Ser Ala Ile Leu Ser Arg Gln IIe Asp GIu Arg Gly
230 235
<210> 4
<211> 947
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte ID No: 2469624CD1
<400> 4
Met Glu Glu Met Phe His Lys Lys Ser Glu Ala Val Arg Arg Leu
1 5 10 15
Val Glu Ala Ala Glu Glu Ala His Leu Lys His Glu Phe Asp Ala
20 25 30
Asp Leu Gln Tyr Glu Tyr Phe Asn Ala Val Leu Ile Asn Glu Arg
35 40 45
Asp Lys Asp Gly Asn Phe Leu Glu Leu Gly Lys Glu Phe Ile Leu
50 55 60
Ala Pro Asn Asp His Phe Asn Asn Leu Pro Val Asn Ile Ser Leu
65 70 75
Ser Asp Val Gln Val Pro Thr Asn Met Tyr Asn Lys Gly Ile Lys
80 85 90
Trp Glu Pro Asp Glu Asn Gly VaI Ile AIa Phe Asp Cys Arg Asn
95 100 105
Arg Lys Trp Tyr Ile Gln Ala Ala Thr Ser Pro Lys;Asp Val Val
110 115 120
Ile Leu Val Asp Val Ser Gly Ser Met Lys Gly Leu Arg Leu Thr
125 130 135
Ile Ala Lys Gln Thr Val Ser Ser Ile Leu Asp Thr Leu Gly Asp
140 145 150
Asp Asp Phe Phe Asn Ile Ile Ala Tyr Asn Glu Glu Leu His Tyr
255 160 165
Val Glu Pro Cys Leu Asn Gly Thr Leu Val Gln Ala Asp Arg Thr
170 175 180
Asn Lys Glu His Phe Arg Glu His Leu Asp Lys Leu Phe Ala Lys
4/19
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
185 190 195
Gly Ile Gly Met Leu Asp Ile Ala Leu Asn Glu Ala Phe Asn Ile
200 205 210
Leu Ser Asp Phe Asn His Thr Gly Gln Gly Ser Ile Cys Ser Gln
215 220 225
Ala Ile Met Leu Ile Thr Asp Gly Ala Val Asp Thr Tyr Asp Thr
230 235 240
Ile Phe Ala Lys Tyr Asn Trp Pro Asp Arg Lys Val Arg Ile Phe
245 250 255
Thr Tyr Leu Ile Gly Arg Glu Ala Ala Phe Ala Asp Asn Leu Lys
260 265 270
Trp Met Ala Cys Ala Asn Lys Gly Phe Phe Thr Gln Ile Ser Thr
275 280 285
Leu Ala Asp Val Gln Glu Asn Val Met Glu Tyr Leu His Val Leu
290 295 300
Ser Arg Pro Lys Val Ile Asp Gln GIu His Asp Val Val Trp Thr
305 310 315
Glu Ala Tyr Ile Asp Ser Thr Leu Thr Asp Asp Gln Gly Pro Val
320 325 330
Leu Met Thr Thr Val Ala Met Pro Val Phe Ser Lys Gln Asn Glu
335 340 345
Thr Arg Ser Lys Gly Ile Leu Leu Gly Val Val Gly Thr Asp Val
350 355 360
Pro Val Lys Glu Leu Leu Lys Thr Ile Pro Lys Tyr Lys Leu Gly
365 370 375
Ile His Gly Tyr Ala Phe Ala Ile Thr Asn Asn Gly Tyr Ile Leu
380 385 390
Thr His Pro Glu Leu Arg Leu Leu Tyr Glu Glu Gly Lys Lys Arg
395 400 405
Arg Lys Pro Asn Tyr Ser Ser Val Asp Leu Ser Glu Val Glu Trp
410 425 420
Glu Asp Arg Asp Asp VaI Leu Arg Asn Ala Met VaI Asn Arg Lys
425 430 435
Thr Gly Lys Phe Ser Met Glu Val Lys Lys Thr Val Asp Lys Gly
440 445 450
Lys Arg Val Leu Val Met Thr Asn Asp Tyr Tyr Tyr Thr Asp Ile
455 460 465
Lys Gly Thr Pro Phe Ser Leu Gly Val Ala Leu Ser Arg Gly His
470 475 480
GIy Lys Tyr Phe Phe Arg GIy Asn Val Thr IIe Glu Glu Gly Leu
485 490 495
His Asp Leu Glu His Pro Asp Val Ser Leu Ala Asp Glu Trp Ser
500 505 510
Tyr Cys Asn Thr Asp Leu His Pro GIu His Arg His Leu Ser Gln
515 520 525
Leu Glu Ala Ile Lys Leu Tyr Leu Lys Gly Lys Glu Pro Leu Leu
530 535 540
Gln Cys Asp Lys Glu Leu Ile Gln Glu Val Leu Phe Asp Ala Val
545 550 555
Val Ser Ala Pro Ile Glu Ala Tyr Trp Thr Ser Leu Ala Leu Asn
560 565 570
Lys Ser Glu Asn Ser Asp Lys Gly Val Glu Val Ala Phe Leu Gly
575 580 585
Thr Arg Thr Gly Leu Ser Arg Ile Asn Leu Phe Val Gly Ala Glu
590 595 600
Gln Leu Thr Asn Gln Asp Phe Leu Lys Ala Gly Asp Lys Glu Asn
5/29
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
605 610 615
Ile Phe Asn Ala Asp His Phe Pro Leu Trp Tyr Arg Arg Ala Ala
620 625 630
Glu Gln Ile Pro Gly Ser Phe VaI Tyr Ser Ile Pro Phe Ser Thr
635 640 645
Gly Pro Val Asn Lys Ser Asn Val Val Thr Ala Ser Thr Ser Ile
650 655 660
Gln Leu Leu Asp Glu Arg Lys Ser Pro Val Val Ala Ala Val Gly
665 670 675
Ile Gln Met Lys Leu Glu Phe Phe Gln Arg Lys Phe Trp Thr Ala
680 685 690
Ser Arg Gln Cys Ala Ser Leu Asp Gly Lys Cys Ser Ile Ser Cys
695 700 705
Asp Asp Glu Thr Val Asn Cys Tyr Leu Ile Asp Asn Asn Gly Phe
710 715 720
Ile Leu Val Ser Glu Asp Tyr Thr Gln Thr Gly Asp Phe Phe Gly
725 730 735
Glu Ile Glu Gly Ala Val Met Asn Lys Leu Leu Thr Met Gly Ser
740 745 750
Phe Lys Arg Ile Thr Leu Tyr Asp Tyr Gln Ala Met Cys Arg Ala
755 760 765
Asn Lys Glu Ser Ser Asp Gly Ala His Gly Leu Leu Asp Pro Tyr
770 775 780
Asn Ala Phe Leu Ser Ala Val Lys Trp Ile Met Thr Glu Leu Val
785 790 795
Leu Phe Leu Val Glu Phe Asn Leu Cys Ser Trp Trp His Ser Asp
800 805 810
Met Thr Ala Lys Ala Gln Lys Leu Lys Gln Thr Leu Glu Pro Cys
