CA2458625A1 - Transporters and ion channels - Google Patents

Abstract

Various embodiments of the invention provide human transporters and ion channels (TRICH) and polynucleotides which identify and encode TRICH. Embodiments of the invention also provide expression vectors, host cells, antibodies, agonists, and antagonists. Other embodiments provide methods for diagnosing, treating, or preventing disorders associated with aberrant expression of TRICH.

Description

TRANSPORTERS AND ION CHANNELS
TECHNICAL FIELD
The invention relates to novel nucleic acids, transporters and ion channels encoded by these nucleic acids, and to the use of these nucleic acids and proteins in the diagnosis, treatment, and prevention of transport, neurological, muscle, immunological and cell proliferative disorders. The invention also relates to the assessment of the effects of exogenous compounds on the expression of nucleic acids and transporters and ion channels.
1o BACKGROUND OF THE INVENTION
Eukaryotic cells are surrounded and subdivided into functionally distinct organelles by hydrophobic lipid bilayer membranes which are highly impermeable to most polar molecules. Cells and organelles require transport proteins to import and export essential nutrients and metal ions including K+~ ~+~ pl~ SOøa-~ sugars, and vitamins, as well as various metabolic waste products. Transport proteins also play roles in antibiotic resistance, toxin secretion, ion balance, synaptic neurotransmission, kidney function, intestinal absorption, tumor growth, and other diverse cell functions (Griffith, J. and C.
Sansom (1998) The Transporter Facts Book, Academic Press, San Diego CA, pp. 3-29). Transport can occur by a passive concentration-dependent mechanism, or can be linked to an energy source such as ATP hydrolysis or an ion gradient. Proteins that function in transport include carrier proteins, which bind to a specific solute and undergo a conformational change that translocates the bound solute across the membrane, and channel proteins, which form hydrophilic pores that allow specific solutes to diffuse through the membrane down an electrochemical solute gradient.
Carrier proteins which transport a single solute from one side of the membrane to the other are called uniporters. In contrast, coupled transporters link the transfer of one solute with simultaneous or sequential transfer of a second solute, either in the same direction (symport) or in the opposite direction (antiport). For example, intestinal and kidney epithelium contains a variety of symporter systems driven by the sodium gradient that exists across the plasma membrane. Sodium moves into the cell down its electrochemical gradient and brings the solute into the cell with it. The sodium gradient that provides the driving force for solute uptake is maintained by the ubiquitous Na+/K+ ATPase system. Sodium-coupled transporters include the mammalian glucose transporter (SGLT1), iodide transporter (NIS), and multivitamin transporter (SMVT). All three transporters have twelve putative transmembrane segments, extracellular glycosylation sites, and cytoplasmically-oriented N- and C-termini. NIS plays a crucial role in the evaluation, diagnosis, and treatment of various thyroid pathologies because it is the molecular basis for radioiodide thyroid-imaging techniques and for specific targeting of radioisotopes to the thyroid gland (Levy, O. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:5568-5573). SMVT is expressed in the intestinal mucosa, kidney, and placenta, and is implicated in the transport of the water-soluble vitamins, e.g., biotin and pantothenate (Prasad, P.D. et al. (1998) J. Biol. Chem. 273:7501-7506).
One of the largest families of transporters is the major facilitator superfamily (MFS), also called the uniporter-symporter-antiporter family. MFS transporters are single polypeptide carriers that transport small solutes in response to ion gradients. Members of the MFS are found in all classes of living organisms, and include transporters for sugars, oligosaccharides, phosphates, nitrates, nucleosides, monocarboxylates, and drugs. MFS transporters found in eukaryotes all have a structure comprising 12 transmembrane segments (Pao, S.S. et al. (1998) Microbiol.
Molec. Biol. Rev. 62:1-34).
The largest family of MFS transporters is the sugar transporter family, which includes the seven glucose transporters (GLUT1-GLUT7) found in humans that are required for the transport of glucose and other hexose sugars. These glucose transport proteins have unique tissue distributions and physiological functions. GLUT1 provides many cell types with their basal glucose requirements and transports glucose across epithelial and endothelial barrier tissues; GLUT2 facilitates glucose uptake or efflux from the liver; GLUTS regulates glucose supply to neurons; GLUT4 is responsible for insulin-regulated glucose disposal; and GLUTS regulates fructose uptake into skeletal muscle. Defects in glucose transporters are involved in a recently identified neurological syndrome causing infantile seizures and developmental delay, as well as glycogen storage disease, Fanconi-Bickel syndrome, and non-insulin-dependent diabetes mellitus (Mueckler, M. (1994) Eur. J. Biochem.
219:713-725; Longo, N. and L.J. Blsas (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 K"t 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 kidney, intestine and liver.
Organic anion transporters are selective for hydrophobic, charged molecules with electron-attracting side groups. Organic cation transporters, such as the ammonium transporter, mediate the secretion of a variety of drugs and endogenous metabolites, and contribute to the maintenance of intercellular pH
(Poole, R.C. and A.P. Halestrap (1993) Am. J. Physiol. 264:C761-C782; Price, N.T. et al. (1998) Biochem. J. 329:321-328; and Martinelle, K. and I. Haggstrom (1993) J.
Biotechnol. 30:339-350).
ATP binding cassette (ABC) transporters are members of a superfamily of membrane proteins that transport substances ranging from small molecules such as ions, sugars, amino acids, peptides, and phospholipids, to lipopeptides, large proteins, and complex hydrophobic drugs. ABC
transporters consist of four modules: two nucleotide-binding domains (NBD), which hydrolyze ATP to supply the energy required for transport, and two membrane-spanning domains (MSD), each containing six putative transmembrane segments. These four modules may be encoded by a single gene, as is the case for the cystic fibrosis transmembrane regulator (CFTR), or by separate genes.
When encoded by separate genes, each gene product contains a single NBD and MSD. These "half molecules" form homo- and heterodimers, such as Tap1 and Tap2, the endoplasmic reticulum based major lustocompatibility (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 trausport appears to occur via a high affinity, low capacity protein-mediated transport process.
