Note: Descriptions are shown in the official language in which they were submitted.
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TRANSPORTERS AND ION CHANNELS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of
transporters and ion
channels and to the use of these sequences in the diagnosis, treatment, and
prevention of transport,
neurological, muscle, immunological and cell proliferative disorders, and in
the assessment of the
effects of exogenous compounds on the expression of nucleic acid and amino
acid sequences of
transporters and ion channels.
l0 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;, SO42-, sugars, and vitamins, as well as various
metabolic waste products.
Transport proteins also play roles in antibiotic resistance, toxin secretion,
ion balance, synaptic
neurotransmission, kidney function, intestinal absorption, tumor growth, and
other diverse cell
functions (Griffith, J. and C. Sansom (1998) The Transporter Facts Book,
Academic Press, San Diego
CA, pp. 3-29). Transport can occur by a passive concentration-dependent
mechanism, or can be
linked to an energy source such as ATP hydrolysis or an ion gradient. Proteins
that function in
transport include carrier proteins, which bind to a specific solute and
undergo a conformational
change that translocates the bound solute across the membrane, and channel
proteins, which form
hydrophilic pores that allow specific solutes to diffuse through the membrane
down an
electrochemical solute gradient.
Carrier proteins which transport a single solute from one side of the membrane
to the other
are called uniporters. In contrast, coupled transporters link the transfer of
one solute with
simultaneous or sequential transfer of a second solute, either in the same
direction (symport) or in the
opposite direction (antiport). For example, intestinal and kidney epithelium
contains a variety of
symporter systems driven by the sodium gradient that exists across the plasma
membrane. Sodium
moves into the cell down its electrochemical gradient and brings the solute
into the cell with it. The
sodium gradient that provides the driving force for solute uptake is
maintained by the ubiquitous
Na+/K~ ATPase system. Sodium-coupled transporters include the mammalian
glucose transporter
(SGLTl), iodide transporter (NIS), and multivitamin transporter (SMVT). All
three transporters have
twelve putative transmembrane segments, extracellular glycosylation sites, and
cytoplasmically-
oriented N- and C-termini. NIS plays a crucial role in the evaluation,
diagnosis, and treatment of
various thyroid pathologies because it is the molecular basis for radioiodide
thyroid-imaging
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techniques and for specific targeting of radioisotopes to the thyroid gland
(Levy, O. et al. (1997)
Proc. Natl. Acad. Sci. USA 94:5568-5573). SMVT is expressed in the intestinal
mucosa, kidney, and
placenta, and is implicated in the transport of the water-soluble vitamins,
e.g., biotin and pantothenate
(Prasad, P.D. et al. (1998) J. Biol. Chem. 273:7501-7506).
One of the largest families of transporters is the major facilitator
superfamily (MFS), also
called the uniporter-symporter-antiporter family. MFS transporters are single
polypeptide carriers
that transport small solutes in response to ion gradients. Members of the MFS
are found in all classes
of living organisms, and include transporters for sugars, oligosaccharides,
phosphates, nitrates,
nucleosides, monocarboxylates, and drugs. MFS transporters found in eukaryotes
all have a structure
comprising 12 transmembrane segments (Pao, S.S. et al. (1998) Microbiol.
Molec. Biol. Rev. 62:1-
34). The largest family of MFS transporters is the sugar transporter family,
which includes the seven
glucose transporters (GLUTl-GLUT7) found in humans that are required for the
transport of glucose
and other hexose sugars. These glucose transport proteins have unique tissue
distributions and
physiological functions. GLUT1 provides many cell types with their basal
glucose requirements and
transports glucose across epithelial and endothelial barrier tissues; GLUT2
facilitates glucose uptake
or efflux from the liver; GLUT3 regulates glucose supply to neurons; GLUT4 is
responsible for
insulin-regulated glucose disposal; and GLUT5 regulates fructose uptake into
skeletal muscle.
Defects in glucose transporters are involved in a recently identified
neurological syndrome causing
infantile seizures and developmental delay, as well as glycogen storage
disease, Fanconi-Bickel
syndrome, and non-insulin-dependent diabetes mellitus (Mueckler, M. (1994)
Eur. J. Biochem.
219:713-725; Longo, N. and L.J. Elsas (1998) Adv. Pediatr. 45:293-313).
Monocarboxylate anion transporters are proton-coupled symporters with a broad
substrate
specificity that includes L-lactate, pyruvate, and the ketone bodies acetate,
acetoacetate, and
beta-hydroxybutyrate. At least seven isoforms have been identified to date.
The isoforms are
predicted to have twelve transmembrane (TM) helical domains with a large
intracellular loop between
TM6 and TM7, and play a critical role in maintaining intracellular pH by
removing the protons that
are produced stoichiometrically with lactate during glycolysis. The best
characterized
H+-monocarboxylate transporter is that of the erythrocyte membrane, which
transports L-lactate and a
wide range of other aliphatic monocarboxylates. Other cells possess H+-linked
monocarboxylate
transporters with differing substrate and inhibitor selectivities. In
particular, cardiac muscle and
tumor cells have transporters that differ in their Km values for certain
substrates, including
stereoselectivity for L- over D-lactate, and in their sensitivity to
inhibitors. There are
Na+-monocarboxylate cotransporters on the luminal surface of intestinal and
kidney epithelia, which
allow the uptake of lactate, pyruvate, and ketone bodies in these tissues. In
addition, there are
specific and selective transporters for organic cations and organic anions in
organs including the
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kidney, intestine and liver. Organic anion transporters are selective for
hydrophobic, charged
molecules with electron-attracting side groups. Organic canon transporters,
such as the ammonium
transporter, mediate the secretion of a variety of drugs and endogenous
metabolites, and contribute to
the maintenance of intercellular pH (Poole, R.C. and A.P. Halestrap (1993) Am.
J. Physiol.
264:C761-C782; Price, N.T. et al. (1998) Biochem. J. 329:321-328; and
Martinelle, K. and I.
Haggstrom (1993) J. Biotechnol. 30:339-350).
ATP-binding cassette (ABC) transporters are members of a superfamily of
membrane
proteins that transport substances ranging from small molecules such as ions,
sugars, amino acids,
peptides, and phospholipids, to lipopeptides, large proteins, and complex
hydrophobic drugs. ABC
transporters consist of four modules: two nucleotide-binding domains (NBD),
which hydrolyze ATP
to supply the energy required for transport, and two membrane-spanning domains
(MSD), each
containing six putative transmembrane segments. These four modules may be
encoded by a single
gene, as is the case for the cystic fibrosis transmembrane regulator (CFTR),
or by separate genes.
When encoded by separate genes, each gene product contains a single NBD and
MSD. These "half
molecules" form homo- and heterodimers, such as Tap1 and Tap2, the endoplasmic
reticulum-based
major histocompatibility (MHC) peptide transport system. Several genetic
diseases are attributed to
defects in ABC transporters, such as the following diseases and their
corresponding proteins: cystic
fibrosis (CFTR, an ion channel), adrenoleukodystrophy (adrenoleukodystrophy
protein, ALDP),
Zellweger syndrome (peroxisomal membrane protein-70, PMP70), and
hyperinsulinemic
hypoglycemia (sulfonylurea receptor, SUR). Overexpression of the multidrug
resistance (MDR)
protein, another ABC transporter, in human cancer cells makes the cells
resistant to a variety of
cytotoxic drugs used in chemotherapy (Taglicht, D. and S. Michaelis (1998)
Meth. Enzymol.
292:130-162).
A number of metal ions such as iron, zinc, copper, cobalt, manganese,
molybdenum,
selenium, nickel, and chromium are important as cofactors for a number of
enzymes. For example,
copper is involved in hemoglobin synthesis, connective tissue metabolism, and
bone development, by
acting as a cofactor in oxidoreductases such as superoxide dismutase,
ferroxidase (ceruloplasmin),
and lysyl oxidase. Copper and other metal ions must be provided in the diet,
and are absorbed by
transporters in the gastrointestinal tract. Plasma proteins transport the
metal ions to the liver and
other target organs, where specific transporters move the ions into cells and
cellular organelles as
needed. Imbalances in metal ion metabolism have been associated with a number
of disease states
(Darks, D.M. (1986) J. Med. Genet. 23:99-106).
Transport of fatty acids across the plasma membrane can occur by diffusion, a
high capacity,
low affinity process. However, under normal physiological conditions a
significant fraction of fatty
acid transport appears to occur via a high affinity, low capacity protein-
mediated transport process.
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Fatty acid transport protein (FATP), an integral membrane protein with four
transmembrane
segments, is expressed in tissues exhibiting high levels of plasma membrane
fatty acid flux, such as
muscle, heart, and adipose. Expression of FATP is upregulated in 3T3-L1 cells
during adipose
conversion, and expression in COS7 fibroblasts elevates uptake of long-chain
fatty acids (Hui, T.Y. et
al. (1998) J. Biol. Chem. 273:27420-27429).
The lipocalin superfamily constitutes a phylogenetically conserved group of
more than forty
proteins that function as extracellular ligand-binding proteins which bind and
transport small
hydrophobic molecules. Members of this family function as carriers of
retinoids, odorants,
chromophores, pheromones, allergens, and sterols, and in a variety of
processes including nutrient
transport, cell growth regulation, immune response, and prostaglandin
synthesis. A subset of these
proteins may be multifunctional, serving as either a biosynthetic enzyme or as
a specific enzyme
inhibitor. (Tanaka, T. et al. (1997) J. Biol. Chem. 272:15789-15795; and van't
Hof, W. et al. (1997)
J. Biol. Chem. 272:1837-1841.)
Members of the lipocalin family display unusually low levels of overall
sequence
conservation. Pairwise sequence identity often falls below 20%. Sequence
similarity between family
members is limited to conserved cysteines which form disulfide bonds and three
motifs which form a
juxtaposed cluster that functions as a target cell recognition site. The
lipocalins share an eight
stranded, anti-parallel beta-sheet which folds back on itself to form a
continuously hydrogen-bonded
beta-barrel. The pocket formed by the barrel functions as an internal ligand
binding site. Seven loops
(L1 to L7) form short beta-hairpins, except loop L1 which is a large omega
loop that forms a lid to
partially close the internal ligand-binding site (Flower (1996) Biochem. J.
318:1-14).
Lipocalins are important transport molecules. Each lipocalin associates with a
particular
ligand and delivers that ligand to appropriate target sites within the
organism. Retinol-binding
protein (RBP), one of the best characterized lipocalins, transports retinol
from stores within the liver
to target tissues. Apolipoprotein D (apo D), a component of high density
lipoproteins (HDLs) and
low density lipoproteins (LDLs), functions in the targeted collection and
delivery of cholesterol
throughout the body. Lipocalins are also involved in cell regulatory
processes. Apo D, which is
identical to gross-cystic-disease-fluid protein (GCDFP)-24, is a
progesterone/pregnenolone-binding
protein expressed at high levels in breast cyst fluid. Secretion of apo D in
certain human breast
cancer cell lines is accompanied by reduced cell proliferation and progression
of cells to a more
differentiated phenotype. Similarly, apo D and another lipocalin, al-acid
glycoprotein (AGP), are
involved in nerve cell regeneration. AGP is also involved in anti-inflammatory
and
immunosuppressive activities. AGP is one of the positive acute-phase proteins
(APP); circulating
levels of AGP increase in response to stress and inflammatory stimulation. AGP
accumulates at sites
of inflammation where it inhibits platelet and neutrophil activation and
inhibits phagocytosis. The
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immunomodulatory properties of AGP are due to glycosylation. AGP is 40%
carbohydrate, making it
unusually acidic and soluble. The glycosylation pattern of AGP changes during
acute-phase
response, and deglycosylated AGP has no immunosuppressive activity (Flower
(1994) FEBS Lett.
354:7-11; Flower (1996) supra).
The lipocalin superfamily also includes several animal allergens, including
the mouse major
urinary protein (mMUP), the rat a-2-microgloobulin (rA2U), the bovine (3-
lactoglobulin ([31g), the
cockroach allergen (Bla g4), bovine dander allergen (Bos d2), and the major
horse allergen,
designated Equus caballus allergen 1 (Equ c1). Equ cl is a powerful allergen
responsible for about
80% of anti-horse IgE antibody response in patients who are chronically
exposed to horse allergens.
It appears that lipocalins may contain a common structure that is able to
induce the IgE response
(Gregoire, C, et al., (1996) J. Biol. Chem. 271:32951-32959).
Lipocalins are used as diagnostic and prognostic markers in a variety of
disease states. The
plasma level of AGP is monitored during pregnancy and in diagnosis and
prognosis of conditions
including cancer chemotherapy, renal disfunction, myocardial infarction,
arthritis, and multiple
sclerosis. RBP is used clinically as a marker of tubular reabsorption in the
kidney, and apo D is a
marker in gross cystic breast disease (Flower (1996) supra). Additionally, the
use of lipocalin animal
allergens may help in the diagnosis of allergic reactions to horses (Gregoire
supra), pigs, cockroaches,
mice and rats.
Mitochondrial carrier proteins are transmembrane-spanning proteins which
transport ions and
charged metabolites between the cytosol and the mitochondrial matrix. Examples
include the ADP,
ATP carrier protein; the 2-oxoglutarate/malate carrier; the phosphate carrier
protein; the pyruvate
carrier; the dicarboxylate carrier which transports malate, succinate,
fumarate, and phosphate; the
txicarboxylate carrier which transports citrate and malate; and the Grave's
disease carrier protein, a
protein recognized by IgG in patients with active Grave's disease, an
autoimmune disorder resulting
in hyperthyroidism. Proteins in this family consist of three tandem repeats of
an approximately 100
amino acid domain, each of which contains two transmembrane regions (Stryer,
L. (1995)
Biochemistry, W.H. Freeman and Company, New York NY, p. 551; PROSITE PDOC00189
Mitochondrial energy transfer proteins signature; Online Mendelian Inheritance
in Man (OMIM)
*275000 Graves Disease).
This class of transporters also includes the mitochondria) uncoupling
proteins, which create
proton leaks across the inner mitochondria) membrane, thus uncoupling
oxidative phosphorylation
from ATP synthesis. The result is energy dissipation in the form of heat.
Mitochondria) uncoupling
proteins have been implicated as modulators of thermoregulation and metabolic
rate, and have been
proposed as potential targets for drugs against metabolic diseases such as
obesity (Ricquier, D. et al.
(1999) J. Int. Med. 245:637-642).
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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 ion against the ion's concentration gradient. Gated ion channels
allow passive flow of an
ion down the ion's electrochemical gradient under restricted conditions.
Together, these types of ion
channels generate, maintain, and utilize an electrochemical gradient that is
used in 1) electrical
impulse conduction down the axon of a nerve cell, 2) transport of molecules
into cells against
concentration gradients, 3) initiation of muscle contraction, and 4) endocrine
cell secretion.
Ion Transporters
Ion transporters generate and maintain the resting electrical potential of a
cell. Utilizing the
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, Caz+-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 transportexs
utilize a proton gradient to
generate ATP from ADP and inorganic phosphate (P;).
The P-ATPases are hexamers of a 100 kD subunit with ten transmembrane domains
and
several large cytoplasmic regions that may play a role in ion binding
(Scarborough, G.A. (1999) Curr.
Opin. Cell Biol. 11:517-522). The V-ATPases are composed of two functional
domains: the V 1
domain, a peripheral complex responsible for ATP hydrolysis; and the V o
domain, an integral
complex responsible for proton translocation across the membrane. The F-
ATPases are structurally
and evolutionarily related to the V-ATPases. The F-ATPase Fo domain contains
12 copies of the c
subunit, a highly hydrophobic protein composed of two transmembrane domains
and containing a
single buried carboxyl group in TM2 that is essential for proton transport.
The V-ATPase V o domain
contains three types of homologous c subunits with four or five transmembrane
domains and the
essential carboxyl group in TM4 or TM3. Both types of complex also contain a
single a subunit that
may be involved in regulating the pH dependence of activity (Forgac, M. (1999)
J. Biol. Chem.
274:12951-12954).
The resting potential of the cell is utilized in many processes involving
carrier proteins and
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gated ion channels. Carrier proteins utilize the resting potential to
transport molecules into and out of
the cell. Amino acid and glucose transport into many cells is linked to sodium
ion co-transport
(symport) so that the movement of Na+ down an electrochemical gradient drives
transport of the other
molecule up a concentration gradient. Similarly, cardiac muscle links transfer
of Ca z+ out of the cell
with transport of Na+ into the cell (antiport).