815 820 825
Asp Thr Glu Tyr Pro Ala Phe Val Ser Glu Arg Thr Ile Lys Glu
830 835 840
Thr Thr Gly Asn Ile Ala Cys Glu Asp Cys Ser Lys Ser Phe Val
845 850 855
Ile Gln Gln Ile Pro Ser Ser Asn Leu Fhe Met Val Val Val Asp
860 865 870
Ser Ser Cys Leu Cys Glu Ser Val Ala Pro Tle Thr Met Ala Pro
875 880 885
Ile Glu Ile Arg Tyr Asn Glu Ser Leu Lys Cys Glu Arg Leu Lys
890 895 900
Ala Gln Lys Ile Arg Arg Arg Pro Glu Ser Cys His Gly Phe His
905 910 915
Pro Glu Glu Asn Ala Arg Glu Cys Gly Gly Ala Pro Ser Leu Gln
920 925 930
Ala Gln Thr Val Leu Leu Leu Leu Pro Leu Leu Leu Met Leu Phe
935 940 945
Ser Arg
<210> 5
<211> 461
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7488292CD1
6/19
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
<400> 5
Met Asp Ile Leu Leu Asp Ala Glu Glu Trp Glu Asp Phe Glu Ser
1 5 10 15
Ser Pro Leu Leu Pro Glu Pro Leu Ser Ser Arg Tyr Lys Leu Tyr
20 25 30
Glu Ala Glu Phe Thr Ser Pro Ser Trp Pro Ser Thr Ser Pro Asp
35 40 45
Thr His Pro Ala Leu Pro Leu Leu Glu Met Pro Glu Glu Lys Asp
50 55 60
Leu Arg Ser Ser Asn Glu Asp Ser His Ile Val Lys Ile Glu Lys
65 70 75
Leu Asn Glu Arg Ser Lys Arg Lys Asp Asp Gly Val Ala His Arg
80 85 90
Asp Ser Ala Gly Gln Arg Cys Ile Cys Leu Ser Lys Ala Val Gly
95 100 105
Tyr Leu Thr Gly Asp Met Lys Glu Tyr Arg Ile Trp Leu Lys Asp
110 115 120
Lys His Leu Ala Leu Gln Phe Ile Asp Trp Val Leu Arg Gly Thr
125 130 135
Ala Gln Val Met Phe Ile Asn Asn Pro Leu Ser Gly Leu Ile Ile
140 145 150
Phe Ile Gly Leu Leu Ile Gln Asn Pro Trp Trp Thr Ile Thr Gly
155 160 165
Gly Leu Gly Thr Val Val Ser Thr Leu Thr Ala Leu Ala Leu Gly
170 175 180
Gln Asp Arg Ser Ala.Ile Ala Ser Gly Leu His Gly Tyr Asn Gly
185 190 195
Met Leu Val Gly Leu Leu Met Ala VaI Phe Ser Glu Lys Leu Asp
200 205 210
Tyr Tyr Trp Trp Leu Leu Phe Pro Val Thr Phe Thr Ala Met Ser
215 220 225
Cys Pro Val Leu Ser Ser Ala Leu Asn Ser Ile Phe Ser Lys Trp
230 235 240
Asp Leu Pro Val Phe Thr Leu Pro Phe Asn IIe Ala Val Thr Leu
245 250 255
Tyr Leu Ala Ala Thr Gly His Tyr Asn Leu Phe Fhe Pro Thr Thr
260 265 270
Leu Val Glu Pro Val Ser Ser Val Pro Asn Ile Thr Trp Thr Glu
275 280 . 285
Met Glu Met Pro Leu Leu Leu Gln Ala Ile Pro Val Gly Val Gly
290 295 300
Gln Val Tyr Gly Cys Asp Asn Pro Trp Thr Gly Gly Val Phe Leu
305 310 315
Val Ala Leu Phe Ile Ser Ser Pro Leu Ile Cys Leu His Ala Ala
320 . 325 330
Ile Gly Ser Ile Val Gly Leu Leu Ala Ala Leu Ser Val Ala Thr
335 340 345
Pro Phe Glu Thr Ile Tyr Thr Gly Leu Trp Ser Tyr Asn Cys Val
350 355 360
Leu Sex Cys Ile Ala Ile Gly Gly Met Phe Tyr Ala Leu Thr Trp
365 370 375
Gln Thr His Leu Leu Ala Leu Ile Cys Ala Leu Phe Cys Ala Tyr
380 385 390
Met Glu Ala Ala Ile Ser Asn Ile Met Ser Val Val Gly Val Pro
395 400 405
Pro Gly Thr Trp Ala Phe Cys Leu Ala Thr Ile Ile Phe Leu Leu
7/19
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
410 415 420
Leu Thr Thr Asn Asn Pro Ala Ile Phe Arg Leu Pro Leu Ser Lys
425 430 435
Val Thr Tyr Pro Glu Ala Asn Arg Ile Tyr Tyr Leu Thr Val Lys
440 445 450
Ser Gly Glu Glu Glu Lys Ala Pro Ser Gly Glu
455 460
<210> 6
<211> 555
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7236815CD1
<400> 6
Met Ser Gly Ile Gln Gly Thr Arg Thr Tyr Pro Gly Ala Gly Asp
1 5 20 15
Thr Ser Asp Leu Lys Tyr Pro Leu Ala Thr Arg Leu Arg Glu Ala
20 25 30
Leu Thr Glu Ala Arg Phe His Gln Leu Phe Arg Gly Glu Glu Gln
35 40 45
Glu Pro Glu Leu Pro Glu Glu Arg Gly Phe Pro Arg Leu Phe Gly
50 55 60
Leu Trp Arg Leu Arg Ala Arg Ala Cys Ser Gly Thr Gly Ala Trp
65 70 75
Arg Leu Leu Leu Ala Arg Leu Pro Ala Leu His Trp Leu Pro His
80 85 90
Tyr Arg Trp Arg Ala Trp Leu Leu Gly Asp Ala Val Ala Gly Val
95 100 105
Thr Val Gly Ile Val His Val Pro Gln Gly Met Ala Phe Ala Leu
110 115 120
Leu Ala Ser Val Pro Pro VaI Phe Gly Leu Tyr Thr Ser Phe Phe
125 130 135
Pro Val Leu Ile Tyr Ser Leu Leu Gly Thr Gly Arg His Leu Ser
140 145 150
Thr Gly Thr Phe Ala Ile Leu Ser Leu Met Thr Gly Ser Ala Val
155 160 165
Glu Arg Leu Val Pro Glu Pro Leu Val Gly Asn Leu Ser Gly Ile
170 175 180
Glu Lys Glu Gln Leu Asp Ala Gln Arg Val Gly Val Ala Ala Ala
185 190 195
Val Ala Phe Gly Ser Gly Ala Leu Met Leu Gly Met Phe Val Leu
200 205 210
Gln Leu Gly Val Leu Ser Thr Phe Leu Ser Glu Pro Val Val Lys
215 220 225