Fatty acid transport protein (FATP), an integral membrane protein with four transmembrane segments, is expressed in tissues exhibiting high levels of plasma membrane fatty acid flux, such as muscle, heart, and adipose. Expression of FATP is upregulated in 3T3-L1 cells during adipose conversion, and expression in COS7 fibroblasts elevates uptake of long-chain fatty acids (Hui, T.Y. et al. (1998) J. Biol. Chem. 273:27420-27429).
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) anal 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 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 (rA2T~, 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 c1 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 tricarboxylate carrier which transports citrate and malate; and the Grave's disease carrier protein, a protein recognized by IgG in patients with active Grave's disease, an autoimmune disorder resulting in hyperthyroidism. Proteins in this family consist of three tandem repeats of an approximately 100 amino~acid domain, each of which contains two transmembrane regions (Stryer, L. (1995) Biochemistry, W.H. Freeman and Company, New York NY, p. 551; PROSITE PDOC00189 Mitochondrial energy transfer proteins signature; Online Mendelian Inheritance in Man (OM1M) *275000 Graves Disease).

This class of trausporters also includes the mitochondrial uncoupling proteins, which create proton leaks across the inner mitochondrial membrane, thus uncoupling oxidative phosphorylation from ATP synthesis. The result is energy dissipation in the form of heat.
Mitochondrial uncoupling proteins have been implicated as modulators of thermoregulation and metabolic rate, and have been proposed as potential targets for drugs against metabolic diseases such as obesity (Ricquier, D. et al. (1999) J.
Int. Med. 245:637-642).
Ion Channels The electrical potential of a cell is generated and maintained by controlling the movement of ions across the plasma membrane. The movement of ions requires ion channels, which form ion-selective pores within the membrane. There are two basic types of ion channels, ion transporters and gated ion channels. Ion trausporters 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 au ion down the ion's electrochemical gradient under restricted conditions.
Together, these types of ion channels generate, maintain, and utilize an electrochemical gradient that is used in 1) electrical impulse conduction down the axon of a nerve cell, 2) trausport of molecules into cells against concentration gradients, 3) initiation of muscle contraction, and 4) endocrine cell secretion.
Ion Trausporters Ion transporters generate and maintain the resting electrical potential of a cell. Utilizing the energy derived from ATP hydrolysis, they transport ions against the ion's concentration gradient.
These transmembrane ATPases are divided into three families. The phosphorylated (P) class ion transporters, including Na+-K+ ATPase, Ca2+-ATPase, and H+-ATPase, are activated by a phosphorylation event. P-class ion transporters are responsible for maintaining resting potential distributions such that cytosolic concentrations of Na+ and Caz+ are low and cytosolic concentration of K~ is high. The vacuolar (V) class of ion trausporters includes H+ pumps on intracellular organelles, such as lysosomes and Golgi. V-class ion transporters are responsible for generating the low pH
within the lumen of these organelles that is required for function. The coupling factor (F) class consists of H+ pumps in the mitochondria. F-class ion transporters utilize a proton gradient to generate ATP from ADP and inorganic phosphate (P;).
The P-ATPases are hexamers of a 100 kD subunit with ten trausmembrane domains and several large cytoplasmic regions that may play a role in ion binding (Scarborough, G.A. (1999) Curr.
Opin. Cell Biol. 11:517-522). The V-ATPases are composed of two functional domains: the Vl domain, a peripheral complex responsible for ATP hydrolysis; and the Vo domain, an integral complex responsible for proton trauslocation across the membrane. The F-ATPases are structurally and evolutionarily related to the V-ATPases. The F-ATPase Fo domain contains 12 copies of the c subunit, a highly hydrophobic protein composed of two transmembrane domains and containing a single buried carboxyl group in TM2 that is essential for proton transport. The V-ATPase Vo domain contains three types of homologous c subunits with four or five transmembrane domains and the essential carboxyl group in TM4 or TM3. Both types of complex also contain a single a subunit that may be involved in regulating the pH dependence of activity (Forgac, M. (1999) J. Biol. Chem.
274:12951-12954).
The resting potential of the cell is utilized in many processes involving carrier proteins and gated ion channels. Carrier proteins utilize the resting potential to transport molecules into and out of the cell. Amino acid and glucose transport into many cells is linked to sodium ion co-transport (symport) so that the movement of Na+ down an electrochemical gradient drives transport of the other molecule up a concentration gradient. Similarly, cardiac muscle links transfer of Caa+ 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 canon channels, and GABA- and glycine-gated chloride channels) open their pores in the presence of a specific ion, nucleotide, or neurotransmitter.
The gating properties of a particular ion channel (i.e., its threshold for and duration of opening and closing) are sometimes modulated by association with auxiliary channel proteins and/or post translational modifications, such as phosphorylation.
Mechanically-gated or mechanosensitive ion channels act as transducers for the senses of touch, hearing, and balance, and also play important roles in cell volume regulation, smooth muscle contraction, and cardiac rhythm generation. A stretch-inactivated channel (SIC) was recently cloned from rat kidney. The SIC channel belongs to a group of channels which are activated by pressure or stress on the cell membrane and conduct both Ca2+ and Na+ (Suzuki, M. et al.
(1999) J. Biol. Chem.
274:6330-6335).
The pore-forming subunits of the voltage-gated cation channels form a superfamily of ion channel proteins. The characteristic domain of these channel proteins comprises six trausmembrane domains (S1-S6), a pore-forming region (P) located between SS and S6, and intracellular amino and carboxy termini. In the Na+ and Ca2+ subfamilies, this domain is repeated four times, while in the K+
channel subfamily, each channel is formed from a tetramer of either identical or dissimilar subunits.
The P region contains information specifying the ion selectivity for the channel. In the case of K+
channels, a GYG tripeptide is involved in this selectivity (Ishii, T.M. et al.
(1997) Proc. Natl. Acad.
Sci. LTSA 94:11651-11656).
Voltage-gated Na+ and K+ channels are necessary for the function of electrically excitable cells, such as nerve and muscle cells. Action potentials, which lead to neurotransmitter release and muscle contraction, arise from large, transient changes in the permeability of the membrane to Na+
and K+ ions. Depolarization of the membrane beyond the threshold level opens voltage-gated Na ~
channels. Sodium ions flow into the cell, further depolarizing the membrane and opening more voltage-gated Na+ channels, which propagates the depolarization down the length of the cell.