Gated Ion Channels
Gated ion channels control ion flow by regulating the opening and closing of
pores. The
ability to control ion flux through various gating mechanisms allows ion
channels to mediate such
diverse signaling and homeostatic functions as neuronal and endocrine
signaling, muscle contraction,
fertilization, and regulation of ion and pH balance. Gated ion channels are
categorized according to
the manner of regulating the gating function. Mechanically-gated channels open
their pores in
response to mechanical stress; voltage-gated channels (e.g., Na+, K+, Ca2+,
and Cl-channels) open
their pores in response to changes in membrane potential; and ligand-gated
channels (e.g.,
acetylcholine-, serotonin-, and glutamate-gated cation channels, and GABA- and
glycine-gated
chloride channels) open their pores in the presence of a specific ion,
nucleotide, or neurotransmitter.
The gating properties of a particular ion channel (i.e., its threshold for and
duration of opening and
closing) are sometimes modulated by association with auxiliary channel
proteins 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 Ca2+ and Na+ (Suzuki, M. et al.
(1999) J. Biol. Chem.
274:6330-6335).
The pore-forming subunits of the voltage-gated cation channels form a
superfamily of ion
channel proteins. The characteristic domain of these channel proteins
comprises six transmembrane
domains (S 1-S6), a pore-forming region (P) located between S5 and S6, and
intracellular amino and
carboxy termini. In the Na+ and Ca2+ subfamilies, this domain is repeated four
times, while in the K+
channel subfamily, each channel is formed from a tetramer of either identical
or dissimilar subunits.
The P region contains information specifying the ion selectivity for the
channel. In the case of K+
channels, a GYG tripeptide is involved in this selectivity (Ishii, T.M. et al.
(1997) Proc. Natl. Acad.
Sci. USA 94:11651-11656).
Voltage-gated Na+ and K~ channels are necessary for the function of
electrically excitable
cells, such as nerve and muscle cells. Action potentials, which lead to
neurotransnnitter release and
muscle contraction, arise from large, transient changes in the permeability of
the membrane to Nay
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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
S outward, which leads to repolarization of the membrane. Voltage-gated
channels utilize charged
residues in the fourth transmembrane segment (S4) to sense voltage change. The
open state lasts only
about 1 millisecond, at which time the channel spontaneously converts into an
inactive state that
cannot be opened irrespective of the membrane potential. Inactivation is
mediated by the channel's
N-terminus, which acts as a plug that closes the pore. The transition from an
inactive to a closed state
requires a return to resting potential.
Voltage-gated Na+ channels are heterotrimeric complexes composed of a 260 kDa
pore-
forming a subunit that associates with two smaller auxiliary subunits, (31 and
(32. The [32 subunit is a
integral membrane glycoprotein that contains an extracellular Ig domain, and
its association with a
and (31 subunits correlates with increased functional expression of the
channel, a change in its gating
1S properties, as well as an increase in whole cell capacitance due to an
increase in membrane surface
area (Isom, L.L. et al. (1995) Cell 83:433-442).
Non voltage-gated Na+ channels include the members of the amiloride-sensitive
Na+
channel/degenerin (NaC/DEG) family. Channel subunits of this family are
thought to consist of two
transmembrane domains flanking a long extracellular loop, with the amino and
carboxyl termini
located within the cell. The NaCIDEG family includes the epithelial Nay
channel (ENaC) involved in
Nay reabsorption in epithelia including the airway, distal colon, cortical
collecting duct of the kidney,
and exocrine duct glands. Mutations in ENaC result in pseudohypoaldosteronism
type 1 and Liddle's
syndrome (pseudohyperaldosteronism). The NaC/DEG family also includes the
recently
characterized H+-gated cation channels or acid-sensing ion channels (ASIC).
ASIC subunits are
2S expressed in the brain and form heteromultimeric Na+-permeable channels.
These channels require
acid pH fluctuations for activation. ASIC subunits show homology to the
degenerins, a family of
mechanically-gated channels originally isolated from C. elegans. Mutations in
the degenerins cause
neurodegeneration. ASIC subunits may also have a role in neuronal function, or
in pain perception,
since tissue acidosis causes pain (Waldmann, R. and M. Lazdunski (1998) Curr.
Opin. Neurobiol.
8:418-424; Eglen, R.M. et al. (1999) Trends Pharmacol. Sci. 20:337-342).
K+ channels are located in all cell types, and may be regulated by voltage,
ATP
concentration, or second messengers such as Caz+ and CAMP. In non-excitable
tissue, I~+ 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
3S potentials and repolariziug membranes, I~+ channels are responsible for
setting the resting membrane
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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
Nay, K+, and Cl-. The
pump actively transports Na+ out of the cell and K+ into the cell in a 3:2
ratio. Ion channels in the
plasma membrane allow K+ and Cl- to flow by passive diffusion. Because of the
high negative charge
within the cytosol, Cl- flows out of the cell. The flow of K~ is balanced by
an electromotive force
pulling K+ into the cell, and a K+ concentration gradient pushing K+ out of
the cell. Thus, the resting
membrane potential is primarily regulated by K+flow (Salkoff, L. and T. Jegla
(1995) Neuron 15:489-
492).
Potassium channel subunits of the Shaker-like superfamily all have the
characteristic six
transmembrane/1 pore domain structure. Four subunits combine as homo- or
heter0tetramers 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)
Curr. Opin. Biotechnol. 9:565-572; Kaczorowski, G.J. and M.L. Garcia (1999)
Curr. Opin. Chem.
Biol. 3:448-458).
A second superfamily of K+ channels is composed of the inward rectifying
channels (Kir).
Kir channels have the property of preferentially conducting K+ currents in the
inward direction.
These proteins consist of a single potassium selective pore domain and two
transmembrane domains ,
which correspond to the fifth and sixth transmembrane domains of voltage-gated
K+ channels. Kir
subunits also 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).
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).
The voltage-gated Ca z+ channels have been classified into several subtypes
based upon their
electrophysiological and pharmacological characteristics. L-type Ca 2+
channels are predominantly
expressed in hears 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
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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 az8, 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 Z+ 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
Cav2 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
z+ c~.ents, which are activated and inactivated more rapidly and at more
negative membrane
potentials than other Ca Z+ 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 Ca 2+-channel may include one or more
Cache
domains. An extracellular Cache domain may be fused to an intracellular
catalytic domain, such as
the histidine kinase, PP2C phosphatase, GGDEF (a predicted diguanylate
cyclase), HD-GYP (a
predicted phosphodiesterase) or adenylyl cyclase domain, or to a noncatalytic
domain, like the
methyl-accepting, DNA-binding winged helix-turn-helix, GAF, PAS or HAMP (a
domain found in
istidine kinases, denylyl cyclases, ethyl-binding proteins and phosphatases).
Small molecules are
bound via the Cache domain and this signal is converted into diverse outputs
depending on the
intracellular domains (Anantharaman, V. and Aravind, L.(2000) Trends Biochem.
Sci. 25:535-537).
The transient receptor family (Trp) of calcium ion channels are thought to
mediate
capacitative calcium entry (CCE). CCE is the Ca2+ influx into cells to
resupply Ca2+ stores depleted
by the action of inositol triphosphate (Il'3) and other agents in response to
numerous hormones and
growth factors. Trp and Trp-like were first cloned from Drosophila and have
similarity to voltage
gated Ca 2+ channels in the S3 through S6 regions. This suggests that Trp
andlor related proteins may
form mammalian CCE channels (Zhu, X. et al. (1996) Cell 85:661-671; Boulay, G.
et al. (1997) J.
Biol. Chem. 272:29672-29680). Melastatin is a gene isolated in both the mouse
and human, whose
CA 02427010 2003-04-25
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expression in melanoma cells is inversely correlated with melanoma
aggressiveness in vivo. The
human cDNA transcript corresponds to a 1533-amino acid protein having homology
to members of
the Trp family. It has been proposed that the combined use of malastatin mRNA
expression status
and tumor thickness might allow for the determination of subgroups of patients
at both low and high
risk for developing metastatic disease (Duncan, L.M. et al (2001) J. Clin.
Oncol. 19:568-576).
Chloride channels are necessary in endocrine secretion and in regulation of
cytosolic and
organelle pH. In secretory epithelial cells, Cl- enters the cell across a
basolateral membrane through
an Na+, K+/Cl- cotransporter, accumulating in the cell above its
electrochemical equilibrium
concentration. Secretion of Cl- from the apical surface, in response to
hormonal stimulation, leads to
flow of Na+ and water into the secretory lumen. The cystic fibrosis
transmembrane conductance
regulator (CFTR) is a chloride channel encoded by the gene for cystic
fibrosis, a common fatal
genetic disorder in humans. CFTR is a member of the ABC transporter family,
and is composed of .
two domains each consisting of six transmembrane domains followed by a
nucleotide-binding site.
Loss of CFTR function decreases transepithelial water secretion and, as a
result, the layers of mucus
that coat the respiratory tree, pancreatic ducts, and intestine are dehydrated
and difficult to clear. The
resulting blockage of these sites leads to pancreatic insufficiency, "meconium
ileus", and devastating
"chronic obstructive pulmonary disease" (Al-Awqati, Q. et al. (1992) J. Exp.
Biol. 172:245-266).
The voltage-gated chloride channels (CLC) are characterized by 10-12
transmembrane
domains, as well as two small globular domains known as CBS domains. The CLC
subunits
probably function as homotetramers. CLC proteins are involved in regulation of
cell volume,
membrane potential stabilization, signal transduction, and transepithelial
transport. Mutations in
CLC-1, expressed predominantly in skeletal muscle, are responsible for
autosomal recessive
generalized myotonia and autosomal dominant myotonia congenita, while
mutations in the kidney
channel CLC-5 lead to kidney stones (Jentsch, T.J. (1996) Curr. Opin.
Neurobiol. 6:303-310).
Ligand-gated channels open their pores when an extracellular or intracellular
mediator binds
to the channel. Neurotransmitter-gated channels are channels that open when a
neurotransmitter
binds to their extracellular domain. These channels exist in the postsynaptic
membrane of nerve or
muscle cells. There are two types of neurotransmitter-gated channels. Sodium
channels open in
response to excitatory neurotransmitters, such as acetylcholine, glutamate,
and serotonin. This
opening causes an influx of Na+ and produces the initial localized
depolarization that activates the
voltage-gated channels and starts the action potential. Chloride channels open
in response to
inhibitory neurotransmitters, such as y-aminobutyric acid (GABA) and glycine,
leading to
hyperpolarization of the membrane and the subsequent generation of an action
potential.
Neurotransmitter-gated ion channels have four transmembrane domains and
probably function as
pentamers (Jentsch, supra). Amino acids in the second transmembrane domain
appear to be important
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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
ifs subunit
composition determined. The oc subunit of the BK channel has seven rather than
six transmembrane
a
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) Curr. Opin. Neurobiol. 8:321-329).
Cyclic nucleotide-gated (CNG) channels are gated by cytosolic cyclic
nucleotides. The best
examples of these are the cAMP-gated Na+ channels involved in olfaction and
the cGMP-gated cation
channels involved in vision. Both systems involve ligand-mediated activation
of a G-protein coupled
receptor which then alters the level of cyclic nucleotide within the cell. CNG
channels also represent
a major pathway for Ca2+ entry into neurons, and play roles in neuronal
development and plasticity.
CNG channels are tetramers containing at least two types of subunits, an a
subunit which can form
functional homomeric channels, and a (3 subunit, which modulates the channel
properties. All CNG
subunits have six transmembrane domains and a pore forming region between the
fifth and sixth
transmembrane domains, similax to voltage-gated K+ channels. A large C-
terminal domain contains a
cyclic nucleotide binding domain, while the N-terminal domain confers
variation among channel
subtypes (Zufall, F. et al. (1997) Curr. Opin. Neurobiol. 7:404-412).
The activity of other types of ion channel proteins may also be modulated by a
variety of
intracellular signalling proteins. Many channels have sites for
phosphorylation by one or more
protein kinases including protein kinase A, protein kinase C, tyrosine kinase,
and casein kinase II, all
of which regulate ion channel activity in cells. Kir channels are activated by
the binding of the G(3~y
subunits of heterotrimeric G-proteins (Reimann, F. and F.M. Ashcroft (1999)
Curr. Opin. Cell. Biol.
11:503-508). Other proteins are involved in the localization of ion channels
to specific sites in the
cell membrane. Such proteins include the PDZ domain proteins known as MAGUKs
(membrane-
associated guanylate kinases) which regulate the clustering of ion channels at
neuronal synapses
(Craven, S.E. and D.S. Bredt (1998) Cell 93:495-498).
Disease Correlation
The etiology of numerous human diseases and disorders can be attributed to
defects in the
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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, supra).
Ion channels have been the target for many drug therapies. Neurotransmitter-
gated channels
have been targeted in therapies for treatment of insomnia, anxiety,
depression, and schizophrenia.
Voltage-gated channels have been targeted in therapies for arrhythmia,
ischemic stroke, head trauma,
and neurodegenerative disease (Taylor, C.P. and L.S. Narasimhan (1997) Adv.
Pharmacol. 39:47-98).
Various classes of ion channels also play an important role in the perception
of pain, and thus are
potential targets for new analgesics. These include the vanilloid-gated ion
channels, which are
activated by the vanilloid capsaicin, as well as by noxious heat. Local
anesthetics such as lidocaine
and mexiletine which blockade voltage-gated Na+ channels have been useful in
the treatment of
neuropathic pain (Eglen, su ra).
Ion channels in the immune system have recently been suggested as targets for
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
13
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immunosuppressants (Cahalan, M.D. and I~.G. Chandy (1997) Curr. Opin.
Biotechnol. 8:749-756).
The discovery of new transporters and ion channels, and the polynucleotides
encoding them,
satisfies a need in the art by providing new compositions which are useful in
the diagnosis,
prevention, and treatment of transport, neurological, muscle, immunological
and cell proliferative
disorders,-and in the assessment of the effects of exogenous compounds on the
expression of nucleic
acid and amino acid sequences of transporters and ion channels.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, transporters and ion channels,
referred to
collectively as "TRICH" and individually as "TRICH-1," "TRICH-2," "TRICH-3,"
"TRICH-4,"
"TRICH-5," "TRICH-6," "TRICH-7," "TRICH-8," "TRICH-9," "TRICH-10," "TRICH-11,"
"TRICH-12," "TRICH-13," "TRICH-14," "TRICH-15," "TRICH-I6," "TRICH-17," "TRICH-
18,"
"TRICH-19," and "TRICH-20." 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:1-20, 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:1-20, c) a biologically active fragment of a
polypeptide having an amino
acid sequence selected from the group consisting of SEQ ~ N0:1-20, and d) an
immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
>D NO:1-20. In one alternative, the invention provides an isolated polypeptide
comprising the amino
acid sequence of SEQ ID NO:1-20.
The invention further provides an isolated polynucleotide encoding a
polypeptide selected
from the group consisting of a) a polypeptide comprising an amino acid
sequence selected from the
group consisting of SEQ ID NO:1-20, 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 N0:1-20, c) a biologically active fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ ID NO:1-20, and d) an immunogenic
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ lD NO:1-20.
In one alternative, the polynucleotide encodes a polypeptide selected from the
group consisting of
SEQ ID NO:1-20. In another alternative, the polynucleotide is selected from
the group consisting of
SEQ ID N0:21-40.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide selected
from the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ID NO:1-20, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
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90% identical to an amino acid sequence selected from the group consisting of
SEQ ID NO; l-20, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID NO: l-20, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ m NO:1-20. 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 NO: l-20, 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:1-20, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ m N0:1-20, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ID NO: l-20. The
method comprises a)
culturing a cell under conditions suitable for expression of the polypeptide,
wherein said cell is
transformed with a recombinant polynucleotide comprising a promoter sequence
operably linked to a
polynucleotide encoding the polypeptide, and b) recovering the polypeptide so
expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid
sequence selected from the group consisting of SEQ m NO:l-20, b) a polypeptide
comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ )D NO:1-20, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ lD
NO:1-20, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID N0:1-20.