Ala Leu Thr Ser Gly Ala Ala Leu His Val Leu Leu Ser Gln Leu
230 235 240
Pro Ser Leu Leu Gly Leu Ser Leu Pro Arg GIn Ile Gly Cys Phe
245 250 255
Ser Leu Phe Lys Thr Leu Ala Ser Leu Leu Thr Thr Leu Pro Arg
260 265 270
Ser Ser Pro Ala GIu Leu Thr Ile Ser Ala Leu Ser Leu Ala Leu
275 280 285
8/19
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
Leu Val Pro VaI Lys Glu Leu Asn Val Arg Phe Arg Asp Arg Leu
290 295 300
Pro Thr Pro Ile Pro Gly Glu Val Val Leu Val Leu Leu Ala Ser
305 310 315
Val Leu Cys Phe Thr Ser Ser Val Asp Thr Arg Tyr Gln Val Gln
320 325 330
Ile Val Gly Leu Leu Pro Gly Gly Phe Pro Gln Pro Leu Leu Pro
335 340 345
Asn Leu Ala Glu Leu Pro Arg Ile Leu Ala Asp Ser Leu Pro Ile
350 355 360
Ala Leu Val Ser Phe Ala Val Ser Ala Ser Leu Ala Ser Ile His
365 370 375
Ala Asp Lys Tyr Ser Tyr Thr Ile Asp Ser Asn Gln Glu Phe Leu
380 385 390
Ala His Gly Ala Ser Asn Leu Ile Ser Ser Leu Phe Ser Cys Phe
395 400 405
Pro Asn Ser Ala Thr Leu Ala Thr Thr Asn Leu Leu Val Asp Ala
410 415 420
Gly Gly Lys Thr Gln Gly Asn Pro Thr Val Ala Phe Lys Val Glu
425 430 435
Val Gly Tyr Lys Thr Gly Glu Leu Glu Gln Trp Thr Ser Thr Arg
440 445 450
Arg Leu Leu Ala Gly Leu Phe Ser Cys Thr Val' Val Leu Ser Val
455 460 ' 465
Leu Leu Trp Leu Gly Pro Phe Phe Tyr Tyr Leu Pro Lys Ala Val
4T0 475 480
Leu Ala Cys Ile Asn Ile Ser Ser Met Arg Gln Val Phe Cys Gln
485 490 495
Met Gln Glu Leu Pro Gln Leu Trp His Ile Ser Arg Val Asp Phe
500 505 . 510
Ala Val Trp Met Val Thr Trp Val Ala Val Val Thr Leu Ser Val
515 520 525
Asp Leu Gly Leu Ala Val Gly Val Val Phe Ser Met Met Thr Val
530 535 540
VaI Cys Arg Thr Arg Ser Ser Ser Arg Ser Arg Gly Ser Ala Ser
545 550 555
<210> 7
<211> 332
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 414046CD1
<400> 7
Met Ala Ala Ala Thr Ala Ala Ala Ala Leu Ala Ala Ala Asp Pro
1 5 l0 l5
Pro Pro Ala Met Pro Gln Ala Ala Gly Ala Gly Gly Pro Thr Thr
20 25 30
Arg Arg Asp Phe Tyr Trp Leu Arg Ser Phe Leu Ala Gly Gly Ile
35 40 45
Ala Gly Cys Cys Ala Lys Thr Thr Val Ala Pro Leu Asp Arg Val
50 55 60
9/19
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
Lys Val Leu Leu Gln Ala His Asn His His Tyr Lys His Leu Gly
65 70 75
Val Phe Ser Ala Leu Arg Ala Val Pro Gln Lys Glu Gly Phe Leu
80 85 90
Gly Leu Tyr Lys Gly Asn Gly Ala Met Met Ile Arg Ile Phe Pro
95 100 105
Tyr Gly Ala Ile Gln Phe Met Ala Phe Glu His Tyr Lys Thr Leu
110 115 120
Ile Thr Thr Lys Leu Gly Ile Ser Gly His Val His Arg Leu Met
125 130 135
Ala Gly Ser Met Ala Gly Met Thr Ala Val Ile Cys Thr Tyr Pro
140 145 150
Leu Asp Met Val Arg Val Arg Leu Ala Phe Gln Val Lys Gly Glu
155 160 165
His Ser Tyr Thr Gly Ile Ile His Ala Phe Lys Thr Ile Tyr Ala
170 175 180
Lys Glu Gly Gly Phe Phe Gly Phe Tyr Arg Gly Leu Met Pro Thr
185 190 195
Ile Leu Gly Met Ala Pro Tyr Ala Gly Val Ser Phe Phe Thr Phe
200 205 210
Gly Thr Leu Lys Ser Val Gly Leu Ser His Ala Pro Thr Leu Leu
215 220 225
Gly Arg Pro Ser Ser Asp Asn Pro Asn Val Leu Val Leu Lys Thr
230 235 240
His Val Asn Leu Leu Cys Gly Gly Val Ala Gly Ala Ile Ala Gln
245 250 255
Thr Ile Ser Tyr Pro Phe Asp Val Thr Arg Arg Arg Met Gln Leu
260 265 270
Gly Thr Val Leu Pro Glu Phe Glu.Lys.Cys Leu Thr Met Arg Asp
275 280 285
Thr Met Lys Tyr Val Tyr Gly His His Gly Ile Arg Lys Gly Leu
290 295 300
Tyr Arg Gly Leu Ser Leu Asn Tyr Ile Arg Cys Ile Pro Ser Gln
305 310 315
Ala Val Ala Phe Thr Thr Tyr Glu Leu Met Lys Gln Phe Phe His
320 325 330
Leu Asn
<210> 8
<211> 296
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6829266CD1
<400> 8
Met Ile Leu Arg Val Thr Leu Arg Asn Pro Gly Ser Ser Gly Arg
1 5 10 15
Lys Glu His Pro Glu Ala Gly Thr Gly Ser Trp Leu Gly Arg Thr
20 25 30
Arg Asn Gln Val Ile Asn Thr Leu Ala Asp His Arg His Arg Gly
35 40 45
Thr Asp Phe Gly Gly Ser Pro Trp Leu Leu Ile Ile Thr Val Phe
10/19
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
50 55 60
Leu Arg Ser Tyr Lys Phe Ala Ile Ser Leu Cys Thr Ser Tyr Leu
65 70 75
Cys Val Ser Phe Leu Lys Thr Ile Phe Pro Ser Gln Asn Gly His
80 85 90
Asp Gly Ser Thr Asp Val Gln Gln Arg Ala Arg Arg Ser Asn Cys
95 100 105
Arg Arg Gln Glu Gly Ile Lys Ile Val Leu Glu Asp Ile Phe Thr
110 115 120
Leu Trp Arg Gln Val Glu Thr Lys Val Arg Ala Lys Ile Arg Lys
125 130 135
Met Lys Val Thr Thr Lys Val Asn Arg His Asp Lys Ile Asn Gly