Depolarization also opens voltage-gated potassium channels. Consequently, potassium ions flow outward, which leads to repolarization of the membrane. Voltage-gated channels utilize charged residues in the fourth trausmembrane segment (S4) to sense voltage change. The open state lasts only about 1 millisecond, at which time the channel spontaneously converts into an inactive state that cannot be opened irrespective of the membrane potential. Inactivation is mediated by the channel's N-terminus, which acts as a plug that closes the pore. The transition from an inactive to a closed state requires a return to resting potential.
Voltage-gated Nay channels are heterotrimeric complexes composed of a 260 kDa pore-forming a subunit that associates with two smaller auxiliary subunits, (31 and (32. The (32 subunit is a integral membrane glycoprotein that contains an extracellular Ig domain, and its association with a and (31 subunits correlates with increased functional expression of the channel, a change in its gating properties, as well as an increase in whole cell capacitance due to an increase in membrane surface area (Isom, L.L. et al. (1995) Cell 83:433-442).
Non voltage-gated Na+ channels include the members of the amiloride-sensitive Na+
channel/degenerin (NaC/DEG) family. Channel subunits of this family are thought to consist of two trausmembrane domains flanking a long extracellular loop, with the amino and carboxyl termini located within the cell. The NaC/DEG family includes the epithelial Na+ channel (ENaC) involved in 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 canon channels or acid-sensing ion channels (ASIC). ASIC subunits are expressed in the brain and form heteromultimeric Na+-permeable channels. These channels require acid pH
fluctuations for activation. ASIC subunits show homology to the degenerins, a family of mechanically-gated channels originally isolated from C. elegans. Mutations in the degenerins cause neurodegeneration. ASIC subunits may also have a role in neuronal function, or in pain perception, since tissue acidosis causes pain (Waldmann, R. and M. Lazdunski (1998) Curr.
Opin. Neurobiol.
8:418-424; Eglen, R.M. et al. (1999) Trends Pharmacol. Sci. 20:337-342).
K+ channels are located in all cell types, and may be regulated by voltage, ATP concentration, or second messengers such as Ca2+ and cAMP. In non-excitable tissue, K+
channels are involved in protein synthesis, control of endocrine secretions, and the maintenance of osmotic equilibrium across membranes. In neurons and other excitable cells, in addition to regulating action potentials and repolarizing membranes, K+ channels are responsible for setting the resting membrane potential. The cytosol contains non-diffusible anions and, to balance this net negative charge, the cell contains a Na~-K+ pump and ion channels that provide the redistribution of Na+, K+, and Cl .
The pump actively transports Na+ out of the cell and K+ into the cell in a 3:2 ratio. Ion channels in the plasma membrane allow K+ and Cl- to flow by passive diffusion. Because of the high negative charge within the cytosol, Cl- flows out of the cell. The flow of K+ is balanced by an electromotive force pulling K+ into the cell, and a K+ concentration gradient pushing K+ out of the cell. Thus, the resting membrane potential is primarily regulated by K+flow (Salkoff, L. and T. Jegla (1995) Neuron 15:489-492).
Potassium channel subunits of the Shaker-like superfamily all have the characteristic six transmembrane/1 pore domain structure. Four subunits combine as homo- or heterotetramers to form 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 dysrythtnia syndrome (Curran, M.E. (1998) Curr. Opin. Biotechnol. 9:565-572; Kaczorowski, G.J. and M.L. Garcia (1999) Curr. Opin. Chem.
Biol. 3 :448-45 8).
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; C~rran, supra).
The recently recognized TW1K K+ channel family includes the mammalian TWIK-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 2+ channels have been classified into several subtypes based upon their electrophysiological and pharmacological characteristics. L-type Ca2+ channels are predominantly expressed in heart and skeletal muscle where they play an essential role in excitation-contraction coupling. T-type channels are important for cardiac pacemaker activity, while N-type and P/Q-type channels are involved in the control of neurotransmitter release in the central and peripheral nervous system. The L-type and N-type voltage-gated Ca 2+ channels have been purified and, though their functions differ dramatically, they have similar subunit compositions. The channels are composed of three subunits. The al subunit forms the membrane pore and voltage sensor, while the a28 and (3 subunits modulate the voltage-dependence, gating properties, and the current amplitude of the channel.
These subunits are encoded by at least six a1, one a28, and four (3 genes. A
fourth subunit, y, has been identified in skeletal muscle (Walker, D. et al. (1998) J. Biol. Chem.
273:2361-2367; McCleskey, E.W. (1994) Curr. Opin. Neurobiol. 4:304-312).
The high-voltage-activated Ca a+ 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 CadZ family of alphal subunits conduct N-type, P/Q-type, and R-type Ca a+ 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 ~+
currents, which are activated and inactivated more rapidly and at more negative membrane potentials than other Ca 2+ current types. The distinct structures and patterns of regulation of these three families of Ca 2+ channels provide an array of Ca 2+ entry pathways in response to changes in membrane potential and a range of possibilities for regulation of Ca 2+ entry by second messenger pathways and interacting proteins (Catterall, W.A. (2000) Annu. Rev. Cell Dev.
Biol. 16:521-555).
to 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 RAMP (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 Caa+ influx into cells to resupply Ca2+ stores depleted by the action of inositol triphosphate (IP3) and other agents in response to numerous hormones and growth factors. Trp and Trp-like were first cloned from Drosophila and have similarity to voltage gated Ca 2+ channels in the S3 through S6 regions. This suggests that Trp and/or 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 expression in melanoma cells is inversely correlated with melanoma aggressiveness i~t vivo. The human cDNA transcript corresponds to a 1533-amino acid protein having homology to members of the Trp family. It has been proposed that the combined use of malastatin mRNA
expression status and tumor thickness might allow for the determination of subgroups of patients at both low and high risk for developing metastatic disease (Duncan, L.M. et al (2001) J. Clip. Oncol.
19:568-576).