The invention further provides an isolated polynucleotide selected from the
group consisting
of a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of
SEQ )D N0:21-40, 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
N0:21-40, 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 >D N0:21-40, b) a polynucleotide comprising a naturally
occurring polynucleotide
CA 02427010 2003-04-25
WO 02/40541 PCT/USO1/46055
sequence at least 90% identical to a polynucleotide sequence selected from the
group consisting of
SEQ m N0:21-40, 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 >D N0:21-40, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at
least 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ m
NO:21-40, c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide
complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
The method
comprises a) amplifying said target polynucleotide or fragment thereof using
polymerase chain
reaction amplification, and b) detecting the presence or absence of said
amplified target
polynucleotide or fragment thereof, and, optionally, if present, the amount
thereof.
The invention further provides a composition comprising an effective amount of
a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid
sequence selected from the group consisting of SEQ ID N0:1-20, 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 N0:1-20, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-20, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-20, and a pharmaceutically acceptable excipient. In
one embodiment, the
composition comprises an amino acid sequence selected from the group
consisting of SEQ m NO:1-
20. 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 NO:1-20, b) a
polypeptide comprising a
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naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-20, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID NO:
l-20, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ll~ NO:1-20. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting agonist activity in the sample. In
one alternative, the
invention provides a composition comprising an agonist compound identified by
the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with decreased expression of
functional TRICH, comprising
administering to a patient in need of such treatment the composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID NO: l-20, 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:1-20, c) a
biologically active fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ m N0:1-20,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID N0:1-20. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting antagonist activity in the sample.
In one alternative, the
invention provides a composition comprising an antagonist compound identified
by the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with overexpression of functional
TRICH, comprising
administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that
specifically binds
to a polypeptide selected from the group consisting of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-20, 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 N0:1-20, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
N0:1-20, and d) an
iixnnunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-20. The method comprises a) combining the
polypeptide with at least
one test compound under suitable conditions, and b) detecting binding of the
polypeptide to the test
compound, thereby identifying a compound that specifically binds to the
polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
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acid sequence selected from the group consisting of SEQ ID NO:1-20, 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: l-20, c) a biologically active
fragment of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-20, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-20. 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 ID N0:21-40,
the method
comprising a) exposing a sample comprising the taxget polynucleotide to a
compound, and b)
detecting altered expression of the target polynucleotide.
The invention further provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound;
b) hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide selected from the group consisting
of i) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:21-40, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:21-40,
iii) a
polynucleotide having a sequence complementary to i), iv) a polynucleotide
complementary to the
polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization
occurs under conditions
whereby a specific hybridization complex is formed between said probe and a
taxget 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
N0:21-40, ii) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ ID N0:21-40,
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
3S hybridization complex in the treated biological sample with the amount of
hybridization complex in
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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 ofthe presentinvention.
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
polypeptides of the invention, along with applicable descriptions, references,
and threshold
parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which
will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof lrnown 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.
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Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods are now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing
the cell lines, protocols, reagents and vectors which are reported in the
publications and which might
be used in connection with the invention. Nothing herein is to be construed as
an admission that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
DEFINITIONS
"TRICH" refers to the amino acid sequences of substantially purified TRICH
obtained from
any species, particularly a mammalian species, including bovine, ovine,
porcine, marine, equine, and
human, and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
TRICH. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of TRICH either by
directly interacting with
TRICH or by acting on components of the biological pathway in which TRICH
participates.
An "allelic variant" is an alternative form of the gene encoding TRICH.
Allelic variants may
result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in
polypeptides whose structure or function may or may not be altered. A gene may
have none, one, or
many allelic variants of its naturally occurring form. Common mutational
changes which give rise to
allelic variants are generally ascribed to natural deletions, additions, or
substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the
others, one or more times
in a given sequence.
"Altered" nucleic acid sequences encoding TRICH include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as TRICH or a
polypeptide with at least one functional characteristic of TRICH. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe
of the polynucleotide encoding TRICH, and improper or unexpected hybridization
to allelic variants,
with a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding
TRICH. The encoded protein may also be "altered," and may contain deletions,
insertions, or
substitutions of amino acid residues which produce a silent change and result
in a functionally
equivalent TRICH. 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 glutamuc 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.
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Amino acids with uncharged side chains having similar hydrophilicity values
may include: leucine,
isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or synthetic
molecules. Where "amino acid sequence" is recited to refer to a sequence of a
naturally occurring
protein molecule, "amino acid sequence" and like terms are not meant to limit
the amino acid
sequence to the complete native amino acid sequence associated with the
recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well
known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of TRICH. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of TRICH either by
directly interacting with TRICH or by acting on components of the biological
pathway in which
TRICH participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, 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 an animal (e.g., a mouse, a rat, or a rabbit)
can be derived from the
translation of RNA, or synthesized chemically, and can be conjugated to a
carrier protein if desired.
Commonly used carriers that are chemically coupled to peptides include bovine
serum albumin,
thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is
then used to immunize
the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures
on the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen '
used to elicit the immune response) for binding to an antibody.
The term "aptamer" refers to a nucleic acid or oligonucleotide molecule that
binds to a
specific molecular target. Aptamers are derived from an in vitro evolutionary
process (e.g., SELEX
(Systematic Evolution of Ligands by EXponential Enrichment), described in U.S.
Patent No.
5,270,263), which selects for target-specific aptamer sequences from large
combinatorial libraries.
Aptamer compositions may be double-stranded or single-stranded, and may
include
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deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other
nucleotide-like molecules.
The nucleotide components of an aptamer may have modified sugar groups (e.g.,
the 2'-OH group of a
ribonucleotide may be replaced by 2'-F or 2'-NHZ), which may improve a desired
property, e.g.,
resistance to nucleases or longer lifetime in blood. Aptamers may be
conjugated to other molecules,
e.g., a high molecular weight carrier to slow clearance of the aptamer from
the circulatory system.
Aptamers may be specifically cross-linked to their cognate ligands, e.g., by
photo-activation of a
cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-
13.)
The term "intramer" refers to an aptamer which is expressed in vivo. For
example, a vaccinia
virus-based RNA expression system has been used to express specific RNA
aptamexs at high levels in
the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl Acad. Sci. USA
96:3606-3610).
The term "spiegelmer" refers to an aptamer which includes L-DNA, L-RNA, or
other left-
handed nucleotide derivatives or nucleotide-like molecules. Aptamers
containing left-handed
nucleotides are resistant to degradation by naturally occurring enzymes, which
normally act on
substrates containing right-handed nucleotides.
The term "antisense" refexs to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA;
RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone
linkages such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
designation "positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic TRICH, or
of any oligopeptide
thereof, to induce a specific immune response .in appropriate animals or cells
and to bind with specific
antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising
a given amino acid sequence" refer broadly to any composition containing the
given polynucleotide
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or amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding TRICH or fragments
of TRICH may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCI), detergents
(e.g., sodium dodecyl
sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk,
salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to repeated
DNA sequence analysis to resolve uncalled bases, extended using the ~,-PCR kit
(Applied
Biosystems, Foster City CA) in the 5' and/or the 3' direction, and
resequenced, or which has been
assembled from one or more overlapping cDNA, EST, or genomic DNA fragments
using a computer
program for fragment assembly, such as the GELVIEW fragment assembly system
(GCG, Madison
WI) or Phrap (University of Washington, Seattle WA). Some sequences have been
both extended and
assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted 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, Isis
Gly Ala
His Asn, Arg, Gln, Glu
lle Leu, Val
Leu lle, 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
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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 noneovalently joined to a
polynucleotide or polypeptide.
"Differential expression" refers to increased or upregulated; or decreased,
downregulated, or
absent gene or protein expression, determined by comparing at least two
different samples. Such
comparisons may be carried out between, for example, a treated and an
untreated sample, or a
diseased and a normal sample.
"Exon shuffling" refers to the recombination of different coding regions
(exons). Since an
exon may represent a structural or functional domain of the encoded protein,
new proteins may be
assembled through the novel reassortment of stable substructures, thus
allowing acceleration of the
evolution of new protein functions.
A "fragment" is a unique portion of TRICH or the polynucleotide encoding TRICH
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example, a
fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10,
15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 25°10 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 N0:21-40 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:21-40, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:21-40 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
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SEQ ID N0:21-40 from related polynucleotide sequences. The precise length of a
fragment of SEQ
ID N0:21-40 and the region of SEQ ID N0:21-40 to which the fragment
corresponds are routinely
determinable by one of ordinary skill in the art based on the intended purpose
for the fragment.
A fragment of SEQ ID NO:1-20 is encoded by a fragment of SEQ ID N0:21-40. A
fragment
of SEQ ID NO:1-20 comprises a region of unique amino acid sequence that
specifically identifies
SEQ ID NO:1-20. For example, a fragment of SEQ ID N0:1-20 is useful as an
immunogenic peptide
for the development of antibodies that specifically recognize SEQ ID N0:1-20.
The precise length of
a fragment of SEQ ID NO:1-20 and the region of SEQ ID NO:1-20 to which the
fragment
corresponds are routinely determinable by one of ordinary skill in the art
based on the intended
purpose for the fragment.
A "full length" polynucleotide sequence is one containing at least a
translation initiation
codon (e.g., methionine) followed by an open reading frame and a translation
termination codon. A
"full length" polynucleotide sequence encodes a "full length" polypeptide
sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between
two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide
sequences, refer
to the percentage of residue matches between at least two polynucleotide
sequences aligned using a
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 Higgins, D.G. et
al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The
"weighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
is provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment
Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410),
which is available
from several sources, including the NCBI, Bethesda, MD, and on the Internet at
http:/lwww.ncbi.nlm.nih.govBLAST/. The BLAST software suite includes various
sequence
analysis programs including "blastn," that is used to align a known
polynucleotide sequence with
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other polynucleotide sequences from a variety of databases. Also available is
a tool called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html.
The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for snatch: 1
Penalty for ~nisrnatch: -2
Open.Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off: SO
Expect: 10
Word Size: 11
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ 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 program (described and referenced above). For pairwise
alignments of
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polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version
2Ø12 (April-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Open Gap: 11 and Extension Gap: 1 penalties
Gap x drop-off. SO
Expect: 10
Word Size: 3
Filter: orc
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 axe 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) axe linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the
stringency of the hybridization process, with more stringent conditions
allowing less non-specific
binding, i.e., binding between pairs of nucleic acid strands that are not
perfectly matched. Permissive
conditions for annealing of nucleic acid sequences are routinely determinable
by one of ordinary skill
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WO 02/40541 PCT/USO1/46055
in the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
1% (w/v) SDS, and about 100 ~,glml 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 (Tm) for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook, J. et al.
(1989) Molecular Cloning: A Laboratory Manual, 2na 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
~,g/ml. Organic
solvent, such as formamide at a concentration of about 35-50% v/v, may also be
used under particular
circumstances, such as for RNA:DNA hybridizations. Useful variations on these
wash conditions
will be readily apparent to those of ordinary skill in the art. Hybridization,
particularly under high
stringency conditions, may be suggestive of evolutionary similarity between
the nucleotides. Such
similarity is strongly indicative of a similar role for the nucleotides and
their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
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An "immunogenic fragment" is a polypeptide or oligopeptide fragment of TRICH
which is
capable of eliciting an immune response when introduced into a living
organism, for example, a
mammal. The term "immunogenic fragment" also includes any polypeptide or
oligopeptide fragment
of TRICH which is useful in any of the antibody production methods disclosed
herein or known in the
art.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides,
polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of TRICH. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
biological, functional, or immunological properties of TRICH.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop
transcript
elongation, and may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an TRICH may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in
the art. These processes may occur synthetically or biochemically. Biochemical
modifications will
vary by cell type depending on the enzymatic milieu of TRICH.
"Probe" refers to nucleic acid sequences encoding TRICH, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents,
and enzymes.
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be
annealed to a target
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polynucleotide by complementary base-pairing. The primer may then be extended
along the target
DNA strand by a DNA polymerase enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence, e.g., by the polymerase chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Bioloav, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to
5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer
selection programs have incorporated additional features for expanded
capabilities. For example, the
PrimOU primer selection program (available to the public from the Genome
Center at University of
Texas South West Medical Center, Dallas TX) is capable of choosing specific
primers from
megabase sequences and is thus useful for designing primers on a genome-wide
scope. The Primer3
primer selection program (available to the public from the Whitehead
Institute/MIT Center for
Genome Research, Cambridge MA) allows the user to input a "mispriming
library," in which
sequences to avoid as primer binding sites are user-specified. Primer3 is
useful, in particular, for the
selection of oligonucleotides fox 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 UI~ Human
Genome Mapping
Project Resource Centre, Cambridge UK) designs primers based on multiple
sequence alignments,
thereby allowing selection of primers that hybridize to either the most
conserved or least conserved
regions of aligned nucleic acid sequences. Hence, this program is useful for
identification of both
unique and conserved oligonucleotides and polynucleotide fragments. The
oligonucleotides and
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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, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter
sequence. Such a recombinant nucleic acid may be part of a vector that is
used, for example, to
transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions
(ITTRs). Regulatory elements interact with host or viral proteins which
control transcription,
translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of the
nitrogenous base thymine are replaced with uracil, and the sugar backbone is
composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing TRICH,
nucleic acids encoding TRICH, or fragments thereof may comprise a bodily
fluid; an extract from a
cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic
DNA, RNA, or
cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natuxal or
synthetic binding composition. The interaction is dependent upon the presence
of a particular
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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 eukaxyotic host cell. The method for
transformation is selected based
on the type of host cell being transformed and may include, but is not limited
to, bacteriophage or
viral infection, electroporation, heat shock, lipofection, and particle
bombardment. The term
"transformed cells" includes stably transformed cells in which the inserted
DNA is capable of
replication either as an autonomously replicating plasmid or as part of the
host chromosome, as well
as transiently transformed cells which express the inserted DNA or RNA for
limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not
limited to
animals and plants, in which one or more of the cells of the organism contains
heterologous nucleic
acid introduced by way of human intervention, such as by transgenic techniques
well known in the
art. The nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor
of the cell, by way of deliberate genetic manipulation, such as by
microinjection or by infection with
a recombinant virus. The term genetic manipulation does not include classical
cross-breeding, or in
vitro fertilization, but rather is directed to the introduction of a
recombinant DNA molecule. The
transgenic organisms contemplated in accordance with the present invention
include bacteria,
cyanobacteria, fungi, plants and animals. The isolated DNA of the present
invention can be
introduced into the host by methods known in the art, for example infection,
transfection,
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transformation or transconjugation. Techniques for transferring the DNA of the
present invention
into such organisms are widely known and provided in references such as
Sambrook et al. (1989),
supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91
%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99% or greater
sequence identity over a certain defined length. A variant may be described
as, for example, an
"allelic" (as defined above), "splice," "species," or "polymorphic" variant. A
splice variant may have
significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternate splicing of exons during mRNA processing. The
corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotide sequences that vary
from one species to
another. The resulting polypeptides will generally have significant amino acid
identity relative to
each other. A polymorphic variant is a variation in the polynucleotide
sequence of a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state. .
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least
92%, at least 93%, at least
94%, at least 95%, at least 96%, at Ieast 97%, at least 98%, or at least 99%
or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human transporters and ion
channels
(TRICH), the polynucleotides encoding TRICH, and the use of these compositions
for the diagnosis,
treatment, or prevention of transport, neurological, muscle, immunological and
cell proliferative
disorders.
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
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sequences of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to a
single Incyte project identification number (Incyte Project )D). Each
polypeptide sequence is denoted
by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:)
and an Incyte
polypeptide sequence number (Incyte Polypeptide ID) as shown. Each
polynucleotide sequence is
denoted by both a polynucleotide sequence identification number
(Polynucleotide SEQ ID NO:) and
an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID)
as shown.
Table 2 shows sequences with homology to the polypeptides of the invention as
identified by
BLAST analysis against the GenBank protein (genpept) database. Columns 1 and 2
show the
polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide )D) for polypeptides of the
invention. Column 3
shows the GenBank identification number (GenBank )D NO:) of the nearest
GenBank homolog.
Column 4 shows the probability scores for the matches between each polypeptide
and its homolog(s).