140 145 150
Lys Arg Lys Thr Ala Lys Glu His Leu Arg Lys Leu Ser Met Lys
155 160 165
Glu Arg Glu His Gly Glu Lys Glu Arg Gln Val Ser Glu Ala Glu
170 175 180
Glu Asn Gly Lys Leu Asp Met Lys Glu Ile His Thr Tyr Met Glu
185 190 195
Met Phe Gln Arg Ala Gln Ala Leu Arg Arg Arg Ala Glu Asp Tyr
200 205 210
Tyr Arg Cys Lys Ile Thr Pro Ser Ala Arg Lys Pro Leu Cys Asn
215 220 225
Arg Val Arg Met Ala Ala Val Glu His Arg His Ser Ser Gly Leu
230 235 240
Pro Tyr Trp Pro Tyr Leu Thr Ala Glu Thr Leu Lys Asn Arg Met
245 250 255
Gly His Gln Pro Pro Pro Pro Thr Gln Gln His Ser Ile Ile Asp
260 265 270
Asn Ser Leu Ser Leu Lys Thr Pro Ser Glu Cys Val.Leu Tyr Pro
275 280 285
Leu Pro Pro Gln Gly Met Ile Ile Ser Arg Asn
290 295
<210> 9
<211> 204
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7486339CD1
<400> 9
Met Glu His Ile Ser Ala Pro Ala Glu Arg Asp Pro Pro Pro Arg
1 5 10 15
Ser Gly Ser Thr Ala His Phe Arg Ser Cys His Arg Leu Ser Asp
20 25 30
Cys Gln Arg Pro Leu Thr Ala Pro Leu Trp Gln Val Arg Gln Asn
35 40 45
Tyr His Pro Asp Cys Asp Ala Ala Val Asn Ser His Val Asn Leu
50 55 60
Glu Leu His Ala Ser Cys Val Tyr Leu Ser Met Ala Phe Tyr Leu
65 70 75
Asp Arg Asp Asp Val Thr Leu Glu Arg Phe Ser Arg Cys Phe Leu
80 85 90
11/19
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
Ser Gln Ser Gln Glu Lys Arg Glu His Ala Gln Lys Leu Ile Met
95 100 105
Leu Gln Asn Leu Arg Gly Gly Arg Ile Cys Leu Pro Asp Ile Trp
110 115 120
Lys Pro Glu Arg Glu Tyr Trp Glu Ser Gly Leu Gln Ala Met Glu
125 130 135
Cys Ala Phe His Leu Glu Glu Ser Val Asn Tyr Ser Leu Leu Glu
140 145 250
Leu His Tyr Leu Ala Met Glu Lys Gly Asp Pro Gln Leu Cys Asp
155 160 165
Phe Leu Glu Ser His Phe Leu Asn Gln Gln Val Lys Ala Ile Lys
170 175 180
Glu Leu Ser Gly Tyr Leu Ser Asn Leu Arg Lys Met Trp Ala Thr
185 190 295
Gly Asn Arg Pro Gly Arg Val Pro Val
200
<210> 10
<211> 2104
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2561248CB1
<400> 20
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
ggtgtcggcg ccgtgttctc actgctattc cacctgggca cccgggagag gcgccggccg 900
catgcggagg agccaggcga gcacaccccc ctgttggccc ctgccacggc ccagcccctg 960
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 atgcagcggc 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 acgctgatga gacctgcacg cagtggctca cagcagcacg 1620
atttgtgaca gcccgaggcg gagaacaccg aacacccagt gaaggtgagg ggatcagcac 1680
ggcgcggcca cccacgcacc cacgcgctgg aatgagactc agccacaagg aggtgcgaag 1740
12/19
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
ctctgaccca ggccacagtg cggatgcacc ttgaggatgt cacgctcagt gagagacacc 1800
agacacagaa gggtacgctg tgatcccact tctatgaaat gtccaggaca gaccaatcca 1860
cagaatcagg gagaggattc gtgggtgccg ggactgggga gggggacctg ggggtgacta 1920
ggtgacataa tggggacagg gctgccttct gggtgatgag aatgttctgg aatcagatgg 1980
gatggctgca cggcgtggtg aaggtactga acgccacctc actgtaagac ggtagatttt 2040
gtattttacc acaataaaca aaacaaaaca aaaccaaacc aaacccaaaa aaaaaaaaaa 2100
aaaa 2104
<210> 11
<211> 2050
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4539525CB1
<400> 11
atgacgtggc ccgagctcag ctgggagaag atgcccacct ctgtcccacc ctaggcccag 60
gggtccggcc ccggctgtgc cacatagtga aagatgaggg tggttttggc ttcagtgtca 120
cccatggcaa tcagggtcct ttctggttgg tgctaagtac tggaggagca gctgagcggg 180
caggggtgcc ccccggggcc cggctgctgg aagtgaatgg ggtcagtgtg gagaagttca 240
ctcacaacca actcaccagg aagctttggc agagtggaca gcaggtgacc ttgctggtgg 300
cagggccaga ggtggaagaa cagtgtcgcc agctgggatt gcccctggct gcacccctgg 360
cagagggctg ggcactgccc accaagcccc gctgcctgca cctggagaaa gggccccagg 420
gttttgggtt cctgctccgg gaggaaaagg gccttgacgg tcgccctggt gagtgggagc 480
cctgggggcg gtgggggaag gtgggccttg gggtgggcac acaagcgtat atacaccttt 540
cagtgcaccg aagaggtgtc cctgtctgag ctctggccct gggccgcctc ttcccgttca 600
ctctggggtc agtcccctgg tgtgtacaca gtggcctagg atagctggag aggagcagtg 660
aggatgtcta tgccccagga cagttcctgt gggaggtgga cccgggactg ccagccaaga 720
aggctgggat gcaggctggg gaccggctgg tggctgtggc tggggagagc gtggaggggc 780
tgggccatga ggagacagtg tccaggatcc aggggcaggg ctcctgtgtc tccctcactg 840
tcgtcgaccc tgaggcggac cgcttcttca gcatggttcg cctgtcccca ctcctcttct 900
tggagaacac agaggctccc gcctcgcccc agggcagcag ctcagcctca ctggttgaga 960
cagaggaccc ttcacttgaa gacacaagcg tgccttctgt ccctcttggc tcccgacagt 1020