Chloride channels are necessary in endocrine secretion and in regulation of cytosolic and organelle pH. In secretory epithelial cells, Cl- enters the cell across a basolateral membrane through an Na+, K+/Cl- cotransporter, accumulating in the cell above its electrochemical equilibrium concentration. Secretion of Cl- from the apical surface, in response to hormonal stimulation, leads to flow of Na+ and water into the secretory lumen. The cystic fibrosis 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 in determining channel permeation and selectivity (Sather, W.A. et al. (1994) Curr. Opin. Neurobiol. 4:313-323).
Ligand-gated channels can be regulated by intracellular second messengers. For example, calcium-activated K+ channels are gated by internal calcium ions. In nerve cells, an influx of calcium during depolarization opens K+ channels to modulate the magnitude of the action potential (Ishi et al., supra). The large conductance (BK) channel has been purified from brain and its subunit composition determined. The a subunit of the BK channel has seven rather than six transmembrane domains in contrast to voltage-gated K+ channels. The extra transmembrane domain is located at the subunit N-terminus. A 28-amino-acid stretch in the C-terminal region of the subunit (the "calcium bowl" region) contains many negatively charged residues and is thought to be the region responsible for calcium binding. The (3 subunit consists of two transmembrane domains connected by a glycosylated extracellular loop, with intracellular N- and C-termini (Kaczorowski, supf~a;
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, similar to voltage-gated K+ channels. A large C-terminal domain contains a cyclic nucleotide binding domain, while the N-terminal domain confers variation among channel subtypes (Zufall, F. et al. (1997) Curr. Opin. Neurobiol. 7:404-412).
The activity of other types of ion channel proteins may also be modulated by a variety of intracellular signaling 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 1I, all of which regulate ion channel activity in cells. Kir channels are activated by the binding of the G(3y subunits of heterotrimeric G-proteins (Reimann, F. and F.M. Ashcroft (1999) Curr. Opin.
Cell. Biol. 11:503-508).
Other proteins are involved in the localization of ion channels to specific sites in the cell membrane.
Such proteins include the PDZ domain proteins known as MAGUKs (membrane-associated guanylate kinases) which regulate the clustering of ion channels at neuronal synapses (Craven, S.E. and D.S.
Bredt (1998) Cell 93:495-498).
Disease Correlation The etiology of numerous human diseases and disorders can be attributed to defects in the transport of molecules across membranes. Defects in the trafficking of membrane-bound transporters and ion channels are associated with several disorders, e.g., cystic fibrosis, glucose-galactose malabsorption syndrome, hypercholesterolemia, von Gierke disease, and certain forms of diabetes mellitus. Single-gene defect diseases resulting in an inability to transport small molecules across membranes include, e.g., cystinuria, iminoglycinuria, Hartup disease, and Fanconi disease (van't Hoff, W.G. (1996) Exp. Nephrol. 4:253-262; Talente, G.M. et al. (1994) Ann. Intern.
Med. 120:218-226;
and Chillon, M. et al. (1995) New Engl. J. Med. 332:1475-1480).
Human diseases caused by mutations in ion channel genes include disorders of skeletal muscle, cardiac muscle, and the central nervous system. Mutations in the pore-forming subunits of sodium and chloride channels cause myotonia, a muscle disorder in which relaxation after voluntary contraction is delayed. Sodium channel myotonias have been treated with channel 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, supt~a).
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 I~vl.3 was found to suppress delayed-type hypersensitivity and allogenic responses in pigs, validating the idea of channel blockers as safe and efficacious immunosuppressants (Cahalan, M.D. and K.G. Chandy (1997) Curr. Opin.
Biotechnol. 8:749-756).
Expression profiling Microarrays are analytical tools used in bioanalysis. A microarray has a plurality of molecules spatially distributed over, and stably associated with, the surface of a solid support. Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry.
One area in particular in which microarrays hnd use is in gene expression analysis. Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants.
When an expression profile is examined, arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling 14 .

cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder.
The potential application of gene expression profiling is relevant to improving the diagnosis, prognosis, and treatment of cancers, such as breast cancer, lung cancer, prostate cancer, ovarian cancer, and bone cancer, as well as the treatment of vascular inflammation and immune responses, liver toxicity, and neurological disorders.
Breast cancer More than 180,000 new cases of breast cancer are diagnosed each year, and the mortality rate for breast cancer approaches 10% of all deaths in females between the ages of 45-54 (Gish, I~.
(1999) AWIS Magazine 28:7-10). However the survival rate based on early diagnosis of localized breast cancer is extremely high (97%), compared with the advanced stage of the disease in which the tumor has spread beyond the breast (22%). Current procedures for clinical breast examination are lacking in sensitivity and specificity, and efforts are underway to develop comprehensive gene expression profiles for breast cancer that may be used in conjunction with conventional screening methods to improve diagnosis and prognosis of this disease (Perou, C.M. et al.
(2000) Nature 406:747-752).
Mutations in two genes, BRCA1 and BRCA2, are known to greatly predispose a woman to breast cancer and may be passed on from parents to children (Gish, supra).
However, this type of hereditary breast cancer accounts for only about 5% to 9% of breast cancers, while the vast majority of breast cancer is due to non-inherited mutations that occur in breast epithelial cells.
The relationship between expression of epidermal growth factor (EGF) and its receptor, EGFR, to human mammary carcinoma has been particularly well studied. (See I~hazaie, K. et al.
(1993) Cancer and Metastasis Rev. 12:255-274, and references cited therein for a review of this area.) Overexpression of EGFR, particularly coupled with down-regulation of the estrogen receptor, is a marker of poor prognosis in breast cancer patients. In addition, EGFR
expression in breast tumor metastases is frequently elevated relative to the primary tumor, suggesting that EGFR is involved in tumor progression and metastasis. This is supported by accumulating evidence that EGF has effects on cell functions related to metastatic potential, such as cell motility, chemotaxis, secretion and differentiation. Changes in expression of other members of the erbB receptor family, of which EGFR
is one, have also been implicated in breast cancer. The abundance of erbB
receptors, such as HER-2/neu, HER-3, and HER-4, and their ligands in breast cancer points to their functional importance in the pathogenesis of the disease, and may therefore provide targets for therapy of the disease (Bacus, S.S. et al. (1994) Am. J. Clin. Pathol. 102:513-S24). Other known markers of breast cancer include a human secreted frizzled protein mRNA that is downregulated in breast tumors;
the matrix G1a protein which is overexpressed in human breast carcinoma cells; Drg1 or RTP, a gene whose expression is diminished in colon, breast, and prostate tumors; maspin, a tumor suppressor gene downregulated in invasive breast carcinomas; and CaNl9, a member of the S 100 protein family, all of which are down-regulated in mammary carcinoma cells relative to normal mammary epithelial cells (Zhou, Z. et al.