Column 5 shows the annotation of the GenBank homologs along with relevant
citations where
applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the
invention. Columns 1
and 2 show the polypeptide sequence identification number (SEQ ID NO:) and the
corresponding
Incyte polypeptide sequence number (Incyte Polypeptide ID) for each
polypeptide of the invention.
Column 3 shows the number of amino acid residues in each polypeptide. Column 4
shows potential
phosphorylation sites, and column 5 shows potential glycosylation sites, as
determined by the
MOTIFS program of the GCG sequence analysis software package (Genetics
Computer Group,
Madison WI). Column 6 shows amino acid residues comprising signature
sequences, domains, and
motifs. Column 7 shows analytical methods for protein structure/function
analysis and in some cases,
searchable databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the
invention, and these
properties establish that the claimed polypeptides are transporters and ion
channels. For example,
SEQ >D NO:S is 61% identical to Drosophila sodium-hydrogen exchanger NHE1
(GenBank ll~
g4894991) as determined by the Basic Local Alignment Search Tool (BLAST). (See
Table 2.) The
BLAST probability score is 6.Oe-139, which indicates the probability of
obtaining the observed
polypeptide sequence alignment by chance. SEQ >D N0:5 also contains a
sodium/hydrogen
exchanger family 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 BLI1VVIPS analysis provides further corroborative evidence
that SEQ )D N0:5 is a
sodium/hydrogen exchanger. In an alternative example, SEQ ID N0:6 is about 50%
identical to
human citrin, the adult-onset type II citrullinemia protein, (GenBank )D
g5052319) as determined by
the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST
probability score is
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6.Oe-51, which indicates the probability of obtaining the observed polypeptide
sequence alignment by
chance. SEQ ID N0:6 also contains mitochondria) earner protein domains as
determined by
searching for statistically significant matches in the hidden Markov model
(HIVIM)-based PFAM
database of conserved protein family domains. (See Table 3.) Data from
BLIIVVIPS, MOTIFS, and
PROFILESCAN analyses provide further corroborative evidence that SEQ ID N0:6
is a
mitochondria) carrier protein. In an alternative example, SEQ ID N0:7 is 27%
identical to
Synechocystis sp. melibiose carrier protein (GenBank ID g1653342) as
determined by the Basic
Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability
score is 1.8e-16,
which indicates the probability of obtaining the observed polypeptide sequence
alignment by chance.
Additional BLAST data from DOMO and PRODOM analyses provide further
corroborative evidence
that SEQ ID N0:7 is a symporter protein. In an alternative example, SEQ m N0:9
is 26% identical
to an Arabidopsis ABC transporter (GenBank ID g4262239) and is 99% identical,
from residue M1 to
residue W374, to human sterolin-2 (GenBank ID g15146444) as determined by the
Basic Local
Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability scores are
4.1e-25 and 0.0
respectively, which indicate the probabilities of obtaining the observed
polypeptide sequence
alignments by chance. SEQ ID N0:9 contains two transmembrane domains as
determined by hidden
Markov model (HMM) analysis, as well as a white/scarlet ABC transporter
domain. (See Table 3.)
These data provide further corroborative evidence that SEQ ID N0:9 is an ABC
transporter. In an
alternative example, SEQ ID N0:12 is 93% identical to rat neuronal glutamine
transporter (GenBank
ID g6978016) as determined by the Basic Local Alignment Search Tool (BLAST).
(See Table 2.)
The BLAST probability score is 4.4e-239, which indicates the probability of
obtaining the observed
polypeptide sequence alignment by chance. SEQ ID N0:12 also contains a
transmembrane amino
acid transporter domain as determined by searching for statistically
significant matches in the hidden
Markov model (HMM)-based PFAM database of conserved protein family domains.
(See Table 3.)
These data provide corroborative evidence that SEQ ID N0:12 is an amino acid
transporter protein.
In an alternative example, SEQ ID N0:14 is 52% identical to mouse multidrug
resistance protein
(GenBank ID g387426) as determined by the Basic Local Alignment Search Tool
(BLAST). (See
Table 2.) The BLAST probability score is 0.0, which indicates the probability
of obtaining the
observed polypeptide sequence alignment by chance. SEQ ID NO:14 also contains
an ABC
transporter domain and an ABC transporter transmembrane region 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,
MOTIFS, and
PROFILESCAN analyses provide further corroborative evidence that SEQ ID N0:14
is a multidrug
resistance ABC transporter. In an alternative example, SEQ ID N0:18 is 41%
identical to
Arabid~sis putative membrane transporter (GenBank ID g2289003) and is 99%
identical, from
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residue M20 to residue E648, to human proton myo-inositol transporter (GenBank
ID g152I1933) as
determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.)
The BLAST
probability scores are 1.4e-94 and 0.0 respectively, which indicate the
probabilities of obtaining the
observed polypeptide sequence alignments by chance. SEQ ID N0:18 also contains
a sugar (and
other) transporter domain as determined by searching for statistically
significant matches in the
hidden Markov model (HMM)-based PFAM database of conserved protein family
domains. (See
Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses provide further
corroborative
evidence that SEQ ID N0:18 is a sugar transporter. SEQ ID NO: l-4, SEQ ID
N0:8, SEQ ID N0:10-
l l, SEQ ID N0:13, SEQ ID N0:15-17, and SEQ TD N0:19-20 were analyzed and
annotated in a
similar manner. The algorithms and parameters for the analysis of SEQ ID NO:1-
20 are described in
Table 7.
As shown in Table 4, the full length polynucleotide sequences of the present
invention were
assembled using cDNA sequences or coding (exon) sequences derived from genomic
DNA, or any
combination of these two types of sequences. Columns l and 2 list the
polynucleotide sequence
identification number (Polynucleotide SEQ ID NO:) and the corresponding Incyte
polynucleotide
consensus sequence number (Incyte Polynucleotide ID) for each polynucleotide
of the invention.
Column 3 shows the length of each polynucleotide sequence in basepairs. Column
4 lists fragments
of the polynucleotide sequences which are useful, for example, in
hybridization or amplification
technologies that identify SEQ ID N0:21-40 or that distinguish between SEQ ID
N0:21-40 and
related polynucleotide sequences. Column 5 shows identification,numbers
corresponding to cDNA
sequences, coding sequences (exons) predicted from genomic DNA, and/or
sequence assemblages
comprised of both cDNA and genomic DNA. These sequences were used to assemble
the full length
polynucleotide sequences of the invention. Columns 6 and 7 of Table 4 show the
nucleotide start (5')
and stop (3') positions of the cDNA and/or genomic sequences in column 5
relative to their respective
full length sequences.
The identification numbers in Column 5 of Table 4 may refer specifically, for
example, to
Incyte cDNAs along with their corresponding cDNA libraries. For example,
6122382H1 is the
identification number of an Incyte cDNA sequence, and BRAHNON05 is the cDNA
library from
which it is derived. Incyte cDNAs for which cDNA libraries are not indicated
were derived from
pooled cDNA libraries (e.g., 72008374V 1). Alternatively, the identification
numbers in column 5
may refer to GenBank cDNAs or ESTs (e.g., g2077361) which contributed to the
assembly of the full
length polynucleotide sequences. In addition, the identification numbers in
column 5 may identify
sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database
(i. e., those
sequences including the designation "ENST"). Alternatively, the identification
numbers in column 5
may be derived from the NCBI RefSeq Nucleotide Sequence Records Database
(i.e., those sequences
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including the designation "NM" or "NT") or the NCBI RefSeq Protein Sequence
Records (i.e., those
sequences including the designation "NP"). Alternatively, the identification
numbers in column 5
may refer to assemblages of both cDNA and Genscan-predicted exons brought
together by an "exon
stitching" algorithm. For example, FL_;XXX NI Nz_YYYYY N3 IV4 represents a
"stitched"
sequence in which XXXXX11 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
N~,2,3_.., if present, represent specific exons that may have been manually
edited during analysis (See
Example V). Alternatively, the identification numbers in column 5 may refer to
assemblages of
exons brought together by an "exon-stretching" algorithm. For example,
FLXXXXXX_gAAAAA_gBBBBB_1 N is the identification number of a "stretched"
sequence, with
XXX~YXX being the Incyte project identification number, gAAAAA being the
GenBank identification
number of the human genomic sequence to which the "exon-stretching" algorithm
was applied,
gBBBBB being the GenBank identification number or NCBI RefSeq identification
number of the
nearest GenBank protein homolog, and N referring to specific exons (See
Example V). In instances
where a RefSeq sequence was used as a protein homolog for the "exon-
stretching" algorithm, a
RefSeq identifier (denoted by "NM," "NP," or "NT") may be used in place of the
GenBank identifier
(i.e., gBBBBB).
Alternatively, a prefix identifies component sequences that were hand-edited,
predicted from
genomic DNA sequences, or derived from a combination of sequence analysis
methods. The
following Table lists examples of component sequence prefixes and
corresponding sequence analysis
methods associated with the prefixes (see Example IV and Example V).
Prefix Type of analysis and/or examples of programs
GNN, GFG,Exon prediction from genomic sequences using,
for example,
ENST GENSCAN (Stanford University, CA, USA) or
FGENES
(Computer Genomics Group, The Sanger Centre,
Cambridge, UK).
GBI Hand-edited analysis of genomic sequences.
FL Stitched or stretched genomic sequences (see
Example V).
INCY Full length transcript and 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
column 5 was obtained to confirm the final consensus polynucleotide sequence,
but the relevant
Incyte cDNA identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length
polynucleotide
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sequences which were assembled using Incyte cDNA sequences. The representative
cDNA library is
the Incyte cDNA library which is most frequently represented by the Incyte
cDNA sequences which
were used to assemble and confirm the above polynucleotide sequences. The
tissues and vectors
which were used to construct the cDNA libraries shown in Table S are described
in Table 6.
The invention also encompasses TRICH variants. A preferred TRICH variant is
one which
has at least about 80%, or alternatively at Ieast about 90%, or even at least
about 95% amino acid
sequence identity to the TRICH amino acid sequence, and which contains at
least one functional or
structural characteristic of TRICH.
The invention also encompasses polynucleotides which encode TRICH. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ m N0:21-40, which encodes TRICH. The
polynucleotide
sequences of SEQ ID N0:21-40, 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
N0:21-40 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 N0:21-40. 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.
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It will be appreciated by those skilled in the art that as a result of the
degeneracy of the
genetic code, a multitude of polynucleotide sequences encoding TRICH, some
bearing minimal
similarity to the polynucleotide sequences of any known and naturally
occurring gene, may be
produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
sequence that could be made by selecting combinations based on possible codon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring TRICH, and all such variations
are to be considered
as being specifically disclosed.
Although nucleotide sequences which encode TRICH and its variants are
generally capable of
hybridizing to the nucleotide sequence of the naturally occurring TRICH under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding TRICH
or its derivatives possessing a substantially different codon usage, e.g.,
inclusion of non-naturally
occurring codons. Codons may be selected to increase the rate at which
expression of the peptide
occurs in a particular prokaryotic or eukaryotic host in accordance with the
frequency with which
particular codons are utilized by the host. Other reasons for substantially
altering the nucleotide
sequence encoding TRICH and its derivatives without altering the encoded amino
acid sequences
include the production of RNA transcripts having more desirable properties,
such as a greater
half life, than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode TRICH
and
TRICH derivatives, or fragments thereof, entirely by synthetic chemistry.
After production, the
synthetic sequence may be inserted into any of the many available expression
vectors and cell
systems using reagents well known in the art. Moreover, synthetic chemistry
may be used to
introduce mutations into a sequence encoding TRICH or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
N0:21-40 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods
Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash
conditions, are described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
I~lenow fragment
of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(Applied
Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerases and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
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automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
(Applied Biosystems). Sequencing is then carried out using either the ABI 373
or 377 DNA
sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing
system
(Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The
resulting sequences
are analyzed using a variety of algorithms which are well known in the art.
(See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Bioloev, John Wiley & Sons, New York NY,
unit 7.7; Meyers,
R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp.
856-853.)
The nucleic acid sequences encoding TRICH may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
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% 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
CA 02427010 2003-04-25
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the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof
which encode TRICH may be cloned in recombinant DNA molecules that direct
expression of
TRICH, or 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 axe
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 occurnng genes in a
directed and controllable
41
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WO 02/40541 PCT/USO1/46055
manner.
In another embodiment, sequences encoding TRICH may be synthesized, in whole
or in part,
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
al. (1980) Nucleic Acids
Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.)
Alternatively, TRICH itself or a fragment thereof may be synthesized using
chemical methods. For
example, peptide synthesis can be performed using various solution-phase or
solid-phase techniques.
(See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular
Properties, WH Freeman, New
York NY, pp. 55-60; and Roberge, J.Y. et al. (I995) Science 269:202-204.)
Automated synthesis
may be achieved using the ABI 431A peptide synthesizer (Applied Biosystems).
Additionally, the
amino acid sequence of TRICH, or any part thereof, may be altered during
direct synthesis and/or
combined with sequences from other proteins, or any part thereof, to produce a
variant polypeptide or
a polypeptide having a sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by
sequencing. (See, e.g., Creighton, supra, pp. 28-53.)
In order to express a biologically active TRICH, the nucleotide sequences
encoding TRICH
or derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which
contains the necessary elements for transcriptional and translational control
of the inserted coding
sequence in a suitable host. These elements include regulatory sequences, such
as enhancers,
constitutive and inducible promoters, and 5' and 3' untranslated regions in
the vector and in
polynucleotide sequences encoding TRICH. Such elements may vary in their
strength and specificity.
Specific initiation signals may also be used to achieve more efficient
translation of sequences
encoding TRICH. Such signals include the ATG initiation codon and adjacent
sequences, e.g. the
Kozak sequence. In cases where sequences encoding TRICH and its initiation
codon and upstream
regulatory sequences are inserted into the appropriate expression vector, no
additional transcriptional
or translational control signals may be needed. However, in cases where only
coding sequence, or a
fragment thereof, is inserted, exogenous translational control signals
including an in-frame ATG
initiation codon should be provided by the vector. Exogenous translational
elements and initiation
codons may be of various origins, both natural and synthetic. The efficiency
of expression may be
enhanced by the inclusion of enhancers appropriate for the particular host
cell system used. (See,
e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding TRICH and appropriate transcriptional
and translational
control elements. These methods include in vitro recombinant DNA techniques,
synthetic techniques,
42
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WO 02/40541 PCT/USO1/46055
and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989)
Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-
17; Ausubel, F.M. et
al. (1995) Current Protocols in Molecular Biolo~y, John Wiley & Sons, New York
NY, ch. 9, 13, and
16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding TRICH. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV,
or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti
or pBR322 plasmids); or
animal cell systems. (See, e.g., Sambrook, supra; Ausubel, supra; Van Heeke,
G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.I~. et al. (1994) Proc.
Natl. Acad. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu,
N. (1987) EMBO
J. 6:307-311; The McGraw Hill Yearbook of Science and Technoloay (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. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature
389:239-242.)
The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding TRICH. For
example, routine cloning,
subcloning, and propagation of polynucleotide sequences encoding TRICH can be
achieved using a
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORT1
plasmid (Life Technologies). Ligation of sequences encoding TRICH into the
vector's multiple
cloning site disrupts the lacZ gene, allowing a colorimetric screening
procedure for identification of
transformed bacteria containing recombinant molecules. In addition, these
vectors may be useful for
in vitro transcription, dideoxy sequencing, single strand rescue with helper
phage, and creation of
nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M.
Schuster (1989) J. Biol.
Chem. 264:5503-5509.) When large quantities of TRICH are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of TRICH may be used.
For example, vectors
containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of TRICH. A number of
vectors
43
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WO 02/40541 PCT/USO1/46055
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable
integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel,
1995, supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and
Scorer, C.A. et al. (1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of TRICH. Transcription of
sequences
encoding TRICH may be driven by viral promoters, e.g., the 35S and 19S
promoters of CaMV used
alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-
1680; Broglie, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105.)
These constructs can be introduced into plant cells by direct DNA
transformation or
pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of
Science and Technolo~y
(1992) McGraw Hill, New York NY, pp. 191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding TRICH
may be ligated into
an adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses TRICH in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus ('RSV) enhancer, may be used to increase expression in mammalian host
cells. SV40 or EBV-
based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. (1997) Nat. Genet.