gcttcctgta ccctgggcct ggtggcagct atggcttccg actcagttgt gtggccagtg 1080-
ggcctcgtct cttcatctcc caggtgactc caggaggctc agctgcccgg gctgggctgc 1140
aagtgggaga cgtgattctg gaagtgaacg ggtatcctgt tgggggacag aatgacctgg 1200
agaggcttca gcagctgcct gaggctgagc cacccctctg cctgaagctg gcagccaggt 1260
ctctgcgggg cttggaagcc tggattcccc ctggggctgc agaggactgg gctctggcct 1320
cggatctact gtagagcacc cctgcttggt acagacatac tcaggggcta ccgtgtcttc 1380
actctccagc ctgaggtggt gaaggcagga tgctctctct aagccagacc agagggactc 1440
agacaccacc gatcacaggc tggcccaggt gctccctccc ttcctgcagg cccacctgcc 1500
agcagagggt gtggttggag gcctcagaca ggtccctgaa ggagtctgag gctccagagg 1560
atgtcatatg ggagttttag agagctgtgt cccaaggatg aaggtgtggc tgtgggtctg 1620
gctaggattg aagccatctg gaccttttct agatatgact ccaggaccct tgagtgtaat 1680
gcaaaaattt ggagaccagc tatgcctgcc ctctgtgggt gccttagcat tgcgggaggg 1740
tggtgcttgg tcaccgttgc atttgttata gaaatggcca ttcgccataa atctgactgc 1800
ctgtgtttgt gttggtgggg gtaaggggca gtggtgtgaa gggaccaaaa gggcctcagg 1860
ctcaaggggt gggatgcggc tcctgcagga gagaggttga gacctggtca aatttatttc 1920
ctatcaatca ctgaatctca gggataatgg gtcaacccag aactgagatg tctgtatgac 1980
agccactcct aaaaataaac aacaacaaaa acaaaaaaag aagaaaacta aataaaaaaa 2040
aaaaaaaaaa 2050
<210> 12
<211> 1293
13!29
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
<212> DNA
<213> Homo Sapiens
<220>
<221> misc-feature
<223> Incyte ID No: 72210802CB1
<400> 12
gtctgctctt agctcagtct agatccctcg ttgtgtgtcc cccatggtgt ggtcctacct 60
gtgttagccg cgttggcttg tcggtggctc gttgtgagtc aaccggttaa ctcgactttc 120
tccagtgcac caccacggta cgagagtacc agagccgagt agttagcgtt tcacaggagt 180
ttcttggaaa tgtggtgcgc taactacggc tacactagaa gctacagtat tttggtatct 240
gcggctcgtg ctgaagccag ttaggcgtcg gatgaagagt tggtagctct tgatccggcg 300
tactagacca ccgctgtatc gtggttttgt ttgtttgcaa gcagcagata cggcgcagta 360
tatagaagga tctcaagaag atcctttgga tcacagtgat gaagcccgct cattaggcgg 420
tgttaaattc ccgggtatct gctgccgaat tcattaatgc aggttaacct ggcttatcga 480
atgaggggat attgccgatg aatcccgccg atgtggccca gtcgactctg ccactggcat 540
cgagcgatgt gtcgctgatc gcattgtttt ggcaggccca ttgggtcgtc aagtgcgtga 600
tgttgggact tctgtcctgc tcggtgtggg tctgggcgat cgcgatcgac aagatcctgc 660
tctacgcccg caccaagcgt gcgatggaca agttcgagca ggcattctgg tccggccagt 720
cgatcgagga gctctaccgg gccctctcgg ccaagccgac ccagtcgatg gccgcctgtt 780
tcgtggcggc gatgcgggag tggaaacgct ccttcgagag ccagtcgcgg tcctttgccg 840
gcctgcaggc ccggatcgac aaggtcatga~acgtctcgat cgcccgcgag gtggagcggc 900
tggaacggcg gctgctggtg ctggccaccg tcggctcggc cggccccttc gtcggcctgt 960
tcggcaccgt ctggggcatc atgtcgagct tccagtcgat tgctgcctcg aaaaatacct 1020
ccctggccgt ggtggcgccg ggtatcgcgg aagcgctgtt tgccaccgcg atcggtctga 1080
ttgccgcaat tccggcgact attttctaca ataagttcac ttcggaggtg aaccggcagg 1140
ccgcgcgcct ggaggggttc gccgacgagt tctccgccat cctgtcgcgt cagatcgacg 1200
agcggggctg agaccgatga tgatcacgat ggtcactctt gtgagcgcac gatcatggcg 1260
atgagcatgg cagggtccgg tggcggcggc agg 1293
<210> 13
<211> 3382
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2469624CB1
<400> 13
gggtgaagct ctgggcctcg gcttttggtg gggagataaa atccattgct gctaagtact 60
ccggttccca gcttctgcaa aagaaataca aagagtatga gaaagacgtt gccatagaag 120
aaatcgatgg cctccaactg gtaaagaagc tggcaaagaa catggaagag atgtttcaca 180
agaagtctga ggccgtcagg cgtctggtgg aggctgcaga agaagcacac ctgaaacatg 240
aatttgatgc agacttacag tatgaatact tcaatgctgt gctgataaat gaaagggaca 300
aagacgggaa ttttttggag ctgggaaagg aattcatctt agccccaaat gaccatttta 360
ataatttgcc tgtgaacatc agtctaagtg acgtccaagt accaacgaac atgtacaaca 420
aagggattaa atgggaacca gatgagaatg gagtcattgc cttegactgc aggaaccgaa 480
aatggtacat ccaggcagca acttctccga aagacgtggt cattttagtt gacgtcagtg 540
gcagcatgaa aggactccgt ctgactatcg cgaagcaaac agtctcatcc attttggata 600
cacttgggga tgatgacttc ttcaacataa ttgcttataa tgaggagctt cactatgtgg 660
aaccttgcct gaatggaact ttggtgcaag ccgacaggac aaacaaagag cacttcaggg 720
agcatctgga caaacttttc gccaaaggaa ttggaatgtt ggatatagct ctgaatgagg 780
ccttcaacat tctgagtgat ttcaaccaca cgggacaagg aagtatctgc agtcaggcca 840
tcatgctcat aactgatggg gcggtggaca cctatgatac aatctttgca aaatacaatt 900
14/19
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
ggccagatcg aaaggttcgc atcttcacat acctcattgg acgagaggct gcgtttgcag 960