(1998) Int. J. Cancer 78:95-99; Chen, L. et al. (1990) Oncogene 5:1391-1395;
Ulrix, W. et al (1999) FEBS Lett 455:23-26; Sager, R. et al. (1996) Curr. Top. Microbiol. Tmmunol.
213:51-64; and Lee, S.W. et al. (1992) Proc. Natl. Acad. Sci. USA 89:2504-2508).
Cell lines derived from human mammary epithelial cells at various stages of breast cancer provide a useful model to study the process of malignant transformation and tumor progression as it has been shown that these cell lines retain many of the properties of their parental tumors for lengthy culture periods (Wistuba, LI. et al. (1998) Clip. Cancer Res. 4:2931-2938).
Such a model is particularly useful for comparing phenotypic and molecular characteristics of human mammary epithelial cells at various stages of malignant transformation.
Lung cancer Lung cancer is the leading cause of cancer death in the United States, affecting more than 100,000 men and 50,000 women each year. Nearly 90% of the patients diagnosed with lung cancer are cigarette smokers. Tobacco smoke contains thousands of noxious substances that induce carcinogen metabolizing enzymes and covalent DNA adduct formation in the exposed bronchial epithelium. In nearly 80°l0 of patients diagnosed with lung cancer, metastasis has already occurred.
Most commonly lung cancers metastasize to pleura, brain, bone, pericardium, and liver. The decision to treat with surgery, radiation therapy, or chemotherapy is made on the basis of tumor histology, response to growth factors or hormones, and sensitivity to inhibitors or drugs. With current treatments, most patients die within one year of diagnosis. Earlier diagnosis and a systematic approach to identification, staging, and treatment of lung cancer could positively affect patient outcome.
Lung cancers progress through a series of morphologically distinct stages from hyperplasia to invasive carcinoma. Malignant lung cancers are divided into two groups comprising four histopathological classes. The Non Small Cell Lung Carcinoma (NSCLC) group includes squamous cell carcinomas, adenocarcinomas, and large cell carcinomas and accounts for about 70% of all lung cancer cases. Adenocarcinomas typically arise in the peripheral airways and often form mucin secreting glands. Squamous cell carcinomas typically arise in proximal airways. The histogenesis of squamous cell carcinomas may be related to chronic inflammation and injury to the bronchial epithelium, leading to squamous metaplasia. The Small Cell Lung Carcinoma (SCLC) group accounts for about 20% of lung cancer cases. SCLCs typically arise in proximal airways and exhibit a number of paraneoplastic syndromes including inappropriate production of adrenocorticotropin and anti-diuretic hormone.
Lung cancer cells accumulate numerous genetic lesions, many of which are associated with cytologically visible chromosomal aberrations. The high frequency of chromosomal deletions associated with lung cancer may reflect the role of multiple tumor suppressor loci in the etiology of this disease. Deletion of the short arm of chromosome 3 is found in over 90% of cases and represents one of the earliest genetic lesions leading to lung cancer. Deletions at chromosome arms 9p and 17p are also common. Other frequently observed genetic lesions include overexpression of telomerase, activation of oncogenes such as I~-ras and c-myc, and inactivation of tumor suppressor genes such as RB, p53 and CDKN2.
Genes differentially regulated in lung cancer have been identified by a variety of methods.
Using mRNA differential display technology, Manda et al. (1999; Genomics 51:5-14) identified five genes differentially expressed in lung cancer cell lines compared to normal bronchial epithelial cells.
Among the known genes, pulmonary surfactant apoprotein A and alpha 2 macroglobulin were down regulated whereas nm23H1 was upregulated. Petersen et al.. (2000; Int J.
Cancer, 86:512-517) used suppression subtractive hybridization to identify 552 clones differentially expressed in lung tumor derived cell lines, 205 of which represented known genes. Among the known genes, throinbospondin-1, fibronectin, intercellular adhesion molecule 1, and cytokeratins 6 and 18 were previously observed to be differentially expressed in lung cancers. Wang et al. (2000; Oncogene 19:1519-1528) used a combination of microarray analysis and subtractive hybridization to identify 17 genes differentially overexpresssed in squamous cell carcinoma compared with normal lung epithelium. Among the known genes they identified were keratin isoform 6, KOC, SPRC, IGFb2, connexin 26, plakofillin 1 and cytokeratin 13.
Prostate Cancer Prostate cancer is a common malignancy in men over the age of 50, and the incidence increases with age. In the US, there are approximately 132,000 newly diagnosed cases of prostate cancer and more than 33,000 deaths from the disorder each year.
Once cancer cells arise in the prostate, they are stimulated by testosterone to a more rapid growth. Thus, removal of the testes can indirectly reduce both rapid growth and metastasis of the cancer. Over 95 percent of prostatic cancers are adenocarcinomas which originate in the prostatic acini. The remaining 5 percent are divided between squamous cell and transitional cell carcinomas, both of which arise in the prostatic ducts or other parts of the prostate gland.
As with most tumors, prostate cancer develops through a multistage progression ultimately resulting in an aggressive tumor phenotype. The initial step in tumor progression involves the hyperproliferation of normal luminal and/or basal epithelial cells. Androgen responsive cells become hyperplastic and evolve into early-stage tumors. Although early-stage tumors are often androgen sensitive and respond to androgen ablation, a population of androgen independent cells evolve from the hyperplastic population. These cells represent a more advanced form of prostate tumor that may become invasive and potentially become metastatic to the bone, brain, or lung.
A variety of genes may be differentially expressed during tumor progression. For example, loss of heterozygosity (LOH) is frequently observed on chromosome 8p in prostate cancer. Fluorescence in situ hybridization (FISH) revealed a deletion for at least 1 locus on 8p in 29 (69%) tumors, with a significantly higher frequency of the deletion on 8p21.2-p21.1 in advanced prostate cancer than in localized prostate cancer, implying that deletions on 8p22-p21.3 play an important role in tumor differentiation, while 8p21.2-p21.1 deletion plays a role in progression of prostate cancer (Oba, K.
et al. (2001) Cancer Genet. Cytogenet. 124: 20-26).