15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression
of TRICH in cell lines is preferred. For example, sequences encoding TRICH can
be transformed
into cell lines using expression vectors which may contain viral origins of
replication and/or
endogenous expression elements and a selectable marker gene on the same or on
a separate vector.
Following the introduction of the vector, cells may be allowed to grow for
about 1 to 2 days in
enriched media before being switched to selective media. The purpose of the
selectable marker is to
confer resistance to a selective agent, and its presence allows growth and
recovery of cells which
44.
CA 02427010 2003-04-25
WO 02/40541 PCT/USO1/46055
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,
dlzfr confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. (1981)
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and IzisD, which
alter cellular requirements for metabolites. (See, e.g., Haxtman, S.C. and
R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech),13 glucuronidase and its substrate 13-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
confirmed. For example, if the
sequence encoding TRICH is inserted 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.
hnmunological methods for detecting and measuring the expression of TRICH
using either
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques
include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),
and
fluorescence activated cell sorting (FAGS). 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,
CA 02427010 2003-04-25
WO 02/40541 PCT/USO1/46055
e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN,
Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Immunolo~y, Greene
Pub. Associates and
Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical
Protocols, Humana
Press, Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled
hybridization or PCR probes for detecting sequences related to polynucleotides
encoding TRICH
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide.
Alternatively, the sequences encoding TRICH, or any fragments thereof, may be
cloned into a vector
for the production of an mRNA probe. Such vectors are known in the art, are
commercially available,
and may be used to synthesize RNA probes in vitro by addition of an
appropriate RNA polymerase
such as T7, T3, or SP6 and labeled nucleotides. These procedures may be
conducted using a variety
of commercially available kits, such as those provided by Amersham Pharmacia
Biotech, Promega
(Madison WI), and US Biochemical. Suitable reporter molecules or labels which
may be used for
ease of detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic
agents, as well as substrates, cofactors, inhibitors, magnetic particles, and
the like.
Host cells transformed with nucleotide sequences encoding TRICH may be
cultured under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the sequence
and/or the vector used. As will be understood by those of skill in the art,
expression vectors
containing polynucleotides which encode TRICH may be designed to contain
signal sequences which
direct secretion of TRICH through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of
the polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro" or
"pro" form of the protein may also be used to specify protein targeting,
folding, andlor activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are
available from the
American Type Culture Collection (ATCC, Manassas VA) and may be chosen to
ensure the correct
modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding TRICH may be ligated to a heterologous sequence resulting
in translation of a
fusion protein in any of the aforementioned host systems. For example, a
chimeric TRICH protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
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facilitate the screening of peptide libraries for inhibitors of TRICH
activity. Heterologous protein and
peptide moieties may also facilitate purification of fusion proteins using
commercially available
affinity matrices. Such moieties include, but are not limited to, glutathione
S-transferase (GST),
maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide
(CBP), 6-His, FLAG,
c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification of their
cognate fusion proteins on immobilized glutathione,~maltose, phenylarsine
oxide, calmodulin, and
metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable
immunoaffinity
purification of fusion proteins using commercially available monoclonal and
polyclonal antibodies
that specifically recognize these epitope tags. A fusion protein may also be
engineered to contain a
proteolytic cleavage site located between the TRICH encoding sequence and the
heterologous protein
sequence, so that TRICH may be cleaved away from the heterologous moiety
following purification.
Methods for fusion protein expression and purification are discussed in
Ausubel (1995, supra, ch. 10).
A variety of commercially available kits may also be used to facilitate
expression and purification of
fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled TRICH may
be achieved
in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These
systems couple transcription and translation of protein-coding sequences
operably associated with the
T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
precursor, for example, 35S-methionine.
TRICH of the present invention or fragments thereof may be used to screen for
compounds
that specifically bind to TRICH. At least one and up to a plurality of test
compounds may be
screened for specific binding to TRICH. Examples of test compounds include
antibodies,
oligonucleotides, proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the
natural ligand of
TRICH, e.g., a ligand or fragment thereof, a natural substrate, a structural
or functional mimetic, or a
natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current
Protocols in Immunology 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which TRICH
binds, or to at least a fragment of the receptor, e.g., the ligand binding
site. In either ease, the
compound can be rationally designed using known techniques. In one embodiment,
screening for
these compounds involves producing appropriate cells which express TRICH,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or
E. coli. Cells expressing TRICH or cell membrane fractions which contain TRICH
are then contacted
with a test compound and binding, stimulation, or inhibition of activity of
either TRICH or the
compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide,
wherein binding is
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CA 02427010 2003-04-25
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detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable
Iabel. For example,
the assay may comprise the steps of combining at least one test compound with
TRICH, either in
solution or affixed to a solid support, and detecting the binding of TRICH to
the compound.
Alternatively, the assay may detect or measure binding of a test compound in
the presence of a
labeled competitor. Additionally, the assay may be carried out using cell-free
preparations, chemical
libraries, or natural product mixtures, and the test compounds) may be free in
solution or affixed to a
solid support.
TRICH of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of TRICH. Such compounds may include agonists,
antagonists, or partial
or inverse agonists. In one embodiment, an assay is performed under conditions
permissive for
TRICH activity, wherein TRICH is combined with at least one test compound, and
the activity of
TRICH in the presence of a test compound is compared with the activity of
TRICH in the absence of
the test compound. A change in the activity of TRICH in the presence of the
test compound is
indicative of a compound that modulates the activity of TRICH. Alternatively,
a test compound is
combined with an in vitro or cell-free system comprising TRICH under
conditions suitable for
TRICH activity, and the assay is performed. In 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 phosphotransferase
gene (neo; Capecchi, M.R.
(1989) Science 244:1288-1292). The vector integrates into the corresponding
region of the host
genome by homologous recombination. Alternatively, homologous recombination
takes place using
the Cre-loxP system to knockout a gene of interest in a tissue- or
developmental stage-specific
manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al.
(1997) Nucleic Acids
Res. 25:4323-4330). Transformed ES cells are identified and microinjected into
mouse cell
blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are
surgically transferred
to pseudopregnant dams, and the resulting chimeric progeny are genotyped and
bred to produce
heterozygous or homozygous strains. Transgenic animals thus generated may be
tested with potential
therapeutic or toxic agents.
Polynucleotides encoding TRICH may also be manipulated in vitro in ES cells
derived from
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human blastocysts. Human ES cells have the potential to differentiate into at
least eight separate cell
lineages including endoderm, mesoderm, and ectodennal cell types. These cell
lineages differentiate
into, for example, neural cells, hematopoietic lineages, and cardiomyocytes
(Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding TRICH can also be used to create "knockin" humanized
animals
(pigs) or transgenic animals (mice or rats) to model human disease. With
knockin technology, a
region of a polynucleotide encoding TRICH is injected into animal ES cells,
and the injected
sequence integrates into the animal cell genome. Transformed cells are
injected into blastulae, and
the blastulae are implanted as described above. Transgenic progeny or inbred
lines are studied and
treated with potential pharmaceutical agents to obtain information on
treatment of a human disease.
Alternatively, a mammal inbred to overexpress TRICH, e.g., by secreting TRICH
in its milk, may also
serve as a convenient source of that protein (Janne, J. et al. (1998)
Biotechnol. Annu. Rev. 4:55-74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists
between regions of TRICH and transporters and ion channels. In addition, the
expression of TRICH
is closely associated with tumorous tissues such as spleen tumor tissue,
esophageal tumor tissue,
brain tumor tissue, and myxoma from atrium tissue; and normal tissues such as
kidney, liver, nasal
polyp, prostate, thyroid, umbilical cord blood, neuronal, digestive, uterine
endometrial tissue, and
normal brain tissue such as the tissues from striatum, globus pallidus, and
posterior putamen.
Therefore, TRICH appears to play a role in transport, neurological, muscle,
immunological and cell
proliferative disorders. In the treatment of disorders associated with
increased TRICH expression or
activity, it is desirable to decrease the expression or activity of TRICH. In
the treatment of disorders
associated with' decreased TRICH expression or activity, it is desirable to
increase the expression or
activity of TRICH.
Therefore, in one embodiment, TRICH or a fragment or derivative thereof may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of TRICH. Examples of such disorders include, but are not limited to,
a transport disorder
such as akinesia, amyotrophic lateral sclerosis, ataxia telangiectasia, cystic
fibrosis, Becker's
muscular dystrophy, Bell's palsy, Charcot-Marie Tooth disease, diabetes
mellitus, diabetes insipidus,
diabetic neuropathy, Duchenne muscular dystrophy, hyperkalemic periodic
paralysis, normokalemic
periodic paralysis, Parkinson's disease, malignant hyperthermia, multidrug
resistance, myasthenia
gravis, myotonic dystrophy, catatonia, tardive dyskinesia, dystonias,
peripheral neuropathy, cerebral
neoplasms, prostate cancer, cardiac disorders associated with transport, e.g.,
angina, bradyarrythxnia,
tachyarrythmia, hypertension, Long QT syndrome, myocaxditis, cardiomyopathy,
nemaline
myopathy, centronuclear myopathy, lipid myopathy, mitochondria) myopathy,
thyrotoxic myopathy,
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ethanol myopathy, dermatomyositis, inclusion body myositis, infectious
myositis, polymyositis,
neurological disorders associated with transport, e.g., Alzheimer's disease,
amnesia, bipolar disorder,
dementia, depression, epilepsy, Tourette's disorder, paranoid psychoses, and
schizophrenia, and other
disorders associated with transport, e.g., neurofibromatosis, postherpetic
neuralgia, trigeminal
neuropathy, sarcoidosis, sickle cell anemia, Wilson's disease, cataracts,
infertility, pulmonary artery
stenosis, sensorineural autosomal deafness, hyperglycemia, hypoglycemia,
Grave's disease, goiter,
Cushing's disease, Addison's disease, glucose-galactose malabsorption
syndrome, glycogen storage
disease, hypercholesterolemia, adrenoleukodystrophy, Zellweger syndrome,
Menkes disease,
occipital horn syndrome, von Gierke disease, pseudohypoaldosteronism type 1,
Liddle's syndrome,
cystinuria, iminoglycinuria, Hartup disease, Fanconi disease, and Banter
syndrome; a neurological
disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral
neoplasms, Alzheimer's
disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease
and other extrapyramidal
disorders, amyotrophic lateral sclerosis and other motor neuron disorders,
progressive neural
muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis
and other demyelinating
diseases, bacterial and viral meningitis, brain abscess, subdural empyema,
epidural abscess,
suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral
central nervous system
disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and
Gerstmann-
Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and
metabolic diseases of the
nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the central
nervous system including Down syndrome, cerebral palsy, neuroskeletal
disorders, autonomic
nervous system disorders, cranial nerve disorders, spinal cord diseases,
muscular dystrophy and other
neuromuscular disorders, peripheral nervous system disorders, dermatomyositis
and polymyositis,
inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis,
periodic paralysis, mental
disorders including mood, anxiety, and schizophrenic disorders, seasonal
affective disorder (SAD),
akathesia, amnesia, catatonia, diabetic neuropathy, hemiplegic migraine,
tardive dyskinesia,
dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder,
progressive supranuclear
palsy, conicobasal degeneration, and familial frontotemporal dementia; a
muscle disorder such as
cardiomyopathy, myocarditis, Duchenne's muscular dystrophy, Becker's muscular
dystrophy,
myotonic dystrophy, central core disease, nemaline myopathy, centronuclear
myopathy, lipid
myopathy, mitochondrial myopathy, infectious myositis, polymyositis,
dermatomyositis, inclusion
body myositis, thyrotoxic myopathy, ethanol myopathy, angina, anaphylactic
shock, arrhythmias,
asthma, cardiovascular shock, Cushing's syndrome, hypertension, hypoglycemia,
myocardial
infarction, migraine, pheochromocytoma, and myopathies including
encephalopathy, epilepsy,
Kearns-Sayre syndrome, lactic acidosis, myoclonic disorder, ophthalmoplegia,
acid maltase
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deficiency (AMD, also known as Pompe's disease), generalized myotonia, and
myotonia congenita;
an immunological disorder such as acquired immunodeficiency syndrome (AIDS),
Addison's disease,
adult respiratory distress syndrome, allergies, ankylosing spondylitis,
amyloidosis, anemia, asthma,
atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis,
autoimmune
polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis,
cholecystitis, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema,
episodic Iymphopenia 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, sclerodenna, Sjogren's
syndrome, systemic
anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and
extracorporeal
circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic
infections, and trauma; and a
cell proliferative disorder such as actinic keratosis, arteriosclerosis,
atherosclerosis, bursitis, cirrhosis,
hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal
nocturnal
hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and
cancers including
adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in
particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain,
breast, cervix, gall
bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,
ovary, pancreas, parathyroid,
penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and
uterus.
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 T1RICH 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 TRICK including, but not limited to, those listed above.
In a further embodiment, an antagonist of TRICH may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of TRICH.
Examples of such
disorders include, but are not limited to, those transport, neurological,
muscle, immunological and
cell proliferative disorders described above. In one aspect, an antibody which
specifically binds
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TRICH may be used directly as an antagonist or indirectly as a targeting or
delivery mechanism for
bringing a pharmaceutical agent to cells or tissues which express TRICH.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding TRICH may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of TRICH including, but not limited to, those
descxibed above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be administered in combination with
other appropriate
therapeutic agents. Selection of the appropriate agents for use in combination
therapy may be made
by one of ordinary skill in the art, according to conventional pharmaceutical
principles. The
combination of therapeutic agents may act synergistically to effect the
treatment or prevention of the
various disorders described above. Using this approach, one may be able to
achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the potential for
adverse side effects.
An antagonist of TRICH may be produced using methods which are generally known
in the
art. In particular, purified TRICH may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind TRICH.
Antibodies to TRICH may
also be generated using methods that are well known in the art. Such
antibodies may include, but are
not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies,
Fab fragments, and
fragments produced by a Fab expression library. Neutralizing antibodies (i.e.,
those which inhibit
dimer formation) are generally preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, humans,
and others may be immunized by injection with 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
Iysolecithin, platonic
polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among
adjuvants used in
humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are
especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
TRICH have an amino acid sequence consisting of at least about 5 amino acids,
and generally will
consist of at least about 10 amino acids. It is also preferable that these
oligopeptides, peptides, or
fragments are identical to a portion of the amino acid sequence of the natural
protein. Short stretches
of TRICH amino acids may be fused with those of another protein, such as KLH,
and antibodies to
the chimeric molecule may be produced.
Monoclonal antibodies to TRICH may be prepared using any technique which
provides for
the production of antibody molecules by continuous cell lines in culture.
These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma technique, and
the EBV-hybridoma
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technique. (See, e.g., Kohler, G. et al. (1975) Nature 255:495-497; Kozbor, D.
et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; and
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
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')z fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab')2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
TRICH and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies
reactive to two non-interfering TRICH epitopes is generally used, but a
competitive binding assay
may also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay
techniques may be used to assess the affinity of antibodies for TRICH.
Affinity is expressed as an
association constant, Ka, which is defined as the molar concentration of TRICH-
antibody complex
divided by the-molar concentrations of free antigen and. free antibody under
equilibrium conditions.
The Ka determined for a preparation of polyclonal antibodies, which are
heterogeneous in their
affinities for multiple TRICH epitopes, represents the average affinity, or
avidity, of the antibodies
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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 10'2 Llmole are preferred for
use in immunoassays in
which the TRICH-antibody complex must withstand rigorous manipulations. Low-
affinity antibody
preparations with Ka ranging from about 106 to 10' L/mole are preferred for
use in
immunopurification and similar procedures which ultimately require
dissociation of TRICH,
preferably in active form, from the antibody (Catty, D. (1988) Antibodies,
Volume I: A Practical
Ap rp oach, IRL Press, Washington DC; Liddell, J.E. and A. Cryer (1991) A
Practical Guide to
Monoclonal Antibodies, John Wiley & Sons, New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
determine the quality and suitability of such preparations for certain
downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml,
preferably 5-10 mg specific antibody/ml, is generally employed in procedures
requiring precipitation
of TRICH-antibody complexes. Procedures for evaluating antibody specificity,
titer, and avidity, and
guidelines for antibody quality and usage in various applications, axe
generally available. (See, e.g.,
Catty, su ra, and Coligan et al. supra.)