acaatctaaa gtggatggcc tgtgccaaca aaggattttt tacccagatc tccaccttgg 1020
ctgatgtgca ggagaatgtc atggaatacc ttcacgtgct tagccggccc aaagtcatcg 1080
accaggagca tgatgtggtg tggaccgaag cttacattga cagcactctg actgatgatc 1140
agggccccgt cctgatgacc actgtagcca tgcctgtgtt tagtaaacag aacgaaacca 1200
gatcgaaggg cattcttctg ggagtggttg gcacagatgt cccagtgaaa gaacttctga 1260
agaccatccc caaatacaag ttagggattc acggttatgc ctttgcaatc acaaataatg 1320
gatatatcct gacgcatccg gaactcaggc tgctgtacga agaaggaaaa aagcgaagga 1380
aacctaacta tagtagcgtt gacctctctg aggtggagtg ggaagaccga gatgacgtgt 1440
tgagaaatgc tatggtgaat cgaaagacgg ggaagttttc catggaggtg aagaagacag 1500
tggacaaagg gaaacgggtt ttggtgatga caaatgacta ctattataca gacatcaagg 1560
gtactccttt cagtttaggt gtggcgcttt ccagaggtca tgggaaatat ttcttccgag 1620
ggaatgtaac catcgaagaa ggcctgcatg acttagaaca tcccgatgtg tccttggcag 1680
atgaatggtc ctactgcaac actgacctac accctgagca ccgccatctg tctcagttag 1740
aagcgattaa gctctaccta aaaggcaaag aacctctgct ccagtgtgat aaagaattga 1800
tccaagaagt cctttttgac gcggtggtga gtgcccccat tgaagcgtat tggaccagcc 1860
tggccctcaa caaatctgaa aattctgaca agggcgtgga ggttgccttc ctcggcactc 1920
gcacgggcct ctccagaatc aacctgtttg tcggggctga gcagctcacc aatcaggact 1980
tcctgaaagc tggcgacaag gagaacattt ttaacgcaga ccatttccct ctctggtacc 2040
gaagagccgc tgagcagatt ccagggagct tcgtctactc gatcccattc agcactggac 2100
cagtcaataa aagcaatgtg gtgacagcaa gtacatccat ccagctcctg gatgaacgga 2260
aatctcctgt ggtggcagct gtaggcattc agatgaaact tgaatttttc caaaggaagt 2220
tctggactgc cagcagacag tgtgcttccc tggatggcaa atgctccatc agctgtgatg 2280
atgagactgt gaattgttac ctcatagaca ataatggatt tattttggtg tctgaagact 2340
acacacagac tggagacttt tttggtgaga tcgagggagc tgtgatgaac aaattgctaa 2400
caatgggctc ctttaaaaga 'attacccttt atgactacca agccatgtgt agagccaaca 2460
aggaaagcag cgatggcgcc catggcctcc tggatcctta taatgccttc ctctctgcag 2520
taaaatggat catgacagaa cttgtcttgt tcctggtgga atttaacctc tgcagttggt 2586
ggcactccga tatgacagct aaagcccaga aattgaaaca gaccctggag ccttgtgata 2640
ctgaatatcc agcattcgtc tctgagcgca ccatcaagga gactacaggg aatattgctt 2700
gtgaagactg ctccaagtcc tttgtcatcc agcaaatccc aagcagcaac ctgttcatgg 2760
tggtggtgga cagcagctgc ctctgtgaat ctgtggcccc catcaccatg gcacccattg 2820
aaatcaggta taatgaatcc cttaagtgtg aacgtctaaa ggcccagaag atcagaaggc 2880
gcccagaatc ttgtcatggc ttccatcctg aggagaatgc aagggagtgt gggggtgcgc 2940
cgagtctcca agcccagaca gtcctccttc tgctccctct gcttttgatg ctcttctcaa 3000
ggtgacactg actgagatgt tctcttactg actgagatgt tctcttggca tgctaaatca 3060
tggataaact gtgaaccaaa atatggtgca acatacgaga catgaatata gtccaaccat 3120
cagcatctca tcatgatttt aaactgtgcg tgatataaac tcttaaagat atgttgacaa 3180
aaagttatct atcatctttt tactttgcca gtcatgcaaa tgtgagtttg ccacatgata 3240
atcacccttc atcagaaatg ggaccgcaag tggtaggcag tgtcccttct gcttgaaacc 3300
tattgaaacc aatttaaaac tgtgtacttt ttaaataaag tatattaaaa tcataaaaaa 3360
aaaaaaaaaa aaaaaattgc tg 3382
<210> 14
<211> 1476
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7488292CB1
<400> 14
atgaaaggca gagaaaaaac actcagtgat caagagaaaa agagagactt ccattctgac 60
tcagtggtca gggatatctt catcggccaa atggacattc ttctggacgc ggaagaatgg 120
gaggattttg aaagcagtcc tctcctgcca gagccacttt ccagcagata caaactctac 180
25/19
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
gaggcagagt ttaccagccc gagctggccc tcgacatccc cggatactca cccagctctg 240
cccctcctgg aaatgcctga agaaaaggat ctccggtctt ccaatgaaga cagtcacatt 300
gtgaagatcg aaaagctcaa tgaaaggagt aaaaggaaag acgacggggt ggcccatcgg 360
gactcagcag gccaaaggtg catctgcctc tccaaagcag tgggctacct cacgggcgac 420
atgaaggagt acaggatctg gctgaaagac aagcaccttg ccctccagtt catagactgg 480
gtcctgagag ggaccgctca ggtgatgttc atcaacaatc ctctcagcgg cctcatcatc 540
ttcatagggc tgctgatcca gaatccctgg tggacaatca ctgggggcct ggggacagtg 600
gtctcgacct taacagctct cgccttgggc caagacaggt ctgccattgc ctcaggactc 660
catgggtaca acgggatgct ggtgggactg ctgatggceg tgttctcgga gaagttagac 720
tactactggt ggcttctgtt tcctgtgacc ttcacagcca tgtcctgccc agttctttct 780
agtgccttga attccatctt cagcaagtgg gacctcccgg tcttcactct gcccttcaac 840
attgcagtca ccttgtacct tgcagccaca ggccactaca acctcttctt ccccacaaca 900
ctggtagagc ctgtgtcttc agtgcccaat atcacctgga cagagatgga aatgcccctg 960