A primary diagnostic marker for prostate cancer is prostate specific antigen (PSA). PSA is a tissue-specific serine protease almost exclusively produced by prostatic epithelial cells. The quantity of PSA correlates with the number and volume of the prostatic epithelial cells, and consequently, the levels of PSA are an excellent indicator of abnormal prostate growth. Men with prostate cancer exhibit an early linear increase in PSA levels followed by an exponential increase prior to diagnosis.
However, since PSA levels are also influenced by factors such as inflammation, androgen and other growth factors, some scientists maintain that changes in PSA levels are not useful in detecting individual cases of prostate cancer.
Current areas of cancer research provide additional prospects for markers as well as potential therapeutic targets for prostate cancer. Several growth factors have been shown to play a critical role in tumor development, growth, and progression. The growth factors Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), and Tumor Growth Factor alpha (TGFa) are important in the growth of normal as well as hyperproliferative prostate epithelial cells, particularly at early stages of tumor development and progression, and affect signaling pathways in these cells in various ways (Lin, J. et al. (1999) Cancer Res. 59:2891-2897; Putz, T. et al. (1999) Cancer Res. 59:227-233). The TGF-(3 family of growth factors are generally expressed at increased levels in human cancers and the high expression levels in many cases.correlates with advanced stages of malignancy and poor survival (Gold, L.I. (1999) Crit. Rev. Oncog. 10:303-360). Finally, there are human cell lines representing both the androgen-dependent stage of prostate cancer (LNCap) as well as the androgen-independent, hormone refractory stage of the disease (PC3 and DU-145) that have proved useful in studying gene expression patterns associated with the progression of prostate cancer, and the effects of cell treatments on these expressed genes (Chung, T.D. (1999) Prostate 15:199-207).
Ovarian cancer Ovarian cancer is the leading cause of death from a gynecologic cancer. The majority of ovarian cancers are derived from epithelial cells, and 70% of patients with epithelial ovarian cancers present with late-stage disease. As a result, the long-term survival rate for this disease is very low.
Identification of early-stage markers for ovarian cancer would significantly increase the survival rate.
Genetic variations involved in ovarian cancer development include mutation of p53 and microsatellite instability. Gene expression patterns likely vary when normal ovary is compared to ovarian tumors.
Bone cancer Osteosarcoma is the most common malignant bone tumor in children.
Approximately 80% of patients present with non-metastatic disease. After the diagnosis is made by an initial biopsy, treatment involves the use of 3-4 courses of neoadjuvant chemotherapy before definitive surgery, followed by post-operative chemotherapy. With currently available treatment regimens, approximately 30-4.0% of patients with non-metastatic disease relapse after therapy.
Currently, there is no prognostic factor that can be used at the time of initial diagnosis to predict which patients will have a high risk of relapse. The only significant prognostic factor predicting the outcome in a patient with non-metastatic osteosarcoma is the histopathologic response of the primary tumor resected at the time of definitive surgery. The degree of necrosis in the primary tumor is a reflection of the tumor response to neoadjuvant chemotherapy. A higher degree of necrosis (good or favorable response) is associated with a lower risk of relapse and a better outcome. Patients with a lower degree of necrosis (poor or unfavorable response) have a much higher risk of relapse and poor outcome even after complete resection of the primary tumor. Unfortunately, poor outcome cannot be altered despite modification of post-operative chemotherapy to account for the resistance of the primary tumor to neoadjuvant chemotherapy. Thus, there is an urgent need to identify prognostic factors that can be used at the time of diagnosis to recognize the subtypes of osteosarcomas that have various risks of relapse, so that more appropriate chemotherapy can be used at the outset to improve the outcome.
Inflammation and immune responses Atherosclerosis is a pathological condition characterized by a chronic local inflammatory response within the vessel wall of major arteries. Disease progression results in the formation of atherosclerotic lesions, unstable plaques which occasionally rupture;
precipitating a catastrophic thrombotic occlusion of the vessel lumen. Atherosclerosis and the associated coronary artery disease and cerebral stroke represent the most common causes of death in industrialized nations. Although certain key risk factors have been identified, a full molecular characterization that elucidates the causes and identifies all potential therapeutic targets for this complex disease has not been achieved.
Molecular characterization of atherosclerosis requires identification of the genes that contribute to lesion growth, stability, dissolution, rupture and induction of occlusive vessel thrombi.
Blood vessel walls are composed of two tissue layers: au endothelial cell (EC) layer which comprises the lumenal surface of the vessel, and an underlying vascular smooth muscle cell (VSMC) layer. Through dynamic interactions with each other and with surrounding tissues, the vascular endothelium and smooth muscle tissues maintain vascular tone, control selective permeability of the vascular wall, direct vessel remodeling and angiogenesis, and modulate inflammatory and immune responses.
The inflammatory response is a complex vascular reaction mediated by numerous cytokines, chemokines, growth factors, and other signaling molecules expressed by activated ECs, VSMCs and leukocytes. Inflammation protects the organism during trauma and infection, but can also lead to pathological conditions such as atherosclerosis. Activation of vascular endothelium is a central event in a wide range of physiological and disease processes such as vascular tone regulation, coagulation and thrombosis, atherosclerosis, inflammation and some infectious diseases.
The pro-inflammatory cytokines, interleukin (IL)-1 and tumor necrosis factor (TNF), are secreted by a small number of activated macrophages or other cells and can set off a cascade of vascular changes, largely through their ability to alter gene expression patterns in ECs and VSMCs.
These vascular changes include vasodilation and increased permeability of microvasculature, edema, and leukocyte extravasation and transmigration across the vessel wall.
Ultimately, leukocytes, particularly neutrophils and monocytes/macrophages, accumulate iu the extravascular space, where they remove injurious agents by phagocytosis and oxidative killing, a process accompanied by release of toxic factors, such as proteases and reactive oxygen species.