In another embodiment of the invention, the polynucleotides encoding TRICH, or
any
fragment or complement thereof, may be used for therapeutic purposes. In one
aspect, modifications
of gene expression can be achieved by designing complementary sequences or
antisense molecules
(DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory
regions of the gene
encoding TRICH. Such technology is well known in the art, and antisense
oligonucleotides or larger
fragments can be designed from various locations along the coding or control
regions of sequences
encoding TRICH. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics,
Humana Press Inc.,
Totawa NJ.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. (1990) Blood
76:271; Ausubel, supra; 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.
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25(I4):2730-2736.)
In another embodiment of the invention, polynucleotides encoding TRICH may be
used for
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCID)-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) Cel175:207-216; Crystal, R.G. et al.
(1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-
242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites
(e.g., against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore, D.
(1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci.
USA. 93:11395-11399),
hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans
and Paracoccidioides
brasiliensis; and protozoan parasites such as Plasmodium falciparum and
Trypanosoma cruzi). In the
case where a genetic deficiency in TRICH expression or regulation causes
disease, the expression of
TRICH from an appropriate population of transduced cells may alleviate the
clinical manifestations
caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by
deficiencies in
TRICH are treated by constructing mammalian expression vectors encoding TRICH
and introducing
these vectors by mechanical means into TRICH-deficient cells. Mechanical
transfer teclmologies for
use with cells in vivo or ex vitro include (i) direct DNA microinjection into
individual cells, (ii)
ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv)
receptor-mediated gene
transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson
(1993) Annu. Rev.
Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H.
Recipon (1998) Curr.
Opin. Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of TRICH include,
but are not
limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors
(Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSHlPERV (Stratagene, La
Jolla CA),
and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
TRICH
may be expressed using (i) a constitutively active promoter, (e.g., from
cytomegalovirus (CMV),
Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or [3-actin
genes), (ii) an inducible
promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard
(1992) Proc. Natl.
CA 02427010 2003-04-25
WO 02/40541 PCT/USO1/46055
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 KTT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of
these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to TRICH expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding TRICH under the control of an independent promoter or
the retrovirus long
terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and
(iii) a Rev-responsive
element (RRE) along with additional retrovirus cis-acting RNA sequences and
coding sequences
required for efficient vector propagation. Retrovirus vectors (e.g., PFB and
PFBNEO) are
commercially available (Stratagene) and are based on published data (Riviere,
I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The
vector is propagated in
an appropriate vector producing cell line (VPCL) that expresses an envelope
gene with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R.
et al. (1998) J. Virol. 72:9873-9880). U.S. Patent No. 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant")
discloses a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by
reference. Propagation of retrovirus vectors, transduction of a population of
cells (e.g., CD4+ T-
cells), and the return of transduced cells to a patient are procedures well
known to persons skilled in
the art of gene therapy and have been well documented (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
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polynucleotides encoding TRICH to cells which have one or more genetic
abnornialities 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 axt. 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.
169:385-395). The construction of a HSV-1 virus vector has also been disclosed
in detail in U.S.
Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is hereby
incorporated by reference. U.S. Patent No. 5,804,413 teaches the use of
recombinant HSV d92 which
consists of a genome containing at least one exogenous gene to be transferred
to a cell under the
control of the appropriate promoter for purposes including human gene therapy.
Also taught by this
patent are the construction and use of recombinant HSV strains deleted for
ICP4, ICP27 and 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: I52-161, hereby incorporated by reference. The manipulation of
cloned herpesvirus
sequences, the generation of recombinant virus following the transfection of
multiple plasmids
containing different segments of the large herpesvirus genomes, the growth and
propagation of
herpesvirus, and the infection of cells with herpesvirus are techniques well
known to those of
ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding TRICH to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based
on the SFV genome (Garoff, H. and I~.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During
alphavirus RNA replication, a subgenomic RNA is generated that noxmally
encodes the viral capsid
pxoteins. 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
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(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 numbei 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 (BHI~-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 Immunologic Approaches, Futura Publishing, Mt.
Kisco NY, pp. 163-
177.) A complementary sequence or antisense molecule may also be designed to
block translation of
mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding TRICH.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared
by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques
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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 TRTCH. Such DNA sequences may be incorporated into a wide
variety of
vectors with suitable RNA polymerase promoters such as T7 or SP6.
Alternatively, these cDNA
constructs that synthesize complementary RNA, constitutively or inducibly, can
be introduced into
cell lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3'
ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather
than phosphodiesterase
linkages within the backbone of the molecule. This concept is inherent in the
production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as inosine,
queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly
modified forms of adenine,
cytidine, guanine, thymine, and uridine which are not as easily recognized by
endogenous
endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding TRICH.
Compounds which may be effective in altering expression of a specific
polynucleotide may include,
but are not limited to, oligonucleotides, antisense oligonucleotides, triple
helix-forming
oligonucleotides, transcription factors and other polypeptide transcriptional
regulators, and non-
macromolecular chemical entities which are capable of interacting with
specific polynucleotide
sequences. Effective compounds may alter polynucleotide expression by acting
as either inhibitors or
promoters of polynucleotide expression. Thus, in the treatment of disorders
associated with increased
TRICH expression or activity, a compound which specifically inhibits
expression of the
polynucleotide encoding TRICH may be therapeutically useful, and in the
treatment of disorders
associated with decreased TRICH expression or activity, a compound which
specifically promotes
expression of the polynucleotide encoding TRICH may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for
effectiveness in
altering expression of a specific polynucleotide. A test compound may be
obtained by any method
commonly known in the art, including chemical modification of a compound known
to be effective in
altering polynucleotide expression; selection from an existing, commercially-
available or proprietary
library of naturally-occurring or non-natural chemical compounds; rational
design of a compound
based on chemical and/or structural properties of the target polynucleotide;
and selection from a
library of chemical compounds created combinatorially or randomly. A sample
comprising a
polynucleotide encoding TRICH is exposed to at least one test compound thus
obtained. The sample
may comprise, for example, an intact or permeabilized cell, or an in vitro
cell-free or reconstituted
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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 Schizosaccharomyces
pombe gene expression
system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al.
(2000) Nucleic Acids
Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al.
(2000) Biochem. Biophys.
Res. Commun. 268:8-13). A particular embodiment of the present invention
involves screening a
combinatorial library of oligonucleotides (such as deoxyribonucleotides,
ribonucleotides, peptide
nucleic acids, and modified oligonucleotides) for antisense activity against a
specific polynucleotide
sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W.
et al. (2000) U.S.
Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and
equally suitable
for use in vivo, in vitro, and ex vivo. Fox ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.I~. 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
Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such
compositions may
consist of TRICH, antibodies to TRICH, and mimetics, agonists, antagonists, or
inhibitors of TRICH.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, infra-
arterial, intramedullary,
intrathecal, intraventricular, pulmonary, transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
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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 HIV Tat-1 protein. Fusion proteins thus generated
have been found to
transduce into the cells of all tissues, including the brain, in a mouse model
system (Schwarze, S.R. et
al. (1999) Science 285:1569-1572). ,
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs,
monkeys, or pigs. An animal model may also be used to determine the
appropriate concentration
range and route of administration. Such information can then be used to
determine useful doses and
routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example
TRICH or fragments thereof, antibodies of TRICH, and agonists, antagonists or
inhibitors of TRICH,
which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity
may be determined
by standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDSO (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDSO/EDSO ratio. Compositions
which exhibit large
therapeutic indices are preferred. The data obtained from cell culture assays
and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDso
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
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The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and
response to therapy. Long-acting compositions may be administered every 3 to 4
days, every week,
or biweekly depending on the half life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about 0.1 ,ug to 100,000 ,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
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 FAGS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
TRICH expression. Normal
or standard values for TRICH expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibodies to TRICH
under conditions suitable for complex formation. The amount of standard
complex formation may be
quantitated by various methods, such as photometric means. Quantities of TRICH
expressed in
subject, control, and disease samples from biopsied tissues are compared with
the standard values.
Deviation between standard and subject values establishes the parameters for
diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding TRTCH may
be used
for diagnostic purposes. The polynucleotides which may be used include
oligonucleotide sequences,
complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used
to detect
and quantify gene expression in biopsied tissues in which expression of TRICH
may be correlated
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with disease. The diagnostic assay may be used to determine absence, presence,
and excess
expression of TRICH, and to monitor regulation of TRICH levels during
therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding TRICH or closely related
molecules may be used
to identify nucleic acid sequences which encode TRICH. The specificity of the
probe, whether it is
made from a highly specific region, e.g., the 5'regulatory region, or from a
less specific region, e.g., a
conserved motif, and the stringency of the hybridization or amplification will
determine whether the
probe identifies only naturally occurring sequences encoding TRICH, allelic
variants, or related
sequences.
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 ID
N0:21-40 or from
genomic sequences including promoters, enhancers, and introns of the TRICH
gene.
Means for producing specific hybridization probes for DNAs encoding TRICH
include the
cloning of polynucleotide sequences encoding TRICH or TRICH derivatives into
vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and may
be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as 3~P or 355,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding TRICH may be used for the diagnosis of
disorders
associated with expression of TRICH. Examples of such disorders include, but
are not limited to, a
transport disorder such as akinesia, amyotrophic lateral sclerosis, ataxia
telangiectasia, cystic fibrosis,
Becker's muscular dystrophy, Bell's palsy, Charcot-Marie Tooth disease,
diabetes mellitus, diabetes
insipidus, diabetic neuropathy, Duchenne muscular dystrophy, hyperkalemic
periodic paralysis,
normokalemic periodic paralysis, Parkinson's disease, malignant hyperthermia,
multidrug resistance,
myasthenia gravis, myotonic dystrophy, catatonia, tardive dyskinesia,
dystonias, peripheral
neuropathy, cerebral neoplasms, prostate cancer, cardiac disorders associated
with transport, e.g.,
angina, bradyarrythmia, tachyarrythmia, hypertension, Long QT syndrome,
myocarditis,
cardiomyopathy, nemaline myopathy, centronuclear myopathy, lipid myopathy,
mitochondria)
myopathy, thyrotoxic myopathy, ethanol myopathy, dermatomyositis, inclusion
body myositis,
infectious myositis, polymyositis, neurological disorders associated with
transport, e.g., Alzheimer's
disease, amnesia, bipolar disorder, dementia, depression, epilepsy, Tourette's
disorder, paranoid
psychoses, and schizophrenia, and other disorders associated with transport,
e.g., neurofibromatosis,
postherpetic neuralgia, trigeminal neuropathy, sarcoidosis, sickle cell
anemia, Wilson's disease,
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cataracts, infertility, pulmonary artery stenosis, sensorineural autosomal
deafness, hyperglycemia,
hypoglycemia, Grave's disease, goiter, Cushing's disease, Addison's disease,
glucose-galactose
malabsorption syndrome, glycogen storage disease, hypercholesterolemia,
adrenoleukodystrophy,
Zellweger syndrome, Menkes disease, occipital horn syndrome, von Gierke
disease,
pseudohypoaldosteronism type 1, Liddle's syndrome, cystinuria,
iminoglycinuria, Hanup disease,
Fanconi disease, and Banter syndrome; a neurological disorder such as
epilepsy, ischemic
cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease,
Pick's disease,
Huntington's disease, dementia, Parkinson's disease and other extrapyramidal
disorders, amyotrophic
lateral sclerosis and other motor neuron disorders, progressive neural
muscular atrophy, retinitis
pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating
diseases, bacterial and
viral meningitis, brain abscess, subdural empyema, epidural abscess,
suppurative intraeranial
thrombophlebitis, myelitis and radiculitis, viral central nervous system
disease, prion diseases
including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker
syndrome, fatal
familial insomnia, nutritional and metabolic diseases of the nervous system,
neurofibromatosis,
tuberous sclerosis, cerebelloretinal hemangioblastomatosis,
encephalotrigeminal syndrome, mental
retardation and other developmental disorders of the central nervous system
including Down
syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system
disorders, cranial nerve
disorders, spinal cord diseases, muscular dystrophy and other neuromuscular
disorders, peripheral
nervous system disorders, dermatomyositis and polymyositis, inherited,
metabolic, endocrine, and
toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders
including mood, anxiety,
and schizophrenic disorders, seasonal affective disorder (SAD), akathesia,
amnesia, catatonia,
diabetic neuropathy, hemiplegic migraine, tardive dyskinesia, dystonias,
paranoid psychoses,
postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy,
conicobasal
degeneration, and familial frontotemporal dementia; a muscle disorder such as
cardiomyopathy,
myocarditis, Duchenne's muscular dystrophy, Becker's muscular dystrophy,
myotonic dystrophy,
central core disease, nemaline myopathy, centronuclear myopathy, lipid
myopathy, mitochondria)
myopathy, infectious myositis, polymyositis, dermatomyositis, inclusion body
myositis, thyrotoxic
myopathy, ethanol myopathy, angina, anaphylactic shock, arrhythmias, asthma,
cardiovascular shock,
Cushing's syndrome, hypertension, hypoglycemia, myocardial infarction,
migraine,
pheochromocytoma, and myopathies including encephalopathy, epilepsy, Learns-
Sayre syndrome,
lactic acidosis, myoclonic disorder, ophthalinoplegia, acid maltase deficiency
(AMD, also known as
Pompe's disease), generalized myotonia, and myotonia congenita; an
immunological disorder such as
acquired immunodeficiency syndrome (AIDS), Addison's disease, adult
respiratory distress
syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma,
atherosclerosis,
autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune
polyendocrinopathy-
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candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact
dermatitis, Crohn's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema,
episodic lymphopenia
with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic
gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis,
hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's
syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative
colitis, uveitis,
Werner syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral,
bacterial, fungal, parasitic, protozoal, and helminthic infections, and
trauma; and a cell proliferative
disorder such as actinic keratosis, arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis, mixed
connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal
hemoglobinuria,
polycythemia vera, psoriasis, primary thrombocythemia, and cancers including
adenocarcinoma,
leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in
particular, cancers of
the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall
bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas,
parathyroid, penis, prostate,
salivary glands, skin, spleen, testis, thymus, thyroid, and uterus. The
polynucleotide sequences
encoding TRICH may be used in Southern or northern analysis, dot blot, or
other membrane-based
technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-
like assays; and in
microarrays utilizing fluids or tissues from patients to detect altered TRICH
expression. Such
qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding TRICH may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding TRICH may be labeled by standard methods and added to a
fluid or tissue sample
from a patient under conditions suitable for the formation of hybridization
complexes. After a
suitable incubation period, the sample is washed and the signal is quantified
and compared with a
standard value. If the amount of signal in the patient sample is significantly
altered in comparison to
a control sample then the presence of altered levels of nucleotide sequences
encoding TRICH in the
sample indicates the presence of the associated disorder. Such assays may also
be used to evaluate
the efficacy of a particular therapeutic treatment regimen in animal studies,
in clinical trials, or to
monitor the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with
expression of
TRICH, a normal or standard profile for expression is established. This may be
accomplished by
combining body fluids or cell extracts taken from normal subjects, either
animal or human, with a
sequence, or a fragment thereof, encoding TRICH, under conditions suitable for
hybridization or
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amplification. Standard hybridization may be quantified by comparing the
values obtained from
normal subjects with values from an experiment in which a known amount of a
substantially purified
polynucleotide is used. Standard values obtained in this manner may be
compared with values
obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard
values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the
development of the disease, or may provide a means for detecting the disease
prior to the appearance
of actual clinical symptoms. A more definitive diagnosis of this type may
allow health professionals
to employ preventative measures or aggressive treatment earlier thereby
preventing the development
or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding
TRICH may involve the use of PCR. These oligomers may be chemically
synthesized, generated
enzymatically, or produced in vitro. Oligomers will preferably contain a
fragment of a polynucleotide
encoding TRICH, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
TRICH, and will be employed under optimized conditions for identification of a
specific gene or
condition. Oligomers may also be employed under less stringent conditions for
detection or
quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding TRICH may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are
substitutions, insertions and deletions that are a frequent cause of inherited
or acquired genetic
disease in humans. Methods of SNP detection include, but are n'ot 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
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methods, termed in silico SNP (isSNP), are capable of identifying
polymorphisms by comparing the
sequence of individual overlapping DNA fragments which assemble into a common
consensus
sequence. These computer-based methods filter out sequence variations due to
laboratory preparation
of DNA and sequencing errors using statistical models and automated analyses
of DNA sequence
chromatograms. In the alternative, SNPs may be detected and characterized by
mass spectrometry
using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San
Diego CA).