ctgttacaag ccatccctgt tggggtcggc caggtgtatg gctgtgacaa tccctggaca 1020
ggcggcgtgt tcctggtggc tctgttcatc tcctcgccac tcatctgctt gcatgcagcc 1080
attggctcaa tcgtggggct gctagcagcc ctgtcagtgg ccacaccctt cgagaccatc 1140
tacacaggcc tctggagcta caactgcgtc ctctcctgca tcgccatcgg aggcatgttc 1200
tatgccctca cctggcagac tcacctgctg gccctcatct gtgccctgtt ctgtgcatac 1260
atggaagcag ccatctccaa catcatgtca gtggtgggcg tgccaccagg cacctgggcc 1320
ttctgccttg ccaccatcat cttcctgctc ctgacgacaa acaacccagc catcttcaga 1380
ctcccactca gcaaagtcac ctaccccgag gccaaccgca tctactacct gacagtgaaa 1440
agcggtgaag aagagaaggc ccccagcggt gaatag 1476
<210> 15
<211> 2495
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7236815CB1
<400> 15
catttccaca gccaccccag agccagcgat cagatccggc caaggatgtc tgcagaaacg 60
cctaacatag agactctccc tctccaagcc gcagctctcg ccagacccga gaaggtcctc 120
aagcgagggt gacctcaggg ccggattgga ccctgcttcg tgggaggcgg gactcagggc 180
ttagcgggcg gaggagtatt taagaccggg cggagttgga ggtggccaag ggcagaatga 240
gcgggattca gggcaccagg acctacccag gcgcggggga cacctctgac cttaagtacc 300
ccttggcgac caggctcagg gaagctctca ccgaggctcg gttccatcag ctcttcaggg 360
gcgaagagca ggaaccggag ctacctgaag agcgcggctt tccccggctc ttcgggctgt 420
ggaggctgcg ggctcgcgct tgttccggga caggggcgtg gcgcctgctg ctggctcggc 480
tgcccgcgct gcactggctg ccccattacc gctggcgggc ctggctgctc ggagatgcgg 540
tggccggagt gaccgtgggc atcgtgcacg tgccccaggg catggctttt gctctcctgg 600
cctccgtgcc cccggtgttt ggactctaca cttctttctt ccccgtcctc atctacagct 660
tgctaggtac tgggagacac ctgtccacag gaactttcgc catactcagc ctcatgacag 720
gctcggccgt cgagcggctg gtgccggaac ccctcgtggg gaatctgagc ggaatcgaga 780
aggagcagct ggacgctcaa cgggttgggg tagccgcggc cgtggccttc gggagcgggg 840
cgttgatgct ggggatgttc gtgctgcagc tcggcgtctt gtccaccttt ttgtccgagc 900
ctgtggtcaa ggcgctgacc agcggggccg cgctgcacgt gctcttgtcc cagctgccga 960
gcctcttggg gttgtccctc ccgcgccaga tcggctgctt ctctctcttc aagacgctgg 1020
cctccttgct gactacgctg cctcggagca gtccggccga actgaccatc tccgcgctca 1080
gcctggcgct gctcgtgccg gtcaaggaat tgaacgtgag attccgagac cggctaccca 1140
cgccgatccc gggggaagtc gtcttggtgc ttctggcctc cgtgctctgc ttcacctctt 1200
ctgtggacac aagataccaa gtccagatag tggggctgtt gcctggagga tttccccaac 1260
ccctcctccc caacctggct gagctgccca ggattctggc tgactcgctg cccattgcac 1320
tggttagttt tgcggtgtct gcctccctgg cctccatcca tgcagacaag tatagctaca 1380
16/19
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
ctattgactc caaccaggag ttcctggcac atggtgcctc caacctcatc tcctccctct 1440
tctcttgctt tcccaactcg gctacgctgg ccaccaccaa tctactggtg gatgctggtg 1500
ggaaaacaca gggtaaccca acagtggctt ttaaggtgga ggtgggctac aaaactgggg 1560
aacttgaaca atggacatct acaaggagac tgctggcagg cctcttctcc tgcacagtgg 1620
tcctgtcggt gctgctgtgg ctggggccct tcttttacta tctgcccaag gctgtcctgg 1680
cttgcatcaa catctccagc atgcgccagg tgttctgcca gatgcaggaa cttccacaac 1740
tatggcacat cagccgagtg gactttgctg tgtggatggt cacctgggtg gcagtagtga 1800
ccctgagtgt ggatttgggc ctggctgtgg gtgtggtctt ctccatgatg actgtggtct 1860
gccgcacccg gagctcctcc aggtcccggg gctctgcatc ctgagctatc caacaccact 1920
gtactttggg acccgtgggc agtttcgctg caacctggag tggcacctgg ggctcggaga 1980
aggagaaaag gagacttcaa agccagatgg cccaatggtt gcagttgctg agcctgtcag 2040
ggtggtggtc ctagacttca gtggtgtcac ctttgcagat gctgctgggg ccagagaagt 2100
ggtgcagctg gccagccgat gtcgagatgc taggatccgc ctcctcctgg ctcagtgtaa 2160
tgccttggtg caggggacac tgacccgggt aggactcctg gacagggtga ctccagatca 2220
gctgtttgtg agtgtgcagg atgcagctgc ttatgccctg gggagcctgg taaggggcag 2280
tagcaccagg agcgggagcc aggaggcact gggctgcggc aagtgaggca ggggagctca 2340
ctgacccaaa gatttgcacc gtgtgggtct gacctcatca tgtggagtgc agagggccct 2400
gatgacatgt gtgtgatgag gaccatgacc cttgaacccc cttacctaac gtaactaata 2460
aaatgaagct gagagctttg ggaaaaaaaa aaaaa 2495
<210> 16
<211> 1879
<212> bNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte TD No: 414046CB1
<400> 16
atcagccggc gccgcgccgc cgggtgttac tttgccccgc cggcggggcg gtcagcctcc 60
tgtcaccgcc tgttccggct atggtcccgt ccggtgttct gtaagttggc aacctaggct 220
cctgacgcga ccctggtcct gatggcggcg gcgacggccg cggcagccct ggcggcggcc 180
gatccccctc ccgcaatgcc gcaggcggca ggggccggag ggcccacaac ccgcagagac 240
ttctactggc tgcgctcctt tctggccgga ggtattgctg gatgctgtgc caaaacaaca 300
gttgctccat tggatcgagt aaaggtttta ttacaagctc acaatcacca ttacaagcat 360