IL-1 and TNF induce pro-inflammatory, thrombotic, and anti-apoptotic changes in gene expression by signaling through receptors on the surface of ECs and VSMCs;
these receptors activate transcription factors such as NFkB as well as AP-1, IRF-1, and NF-GMa, leading to alterations in gene expression. Genes known to be differentially regulated in EC by 1L-1 and TNF include E
selectin, VCAM-1, ICAM-1, PAF, IkBa, IAP-1, MCP-1, eotaxin, ENA-78, G-CSF, A20, ICE, and complement C3 component. A key event in inflammation, adhesion and transmigration of blood leukocytes across the vascular endothelium, for example, is mediated by increased expression of E
selectin, P selectin, ICAM-1, and VCAM-1 on activated endothelium.
Several investigators have examined changes in vascular cell gene expression associated with various inflammatory diseases or model systems. Examining human umbilical vein endothelial cells (IIUVEC) activated by recombinant TNF-a or conditioned medium from activated human primary monocytes, Horrevoets et al. (1999; Blood 93:3418-3431) identified 106 differentially regulated genes.
In a similar approach, deVries et al. (2000; JBC 275:23939-23947) identified 40 differentially regulated genes in umbilical cord artery-derived smooth muscle cells activated by conditioned media from cultured macrophages after stimulation with oxidized LDL particles. In both studies, many of the identified genes were already known to be involved in inflammation. Comparing expression profiles from inflammatory diseased tissues, cultured macrophages, chondrocyte cell lines, primary chondrocytes, and synoviocytes, Heller et al. (1997; Proc Natl Acad Sci USA
94:2150-2155) identifed candidate genes involved in inflammatory responses, including TNF, IL-1 IL-6, IL-8 G-CSF, RANTES, and V-CAM. From this candidate gene set, tissue inhibitor of metalloproteinase 1, ferritin light chain, and manganese superoxide dismutase were found to be differentially expressed in rheumatoid arthritis (RA) relative to inflammatory bowel disease (IBD).
Further, IL-3, chemokine Groa, and metalloproteinase matrix metallo-elastase were expressed in both RA
and 1BD. Most recently, in an analysis of cultured aortic smooth muscle cells treated with TNF-a, Haley et a1. (2000;
Circulation 102:2185-2189) found a 20-fold increase in eotaxin, an eosinophil chemotactic factor. The overexpression of eotaxin and its receptor CCR3 in atherosclerotic lesions was confirmed by northern analysis.
Human coronary artery endothelial cells (HCAECs) are primary cells derived from the endothelium of a human coronary artery. HCAECs are used as an experimental model for investigating the role of the endothelium in human vascular biology itt vitro.
Human umbilical artery endothelial cells (HUAECs) are primary cells derived from the endothelium of an umbilical artery.
Human uterine myometrium microvascular endothelial cells (UtMVECs) are primary cells derived from the uterine myometrium microvasculature. Human Iliac Artery Endothelial Cells (HIAECs) are primary cells derived from the endothelium of an iliac artery. Human umbilical vein endothelial cells (HWECs) are a primary cell line derived from the endothelium of the human umbilical vein.
ECV304 is a human endothelial line.
Neurological disorders Characterization of region-specific gene expression in the human brain provides a context and background for molecular neurobiology on a variety of neurological disorders.
For example, Alzheimer's disease (AD) is a progressive, neurodestructive process of the human neocortex, characterized by the deterioration of memory and higher cognitive function. A
progressive and irreversible brain disorder, AD is characterized by three major pathogenic episodes involving (a) an aberrant processing and deposition of beta-amyloid precursor protein (betaAPP) to form neurotoxic beta-amyloid (betaA) peptides and an aggregated insoluble polymer of betaA
that forms the senile plaque, (b) the establishment of intraneuronal neuritic tan pathology yielding widespread deposits of agyrophilic neurofibrillary tangles (NFT) and (c) the initiation and proliferation of a brain-specific inflammatory response. These three seemingly disperse attributes of AD
etiopathogenesis are licked by the fact that proinflammatory microglia, reactive astrocytes and their associated cytokines and chemokines are associated with the biology of the microtubule associated protein tan, betaA speciation and aggregation. Missense mutations in the presenilin genes PS 1 and PS2, implicated in early onset familial AD, cause abnormal betaAPP processing with resultant overproduction of betaA42 and related neurotoxic peptides. Specific betaA fragments such as betaA42 can further potentiate proinflammatory mechanisms. Expression of the inducible oxidoreductase cyclooxygenase-2 and cytosolic phospholipase A2 (cPLA2) is strongly activated during cerebral ischemia and trauma, epilepsy and AD, indicating the induction of proinflammatory gene pathways as a response to brain injury. Neurotoxic metals such as aluminum and zinc, both implicated in AD
etiopathogenesis, and arachidonic acid, a major metabolite of brain cPLA2 activity, each polymerize hyperphosphorylated tan to form NFT-like bundles. Studies have identified a reduced risk for AD in patients aged over 70 2o years who were previously treated with non-steroidal anti-inflammatory drugs for non-CNS afflictions that include arthritis. (For a review of the interrelationships between the mechanisms of PS 1, PS2 and betaAPP gene expression, tan and betaA deposition and the induction, regulation and proliferation in AD of the neuroinflammatory response, see Lukiw, W.J, and Bazan, N.G. (2000) Neurochem. Res.
2000 25:1173-1184).
Tumor necrosis factor-alpha (TNF-a) is a pleiotropic cytokine that plays a central role in mediation of the inflammatory response through activation of multiple signal transduction pathways.
TNF-a is produced by activated lymphocytes, macrophages, and other white blood cells, and activates endothelial cells. Interferon-gamma (IFN~y), also known as Type II interferon or immune interferon, is a cytokine produced primarily by T-lymphocytes and natural killer cells.
Mature IFNy exists as noncovalently- licked homodimers. IFNy displays antiviral, antiproliferative, immunoregulatory, and proinflammatory activities and is important in host defense mechanisms. 1FN-y induces the production of cytokines; upregulates the expression of class I and 1I MHC antigens, Fc receptor, and leukocyte adhesion molecule; modulates macrophage effector functions; influences isotype switching; potentiates the secretion of immunoglobulins by B cells; augments TH1 cell expansion; and may be required for TH1 cell differentiation. IFNy exerts its biological activities by binding to specific cell surface receptors, which display high affinity binding sites. The IFNy receptor is present on almost all cell types except mature erythrocytes. Upon binding to its receptor, IFNy triggers the activation of JAK-1 and JAK-2 kinases resulting in the phosphorylation of STAT1. Both IFNY and TNF-a are considered proinflammatory cytokines. Cross-talk can exist between the signal transduction pathways of two cytokines; for example, signal transduction cascades initiated by two different cytokines lead to the activation of NfkB.