Methods which may also be used to quantify the expression of TRICH include
radiolabeling
or biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives
rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described below: The microarray may also be
used to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, to monitor
progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used
to develop a pharmacogenomic profile of a patient in order to select the most
appropriate and
effective treatment regimen for that patient. For example, therapeutic agents
which are highly
effective and display the fewest side effects may be selected for a patient
based on his/her
pharmacogenomic profile.
In another embodiment, TRICH, fragments of TRICH, or antibodies specific for
TRICH may
be used as elements on a microarray. The microarray may be used to monitor or
measure protein-
protein interactions, drug-target interactions, and gene expression profiles,
as described above.
A particular embodiment relates to the use of the polynucleotides of the
present invention to
generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern of
gene expression by a particular tissue or cell type. Global gene expression
patterns are analyzed by
quantifying the number of expressed genes and their relative abundance under
given conditions and at
a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent 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
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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
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
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invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a
proteome can be subjected individually to further analysis. Proteome
expression patterns, or profiles,
are analyzed by quantifying the number of expressed proteins and their
relative abundance under
given conditions and at a given time. A profile of a cell's proteome may thus
be generated by
separating and analyzing the polypeptides of a particular tissue or cell type.
In one embodiment, the
separation is achieved using two-dimensional gel electrophoresis, in which
proteins from a sample are
separated by isoelectric focusing in the first dimension, and then according
to molecular weight by
sodium dodecyl sulfate slab gel electrophoresis in the second dimension
(Steiner and Anderson,
supra). The proteins are visualized in the gel as discrete and uniquely
positioned spots, typically by
staining the gel with an agent such as Coomassie Blue or silver or fluorescent
stains. The optical
density of each protein spot is generally proportional to the level of the
protein in the sample. The
optical densities of equivalently positioned protein spots from different
samples, for example, from
biological samples either treated or untreated with a test compound or
therapeutic agent, are
compared to identify any changes in protein spot density related to the
treatment. The proteins in the
spots are partially sequenced using, for example, standard methods employing
chemical or enzymatic
cleavage followed by mass spectrometry. The identity of the protein in a spot
may be determined by
comparing its partial sequence, preferably of at least 5 contiguous amino acid
residues, to the
polypeptide sequences of the present invention. In some cases, further
sequence data may be
obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for TRICH
to quantify
the levels of TRICH expression. In one embodiment, the antibodies are used as
elements on a
microarray, and protein expression levels are quantified by exposing the
microarray to the sample and
detecting the levels of protein bound to each array element (Lueking, A. et
al. (1999) Anal. Biochem.
270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection
may be performed
by a variety of methods known in the art, for example, by reacting the
proteins in the sample with a
thiol- or amino-reactive fluorescent compound and detecting the amount of
fluorescence bound at
each array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
correlation between transcript and protein abundances for some proteins in
some tissues (Anderson,
N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures may be
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to
rapid degradation of mRNA, so proteomic profiling may be more reliable and
informative in such
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cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
each protein is compared to the amount of the corresponding protein in an
untreated biological
sample. A difference in the amount of protein between the two samples is
indicative of a toxic
response to the test compound in the treated sample. Individual proteins are
identified by sequencing
the amino acid residues of the individual proteins and comparing these partial
sequences to the
polypeptides of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are
incubated with antibodies specific to the polypeptides of the present
invention. The amount of
protein recognized by the antibodies is quantified. The amount of protein in
the treated biological
sample is compared with the amount in an untreated biological sample. A
difference in the amount of
protein between the two samples is indicative of a toxic response to the test
compound in the treated
sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-
2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types
of microarrays are
well known and thoroughly described in DNA Microarrays: A Practical Approach,
M. Schena, ed.
(1999) Oxford University Press, London, hereby expressly incorporated by
reference.
In another embodiment of the invention, nucleic acid sequences encoding TRICH
may be
used to generate hybridization probes useful in mapping the naturally
occurring genomic sequence.
Either coding or noncoding sequences may be used, and in some instances,
noncoding sequences may
be preferable over coding sequences. For example, conservation of a coding
sequence among
members of a mufti-gene family may potentially cause undesired cross
hybridization during
chromosomal mapping. The sequences may be mapped to a particular chromosome,
to a specific
region of a chromosome, or to artificial chromosome constructions, e.g., human
artificial
chromosomes (HACs), yeast artificial chromosomes (PACs), 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
CA 02427010 2003-04-25
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with the inheritance of a particular chromosome region or restriction fragment
length polymorphism
(RFLP). (See, for example, Lander, E.S. and D. Botstein (1986) Proc. Natl.
Acad. Sci. USA 83:7353-
7357.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic
map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-
968.) Examples of genetic
map data can be found in various scientific journals or at the Online
Mendelian Inheritance in Man
(OMIM) World Wide Web site. Correlation between the location of the gene
encoding TRICH on a
physical map and a specific disorder, or a predisposition to a specific
disorder, may help define the
region of DNA associated with that disorder and thus may further positional
cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
may reveal associated markers even if the exact chromosomal locus is not
known. This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely
localized by genetic linkage to a particular genomic region, e.g., ataxia-
telangiectasia to l 1q22-23,
any sequences mapping to that area may represent associated or regulatory
genes for further
investigation. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The
nucleotide sequence of
the instant invention may also be used to detect differences in the
chromosomal location due to
translocation, inversion, etc., among normal, carrier, or affected
individuals.
In another embodiment of the invention, TRICH, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used for screening libraries of compounds in any
of a variety of drug
screening techniques. The fragment employed in such screening may be free in
solution, affixed to a
solid support, borne on a cell surface, or located intracellularly. The
formation of binding complexes
between TRICH and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with TRICH,
or fragments thereof,
and washed. Bound TRICH is then detected by methods well known in the art.
Purified TRICH can
also be coated directly onto plates for use in the aforementioned drug
screening techniques.
Alternatively, non-neutralizing antibodies can be used to capture the peptide
and immobilize it on a
solid support.
In another embodiment, one may use competitive drug screening assays in which
neutralizing
antibodies capable of binding TRICH specifically compete with a test compound
for binding TRICH.
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In this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with TRICH.
In additional embodiments, the nucleotide sequences which encode TRICH may be
used in
any molecular biology techniques that have yet to be developed, provided the
new techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following embodiments are,
therefore, to be construed as merely illustrative, and not limitative of the
remainder of the disclosure
in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below,
including U.S. Ser. No. 60/243,989, U.S. Ser. No. 60/245,904, U.S. Ser. No.
60/249,661, U.S. Ser.
No. 60/247,673, U.S. Ser. No. 60/252,232, and U.S. Ser. No. 601250,790, are
hereby expressly
incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database
(Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. Some tissues
were homogenized
and lysed in guanidinium isothiocyanate, while others were homogenized and
lysed in phenol or in a
suitable mixture of denaturants, such as TRIZOL (Life Technologies), a
monophasic solution of
phenol and guanidine isothiocyanate. The resulting lysates were centrifuged
over CsCI cushions or
extracted with chloroform. RNA was precipitated from the lysates with either
isopropanol or sodium
acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A)+ RNA was isolated
using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
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oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
1000 bp) using SEPHACRYL. S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies),
PCDNA2.1 plasmid
(Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid
(Invitrogen),
PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), or
pINCY (Incyte
Genomics), or derivatives thereof. Recombinant plasmids were transformed into
competent E. coli
cells including XL1-Blue, XL1-BIueMRF, or SOLR from Stratagene or DHSa, DH10B,
or
ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo
excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega); an
AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid,
QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
R.E.A.L. PREP 96
plasmid purification kit from QIAGEN. Following precipitation, plasmids were
resuspended in 0.1
ml of distilled water and stored, with or without lyophilization, at
4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically
using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence
scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation
such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-
200 thermal
cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins
Scientific) or the
MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as
the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied
Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carried out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics); the
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ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction
with standard ABI
protocols and base calling software; or other sequence analysis systems known
in the art. Reading
frames within the cDNA sequences were identified using standard methods
(reviewed in Ausubel,
1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension
using the techniques
disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by
removing
vector, linker, and poly(A) sequences and by masking ambiguous bases, using
algorithms and
programs based on BLAST, dynamic programming, and dinucleotide nearest
neighbor analysis. The
Incyte cDNA sequences or translations thereof were then queried against a
selection of public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases, and
BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo
Sapiens,
Rattus norve~icus, Mus musculus, Caenorhabditis ele~ans, Saccharomyces
cerevisiae,
Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto
CA); and hidden
Markov model (HMM)-based protein family databases such as PFAM. (HMM is a
probabilistic
approach which analyzes consensus primary structures of gene families. See,
for example, Eddy,
S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.) The queries were performed
using programs based
on BLAST, FASTA, BLIMPS, and I3Ml~~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 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, and hidden
Markov
model (HMM)-based protein family databases such as PFAM. Full length
polynucleotide sequences
are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering,
South San
Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide
sequence
alignments are generated using default parameters specified by the CLUSTAL
algorithm as
incorporated into the MEGALIGN multisequence alignment program (DNASTAR),
which also
calculates the percent identity between aligned sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis
and assembly of
Incyte cDNA and full length sequences and provides applicable descriptions,
references, and
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threshold parameters. The first column of Table 7 shows the tools, programs,
and algorithms used,
the second column provides brief descriptions thereof, the third column
presents appropriate
references, all of which are incorporated by reference herein in their
entirety, and the fourth column
presents, where applicable, the scores, probability values, and other
parameters used to evaluate the
strength of a match between two sequences (the higher the score or the lower
the probability value,
the greater the identity between two sequences).
The programs described above for the assembly and analysis of full length
polynucleotide
and polypeptide sequences were also used to identify polynucleotide sequence
fragments from SEQ
m N0:21-40. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization
and amplification technologies are described in Table 4, column 4.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative transporters and ion channels were initially identified by running
the Genscan gene
identification program against public genomic sequence databases (e.g., gbpri
and gbhtg). Genscan is
a general-purpose gene identification program which analyzes genomic DNA
sequences from a
variety of organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-
94, and Burge, C. and
S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354). The program
concatenates predicted exons to
form an assembled cDNA sequence extending from a methionine to a stop codon.
The output of
Genscan is a FASTA database of polynucleotide and polypeptide sequences. The
maximum range of
sequence for Genscan to analyze at once was set to 30 kb. To determine which
of these Genscan
predicted cDNA sequences encode transporters and ion channels, the encoded
polypeptides were
analyzed by querying against PFAM models for transporters and ion channels.
Potential transporters
and ion channels were also identified by homology to Incyte cDNA sequences
that had been
annotated as transporters and ion channels. These selected Genscan-predicted
sequences were then
compared by BLAST analysis to the genpept and gbpri public databases. Where
necessary, the
Genscan-predicted sequences were then edited by comparison to the top BLAST
hit from genpept to
correct errors in the sequence predicted by Genscan, such as extra or omitted
exons. BLAST analysis
was also used to find any Incyte cDNA or public cDNA coverage of the Genscan-
predicted
sequences, thus providing evidence for transcription. When Incyte cDNA
coverage was available,
tlus 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 and/or public cDNA sequences using the assembly process
described in
Example III. Alternatively, full length polynucleotide sequences were derived
entirely from edited or
unedited Genscan-predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
"Stitched" Sequences
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Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification program described in Example IV. Partial cDNAs assembled as
described in Example
III were mapped to genomic DNA and parsed into clusters containing related
cDNAs and Genscan
exon predictions from one or more genomic sequences. Each cluster was analyzed
using an algorithm
based on graph theory and dynamic programming to integrate cDNA and genomic
information,
generating possible splice variants that were subsequently confirmed, edited,
or extended to create a
full length sequence. Sequence intervals in which the entire length of the
interval was present on
more than one sequence in the cluster were identified, and intervals thus
identified were considered to
be equivalent by transitivity. For example, if an interval was present on a
cDNA and two genomic
sequences, then all three intervals were considered to be equivalent. This
process allows unrelated
but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals
thus identified were then "stitched" together by the stitching algorithm in
the order that they appear
along their parent sequences to generate the longest possible sequence, as
well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence
(cDNA to cDNA or
genomic sequence to genomic sequence) were given preference over linkages
which change parent
type (cDNA to genomic sequence). The resultant stitched sequences were
translated and compared
by BLAST analysis to the genpept and gbpri public databases. Incorrect exons
predicted by Genscan
were corrected by comparison to the top BLAST hit from genpept. Sequences were
further extended
with additional cDNA sequences, or by inspection of genomic DNA, when
necessary.
"Stretched" Sequences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example 111 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 clumeric protein was generated by using the resultant high-
scoring segment pairs
(HSPs) to map the translated sequences onto the GenBank protein homolog.
Insertions or deletions
may occur in the chimeric protein with respect to the original GenBank protein
homolog. The
GenBank protein homolog, the chimeric protein, or both were used as probes to
search for
homologous genomic sequences from the public human genome databases. Partial
DNA sequences
were therefore "stretched" or extended by the addition of homologous genomic
sequences. The
resultant stretched sequences were examined to determine whether it contained
a complete gene.
VI. Chromosomal Mapping of TRICH Encoding Polynucleotides
The sequences which were used to assemble SEQ ID N0:21-40 were compared with
sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
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implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ ID N0:21-40 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for
Genome Research (WIGR), and Genethon were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ ID NO:, to that
map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map
position of an interval, in centiMorgans, is measured relative to the terminus
of the chromosome's p-
arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb)
of DNA in
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,
sue, ch. 7; Ausubel
(1995) supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or L1FESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the
computer search can be modified to determine whether any particular match is
categorized as exact or
similar. The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity
5 x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. The product score is a normalized value between
0 and 100, and is
calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
product is divided by (5 times the length of the shorter of the two
sequences). The BLAST score is
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calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by
gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate
the product score. The product score represents a balance between fractional
overlap and quality in a
BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the
entire length of the shorter of the two sequences being compared. A product
score of 70 is produced
either by 100% identity and 70% overlap at one end, or by 88% identity and
100% overlap at the
other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
Alternatively, polynucleotide sequences encoding TRICH are analyzed with
respect to the
tissue sources from which they were derived. For example, some full length
sequences are
assembled, at least in part, with overlapping Incyte cDNA sequences (see
Example III). Each cDNA
sequence is derived from a cDNA library constructed from a human tissue. Each
human tissue is
classified into one of the following~organ/tissue categories: cardiovascular
system; connective tissue;
digestive system; embryonic structures; endocrine system; exocrine glands;
genitalia, female;
genitalia, male; germ cells; heroic and immune system; liver; musculoskeletal
system; nervous
system; pancreas; respiratory system; sense organs; skin; stomatognathic
system; unclassified/mixed;
or urinary tract. The number of libraries in each category is counted and
divided by the total number
of libraries across all categories. Similarly, each human tissue is classified
into one of the following
disease/condition categories: cancer, cell line, developmental, inflammation,
neurological, trauma,
cardiovascular, pooled, and other, and the number of libraries in each
category is counted and divided
by the total number of libraries across all categories. The resulting
percentages reflect the tissue- and
disease-specific expression of cDNA encoding TRICH. cDNA sequences and cDNA
library/tissue
information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto
CA).
VIII. Extension of TRICH Encoding Polynucleotides
Full length 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 desixed, additional or nested sets of primers were
designed.
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High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+, (NH~)zS04,
and 2-mercaptoethanol, Taq DNA polymerise (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerise (Stratagene), with the following
parameters for primer
pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C,
2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5
min; Step 7: storage at 4°C. In the
alternative, the parameters for primer pair T7 and 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 p,l
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 ~.l of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ,ul to 10 ,ul aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose gel. to determine which reactions were
successful in extending the
sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones
were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18
vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerise (Stratagene) to fill-in
restriction site
overhangs, and transfected into competent E. coli cells. Transformed cells
were selected on
antibiotic-containing media, and individual colonies were picked and cultured
overnight at 37°C in
384-well plates in LB/2x Garb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerise
(Amersham Pharmacia Biotech) and Pfu DNA polymerise (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
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primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the
above procedure or
are used to obtain 5'regulatory sequences using the above procedure along with
oligonucleotides
designed for such extension, and an appropriate genomic library.