ttaggagtat tttctgcatt gcgtgctgtt cctcaaaaag aaggattcct tggattgtat 420
aaaggaaatg gtgcaatgat gattcgaatc tttccctatg gtgcaatcca gtttatggca 480
tttgagcatt ataaaacgtt aattactacg aagctgggaa tttcaggtca tgtgcacaga 540
ttaatggctg gatccatggc aggtatgaca gcagttatct gtacttaccc tcttgacatg 600
gttagggtcc gcctagcatt ccaggtgaaa ggggaacaca gctatacagg aattattcat 660
gctttcaaaa caatttatgc aaaggaaggt ggtttctttg gattttacag aggtctgatg 720
cctactattt taggaatggc tccatatgca ggtgtttcat tttttacttt tggtaccttg 780
aagagtgttg ggctttccca tgctcctacc cttcttggca gaccttcatc agacaatcct 840
aatgtcttag ttttgaaaac tcatgtaaac ttactttgtg gtggtgttgc tggagcaata 900
gcgcagacaa tatcctaccc atttgatgtg actcgtcggc gaatgcaatt aggaactgtt 960
ctgccggaat ttgaaaagtg ccttaccatg cgggatacta tgaagtatgt ctatggacac 1020
catggaattc gaaaaggact ctatcgtggt ttatctctta attacattcg ctgtattccc 1080
tctcaagcag tggcttttac aacatacgaa cttatgaagc agttttttca cctcaactaa 1140
aaaaaaatta tggttggttt ttcttaatac attctcagag ggagaaatga aacattacta 1200
taattgtggg gggaacatta cttgaatggg gatatttacc ctgtcacaag agccactggt 1260
attttagtac ttgattattt tttctttagt cacaaatcag aactgcttac catacttttt 2320
gatgccaaac attatacctt agaacattga agaaaatatt cctaagctga tgctggctaa 1380
accgctttaa agttttattt ggaagtagaa ctagctttaa aacggggttc aagaggttgc 1440
cattagcttt gtcatgctgt tcaaagtttt taattgttat catggttttt aaaagactga 1500
cagtgtttat tattattaaa ataaacaggg ttggttatat tgcaatagaa taatgagaat 1560
17/19
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
tgaattttta agttctatga aacagccagc attgacattt tatttttgtt atctctcttc 1620
tcacaattat gctccactgg ataataggaa aaacacttct ttccttcatt ttttaaataa 1680
aattaatgtt gtatttaaaa agtagccatg tagagacaca aaaataaatg aagaagctgg 1740
acatggtggg atgggcatgt ggtcccagct actctggaag ctgaggtgag aggatcactt 1800
gagccctgga attccatgcc agcctatgca acatcatgaa accccactta ataaatgaat 1860
gaacgactaa aaaaaaaaa 1879
<210> 17
<211> 1127
<212> DNA
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 6829266CB1
<400> 17
ggggtcctgc cgccttggcg cagcttggac tcaagaccct gtgcacctct cagcaggcct 60
ttgctggaca gatgaagagt gacttgtttc tggatgattc taagagtgac cttgaggaac 120
cctgggagct caggaaggaa ggagcaccca gaagcaggga cagggagctg gttggggagg 180
accagaaatc aggttatcaa tactctggct gaccatcgtc atcgtgggac tgactttggt 240
ggaagtcctt ggttacttat cattactgtg tttctgagaa gttataaatt tgccatctcc 3o-0
ctctgcacaa gttacctttg tgtgtctttc ctgaagacta tcttcccgtc tcaaaatgga 360
catgatggat ccacggatgt acagcagaga gccaggaggt ccaactgccg tagacaggaa 420
ggaattaaaa ttgtcctgga agacatcttt actttatgga gacaggtgga aaccaaagtt 480
cgagctaaaa tccgtaagat gaaggtgaca acaaaagtca accgtcatga caaaatcaat 540
ggaaagagga agaccgccaa agaacatctg aggaaactaa gcatgaaaga acgtgagcac 600
ggagaaaagg agaggcaggt gtcagaggca gaggaaaatg ggaaattgga tatgaaagaa 660
atacacacct acatggaaat gtttcaacgt gcgcaagcgt tgcggcggcg ggcagaggac 720
tactacagat gcaaaatcac cccttctgca agaaagcctc tttgcaaccg ggtcagaatg 780
gcggcagtgg agcatcgtca ttcttcagga ttgccctact ggccctacct cacagctgaa 840
actttaaaaa acaggatggg ccaccagcca cctcctccaa ctcaacaaca tctataatt 900
gataactccc tgagcctcaa gacaccttcc gagtgtgtgc tctatcccct tccacctcag 960
gggatgataa tctcaagaaa ctaaggagga ataaataata tataaaataa aaaacaaaaa 1020
agggggggcg cgtaatgagt cgcgacccgg gaatattccg aacggtacgg ggcgtttccg 1080
gcagggggag aaaaaattgg gccccaaggg gatattcgaa gcagtag 1127
<210> 18
<211> 615
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte TD No: 7486339CB1
<400> 18
atggaacata tctcggcccc tgcggagcgc gacccacccc ccagaagcgg ttccactgcc 60
cacttccggt cctgtcacag actcagcgac tgccagcgac cgctgaccgc cccgctatgg 120
caagtgcgcc aaaactacca ccccgactgc gacgccgccg tcaacagcca cgtcaacctg 180
gagctccacg cctcctgtgt gtacctgtcc atggccttct acttagaccg ggacgacgtg 240
accctggagc gtttcagccg ctgcttcctg agccagtcgc aagagaagag ggagcacgcc 300
cagaagctga taatgctgca gaacctgcgc ggtggccgca tctgccttcc tgacatctgg 360
aaaccagagc gtgaatactg ggagagtggg ctccaggcca tggagtgtgc cttccacctg 420
gaggagagtg tcaactacag cctcctggag ctgcac~tacc tggccatgga gaagggtgac 480
ccccagctgt gcgacttcct ggagagccac ttcctgaacc agcaggtcaa ggccatcaaa 540
18/19
CA 02447662 2003-11-18
WO 02/096932 PCT/US02/16446
gagctgagtg gctacctgag caacctgcgc aagatgtggg ccacgggaaa ccggcctggc 600
agagtacctg tgtga 615
19/19