IX. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:21-40 are employed to screen
cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide
fragments. Oligonucleotides are designed using state-of the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 ,uCi of
[y-32P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-based
hybridization analysis of human genomic DNA digested with one of the following
endonucleases:
Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
X. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, supra.),
mechanical microspotting technologies, and derivatives thereof. The substrate
in each of the
aforementioned technologies should be uniform and solid with a non-porous
surface (Schena (1999),
supra). Suggested substrates include silicon, silica, glass slides, glass
chips, and silicon wafers.
Alternatively, a procedure analogous to a dot or slot blot may also be used to
arrange and link
elements to the surface of a substrate using thermal, UV, chemical, or
mechanical bonding
procedures. A typical array may be produced using available methods and
machines well known to
those of ordinary skill in the art and may contain any appropriate number of
elements. (See, e.g.,
Schena, M. et al. (1995) Science 270:467-470; Shalom D. et al. (1996) Genome
Res. 6:639-645;
Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)
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Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The
array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element
on the microarray may be assessed. In one embodiment, microarray preparation
and usage is
described in detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/~,1 oligo-(dT)
primer (2lmer), 1X
first strand buffer, 0.03 units/pl RNase inhibitor, 500 ,uM dATP, 500 ~,M
dGTP, 500 p,M dTTP, 40
~,M dCTP, 40 ~,M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A) ~ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium
hydroxide and
incubated for 20 minutes at 85°C to the stop the reaction and degrade
the RNA. Samples are purified
using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is
then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
NY) and
resuspended in 14 pl 5X SSC/0.2% SDS.
Microarra~Preparation
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification
uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5
~.g. Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia
Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
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slides (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water,
and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides
are cured in a
110°C oven.
Array elements are applied to the coated glass substrate using a procedure
described in U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 ~,1 of the array
element DNA, at an average
concentration of 100 ng/pl, is loaded into the open capillary printing element
by a lugh-speed robotic
apparatus. The apparatus then deposits about 5 nl of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°
C followed by washes in
0.2% SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 ~tl of sample mixture consisting of 0.2 ~,g
each of Cy3 and
Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65°C for 5 minutes and is aliquoted onto the
microarray surface and covered
with an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber
having a cavity just
slightly larger than a microscope slide. The chamber is kept at 100% humidity
internally by the
addition of 140 ~,l of 5X SSC in a corner of the chamber. The chamber
containing the~arrays is
incubated for about 6.5 hours at 60° C. The arrays are washed for 10
min at 45° C in a first wash
buffer (1X SSC, 0.1% SDS), three times for 10 minutes each at 45°C in a
second wash buffer (O.1X
SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores. Appropriate
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filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that
location to be correlated with a weight ratio of hybridizing species of
1:100,000. When two samples
from different sources (e.g., representing test and control cells), each
labeled with a different
fluorophore, are hybridized to a single array for the purpose of identifying
genes that are
differentially expressed, the calibration is done by labeling samples of the
calibrating cDNA with the
two fluorophores and adding identical amounts of each to the hybridization
mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping
emission spectra) between the fluorophores using each fluorophore's emission
spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each
spot is centered in each element of the grid. The fluorescence signal within
each element is then
integrated to obtain a numerical value corresponding to the average intensity
of the signal. The
software used for signal analysis is the GEMTOOLS gene expression analysis
program (Incyte).
XI. Complementary Polynucleotides
Sequences complementary to the TRICH-encoding sequences, or any parts thereof,
axe used
to detect, decrease, or inhibit expression of naturally occurring TRICH.
Although use of
oligonucleotides comprising from about 15 to 30 base pairs is described,
essentially the same
procedure is used with smaller or with larger sequence fragments. Appropriate
oligonucleotides are
designed using OLIGO 4.06 software (National Biosciences) and the coding
sequence of TRICH. To
inhibit transcription, a complementary oligonucleotide is designed from the
most unique 5' sequence
and used to prevent promoter binding to the coding sequence. To inhibit
translation, a
complementary oligonucleotide is designed to prevent ribosomal binding to the
TR1CH-encoding
transcript.
XII. Expression of TRICH
Expression and purification of TRICH is achieved using bacterial or virus-
based expression
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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 Aut~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 ~odo~tera frugiperda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus. (See Engelhard, E.K.
et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al.
(1996) Hum. Gene Ther.
7:1937-1945.)
In most expression systems, TRICH is synthesized as a fusion protein with,
e.g., glutathione
S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His,
permitting rapid, single-step,
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-
kilodalton enzyme from Schistosoma japonicum, enables the purification of
fusion proteins on
immobilized glutathione under conditions that maintain protein activity and
antigenicity (Amersham
Pharmacia Biotech). Following purification, the GST moiety can be
proteolytically cleaved from
TRICH at specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity
purification using commercially available monoclonal and polyclonal anti-FLAG
antibodies (Eastman
Kodak). 6-His, a stretch of six consecutive histidine residues, enables
purification on metal-chelate
resins (QIAGEN). Methods for protein expression and purification are discussed
in Ausubel (1995,
sera, ch. 10 and 16). Purified TRICH obtained by these methods can be used
directly in the assays
shown in Examples XVI, XVII, and XVIII, where applicable.
XIII. Functional Assays
TRICH function is assessed by expressing the sequences encoding TRICH at
physiologically
elevated levels in mammalian cell culture systems. cDNA is subcloned into a
mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice
include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA),
both of which
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
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formulations or electroporation. 1-2 ,ug of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate
the apoptotic state of the cells and other cellular properties. FCM detects
and quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events
include changes in nuclear DNA content as measured by staining of DNA with
propidium iodide;
changes in cell size and granularity as measured by forward light scatter and
90 degree side light
scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as
measured by reactivity with
specific antibodies; and alterations in plasma membrane composition as
measured by the binding of
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York NY.
The influence of TRICH on gene expression can be assessed using highly
purified
populations of cells transfected with sequences encoding TRICH and either CD64
or CD64-GFP.
CD64 and CD64-GFP are expressed on the surface of transfected cells and bind
to conserved regions
of human immunoglobulin G (IgG). Transfected cells are efficiently separated
from nontransfected
cells using magnetic beads coated with either human IgG or antibody against
CD64 (DYNAL, Lake
Success NY). mRNA can be purified from the cells using methods well known by
those of skill in
the art. Expression of mRNA encoding TRICH and other genes of interest can be
analyzed by
northern analysis or microarray techniques.
XIV. Production of TRICH Specific Antibodies
TRICH substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used to
immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the TRICH amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, su ra, ch. 11.)
Typically, oligopeptides of about 15 residues in length axe synthesized using
an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
I~LH (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) to
CA 02427010 2003-04-25
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increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are
immunized with the
oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are
tested for
antipeptide and anti-TRICH activity by, for example, binding the peptide or
TRICH to a substrate,
blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting
with radio-iodinated goat
anti-rabbit IgG.
XV. Purification of Naturally Occurring TRICH Using Specific Antibodies
Naturally occurring or recombinant TRICH is substantially purified by
immunoa~nity
chromatography using antibodies specific for TRICH. An immunoaffinity column
is constructed by
covalently coupling anti-TRICH antibody to an activated chromatographic resin,
such as
CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the
resin is
blocked and washed according to the manufacturer's instructions.
Media containing TRICH are passed over the immunoaffmity column, and the
column is
washed under conditions that allow the preferential absorbance of TRICH (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/TRICH binding (e.g., a buffer of pH 2 to pH 3, or a high
concentration of a chaotrope, such
as urea or thiocyanate ion), and TRICH is collected. ,
XVI. Identification of Molecules Which Interact with TRICH
Molecules which interact with TRICH may include transporter substrates,
agonists or
antagonists, modulatory proteins such as G(3~y proteins (Reimann, supra) or
proteins involved in
TRICH localization or clustering such as MAGUKs (Craven, supra). TRICH, or
biologically active
fragments thereof, are labeled with'zsI 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
mufti-well plate are incubated with the labeled TRICH, washed, and any wells
with labeled TRICH
complex are assayed. Data obtained using different concentrations of TRICH are
used to calculate
values for the number, affinity, and association of TRICH with the candidate
molecules.
Alternatively, proteins that interact with TRICH are isolated using the yeast
2-hybrid system
(Fields, S. and O. Song (1989) Nature 340:245-246). TRICH, or fragments
thereof, are expressed as
fusion proteins with the DNA binding domain of Gal4 or lexA, and potential
interacting proteins are
expressed as fusion proteins with an activation domain. Interactions between
the TRICH fusion
protein and the TRICH interacting proteins (fusion proteins with an activation
domain) reconstitute a
transactivation function that is observed by expression of a reporter gene.
Yeast 2-hybrid systems are
commercially available, and methods for use of the yeast 2-hybrid system with
ion channel proteins
are discussed in Niethammer, M. and M. Sheng (1998, Meth. Enzymol. 293:104-
122).
TRICH may also be used in the PATHCALLING process (CuraGen Corp., New Haven
CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
86
CA 02427010 2003-04-25
WO 02/40541 PCT/USO1/46055
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 XVBI.
XVII. Demonstration of TRICH Activity
Ion channel activity of TRICH is demonstrated using an electrophysiological
assay for ion
conductance. TRICH can be expressed by transforming a mammalian cell line such
as COS7, HeLa
or CHO with a eukaryotic expression vector encoding TRICH. Eukaryotic
expression vectors are
commercially available, and the techniques to introduce them into cells are
well known to those
skilled in the art. A second plasmid which expresses any one of a number of
marker genes, such as 13-
galactosidase, is co-transformed into the cells to allow rapid identification
of those cells which have
taken up and expressed the foreign DNA. The cells are incubated for 48-72
hours after
transformation under conditions appropriate for the cell line to allow
expression and accumulation of
TRICH and 13-galactosidase.
Transformed cells expressing 13-galactosidase are stained blue when a suitable
colorimetric
substrate is added to the culture media under conditions that are well known
in the art. Stained cells
are tested for differences in membrane conductance by electrophysiological
techniques that are well
known in the art. Untransformed cells, and/or cells transformed with either
vector sequences alone or
13-galactosidase sequences alone, are used as controls and tested in parallel.
Cells expressing TRICH
will have higher anion or cation conductance relative to control cells. The
contribution of TRICH to
conductance can be confirmed by incubating the cells using antibodies specific
for TRICH. The
antibodies will bind to the extracellular side of TRICH, thereby blocking the
pore in the ion channel,
and the associated conductance.
Alternatively, ion channel activity of TRICH is measured as current flow
across a TRICH-
containing Xeno us laevis oocyte membrane using the two-electrode voltage-
clamp technique (Isle et
al., supra; Jegla, T. and L. Salkoff (1997) J. Neurosci. 17:32-44). TRICH is
subcloned into an
appropriate Xenopus oocyte expression vector, such as pBF, and 0.5-5 ng of
mRNA is injected into
mature stage IV aocytes. Injected oocytes are incubated at 18 °C for 1-
5 days. Inside-out
macropatches are excised into an intracellular solution containing 116 mM K-
gluconate, 4 mM KCI,
and 10 mM Hepes (pH 7.2). The intracellular solution is supplemented with
varying concentrations
of the TRICH mediator, such as CAMP, cGMP, or Ca+2 (in the form of CaClz),
where appropriate.
Electrode resistance is set at 2-5 MSZ and electrodes are filled with the
intracellular solution lacking
mediator. Experiments are performed at room temperature from a holding
potential of 0 mV.
Voltage ramps (2.5 s) from -100 to 100 mV are acquired at a sampling frequency
of 500 Hz. Current
measured is proportional to the activity of TRICH in the assay.
87
CA 02427010 2003-04-25
WO 02/40541 PCT/USO1/46055
In particular, the activity of TRICH-2 is measured as voltage-gated Caz+ or
Nay conductance,
the activity of TRICH-15 is measured as Caz+ conductance, and the activity of
TRICH-16 is measured
as K+ conductance.
Transport activity of TRICH is assayed by measuring uptake of labeled
substrates into
Xenopus laevis oocytes. Oocytes at stages V and VI are injected with TRICH
mRNA (10 ng per
oocyte) and incubated for 3 days at 18°C in OR2 medium (82.5mM NaCI,
2.5 mM KCI, 1mM CaCI z,
1mM MgCI z, 1mM NazHP04, 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
(100nnM NaCI, 2 mM
KCI, 1mM CaCI z, 1mM MgCI z, 10 mM HepeslTris pH 7.5). Uptake of various
substrates (e.g.,
amino acids, sugars, drugs, ions, and neurotransmitters) is initiated by
adding labeled substrate (e.g.
radiolabeled with 3H, fluorescently labeled with rhodamine, etc.) to the
oocytes. After incubating for
30 minutes, uptake is terminated by washing the oocytes three times in Na+-
free medium, measuring
the incorporated label, and comparing with controls. TRICH activity is
proportional to the level of
internalized labeled substrate. In particular, test substrates include
tricarboxylates for TRICH-1, H+
for TRICH-3, sulfate for TRICH-4, Na+ for TRICH-5, anionic metabolites for
TRICH-6, glucose-6-
phosphate for TRICH-8, and amino acids for TRICH-10.
ATPase activity associated with TRICH can be measured by hydrolysis of
radiolabeled ATP-
[,y-3zP], separation of the hydrolysis products by chromatographic methods,
and quantitation of the
recovered 3zP using a scintillation counter. The reaction mixture contains ATP-
[~ 3zP] and varying
amounts of TRICH in a suitable buffer incubated at 37°C for a suitable
period of time. The reaction
is terminated by acid precipitation with trichloroacetic acid and then
neutralized with base, and an
aliquot of the reaction mixture is subjected to membrane or filter paper-based
chromatography to
separate the reaction products. The amount of 3zP liberated is counted in a
scintillation counter. The
amount of radioactivity recovered is proportional to the ATPase activity of
TRICH in the assay.
Lipocalin activity of TRICH is measured by ligand fluorescence enhancement
spectrofluorometry (Lin et al. (1997) Molecular Vision 3:17). Examples of
ligands include retinol
(Sigma, St. Louis MO) and 16-anthryloxy-palmitic acid (16-AP) (Molecular
Probes Inc., Eugene OR).
Ligand is dissolved in 100% ethanol and its concentration is estimated using
known extinction
coefficents (retinol: 46,000 A!M/cm at 325 nm; 16-AP: 8,200 A!Mlcm at 361 nm).
A 700 ~,1 aliquot
of 1 ~M TRICH in 10 mM Tris (pH 7.5), 2 mM EDTA, and 500 rnM NaCI is placed in
a 1 cm path
length quartz cuvette and 1 ~,l aliquots of ligand solution are added.
Fluorescence is measured 100
seconds after each addition until readings are stable. Change in fluorescence
per unit change in
ligand concentration is proportional to TRICH activity.
XVIII. Identification of TRICH Agonists and Antagonists
TRICH is expressed in a eukaryotic cell line such as CHO (Chinese Hamster
Ovary) or HEK
88
CA 02427010 2003-04-25
WO 02/40541 PCT/USO1/46055
(Human Embryonic Kidney) 293. Ion channel activity of the transformed cells is
measured in the
presence and absence of candidate agonists or antagonists. Ion channel
activity is assayed using
patch clamp methods well known in the art or as described in Example XVII.
Alternatively, ion
channel activity is assayed using fluorescent techniques that measure ion flux
across the cell
membrane (Velicelebi, G. et al. (1999) Meth. Enzymol. 294:20-47; West, M.R.
and C.R. Molloy
(1996) Anal. Biochem. 241:51-58). These assays may be adapted for high-
throughput screening
using microplates. Changes in internal ion concentration are measured using
fluorescent dyes such as
the Caz+ indicator Fluo-4 AM, sodium-sensitive dyes such as SBFI and sodium
green, or the Cl-
indicator MQAE (all available from Molecular Probes) in combination with the
FL1PR 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.
89
CA 02427010 2003-04-25
WO 02/40541 PCT/USO1/46055
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