Liver toxicity The human C3A cell line is a clonal derivative of HepG2/C3 (hepatoma cell line, isolated from a 15-year-old male with liver tumor), which was selected for strong contact inhibition of growth. The use of a clonal population enhances the reproducibility of the cells. C3A
cells have many characteristics of primary human hepatocytes in culture: i) expression of insulin receptor and insulin-like growth factor II receptor; ii) secretion of a high ratio of serum albumin compared with a-fetoprotein,; iii) conversion of ammonia to urea and glutamine; iv) metabolism of aromatic amino acids; and v) proliferation in glucose-free and insulin-free medium. The C3A
cell lice is now well established as an in vitro model of the mature human liver (Mickelson et al.
(1995) Hepatology 22:866-875; Nagendra et al. (1997) Am. J. Physiol. 272:6408-6416).
The potential application of gene expression profiling is relevant to measuring the toxic response to potential therapeutic compounds and of the metabolic response to therapeutic agents. For instance, diseases treated with steroids and disorders caused by the metabolic response to treatment with steroids include adenomatosis, cholestasis, cirrhosis, hemangioma, Henoch-Schonlein purpura, hepatitis, hepatocellular and metastatic carcinomas, idiopathic thrombocytopenic purpura, porphyria, sarcoidosis, and Wilson disease. It is desirable to measure the toxic response to potential therapeutic compounds and of the metabolic response to therapeutic agents.
Steroids are a class of lipid-soluble molecules, including cholesterol, bile acids, vitamin D, and hormones, that share a common four-ring structure based on cyclopentanoperhydrophenanthrene and that carrry out a wide variety of functions. Steroid hormones, produced by the adrenal cortex, ovaries, and testes, include glucocorticoids, mineralocorticoids, androgens, and estrogens. Steroid hormones 3o are widely used for fertility control and in anti-inflammatory treatments fox physical injuries and diseases such as arthritis, asthma, and auto-immune disorders. Progesterone, a naturally occurring progestin, is primarily used to treat amenorrhea, abnormal uterine bleeding, or as a contraceptive.

Medroxyprogesterone (MAH), also known as 6 a -methyl-17 hydroxyprogesterone, is a synthetic progestin with a pharmacological activity about 15 times greater than progesterone. MAH is usually used for the treatment of renal and endometrial carcinomas, amenorrhea, abnormal uterine bleeding, and endometriosis associated with hormonal imbalance. The primary contraceptive effect of exogenous progestins involves the suppression of the midcycle surge of LH. The exact mechanism of action, however, is unknown. At the cellular level, progestins diffuse freely into target cells and bind to the progesterone receptor. Target cells include the female reproductive tract, mammary gland, hypothalamus, and pituitary. Once bound to the receptor, progestins slow the frequency of release of gonadotropin releasing hormone (GnRH) from the hypothalamus and blunt the pre-ovulatory LH surge, thereby preventing follicular maturation and ovulation. Interestingly, the MAH
stimulatory effect on the respiratory centers has been used clinically to treat low blood oxygenation due to sleep apnea, chronic obstructive pulmonary disease, or hypercapnia (excess of CO 2 in blood). Beclomethasone is a synthetic glucocorticoid that is used for treating steroid-dependent asthma, relieving symptoms associated with allergic or nonallergic (vasomotor) rhinitis, or for preventing recurrent nasal polyps following surgical removal. The anti-inflammatory and vasoconstrictive effects of intranasal beclomethasone are 5,000 times greater than those produced by hydrocortisone.
Budesonide is a corticosteroid used to control symptoms associated with allergic rhinitis or asthma. Dexamethasone is a synthetic glucocorticoid used in anti-inflammatory or immunosuppressive compositions. Prednisone is metabolized in the liver to its active form, prednisolone, a glucocorticoid with anti-inflammatory properties. Betamethasone is a synthetic glucocorticoid with antiinflammatory and immunosuppressive activity and is used to treat psoriasis and fungal infections, such as athlete's foot and ringworm. By comparing both the levels and sequences expressed in tissues from subjects exposed to or treated with steroid compounds with the levels and sequences expressed in normal untreated tissue it is possible to determine tissue responses to steroids. Budesonide (Bude) is a corticosteroid used to control symptoms associated with allergic rhinitis or asthma. Budesonide has high topical anti-inflammatory activity but low systemic activity. Prednisone is a corticosteroid that is metabolized in the liver to its active form, prednisolone. Prednisone is roughly four times more potent as a glucocorticoid than hydrocortisone. Prednisone is intermediate between hydrocortisone and dexamethasone in duration of action. Prednisone is used in conditions such as allograft rejection, asthma, systemic lupus erythematosus, and many other inflammatory states.
Glucocorticoids are naturally occurring hormones that prevent or suppress inflammation and immune responses when administered at pharmacological doses. At the molecular level, unbound glucocorticoids readily cross cell membranes and bind with high affinity to specific cytoplasmic receptors. Subsequent to binding, transcription and, ultimately, protein synthesis are affected. The result can include inhibition of leukocyte infiltration at the site of inflammation, interference in the function of mediators of inflammatory response, and suppression of humoral immune responses. The anti-inflammatory actions of corticosteroids are thought to involve phospholipase A2 inhibitory proteins, collectively called lipocortins. Lipocortins, in turn, control the biosynthesis of potent mediators of inflammation such as prostaglandins and leukotrienes by inhibiting the release of the precursor molecule arachidonic.
There is a need in the art for new compositions, including nucleic acids and proteins, for the diagnosis, prevention, and treatment of transport, neurological, muscle, immunological and cell proliferative disorders.
SUMMARY OF THE INVENTION
Various embodiments of the invention provide purified polypeptides, transporters and ion channels, referred to collectively as 'TRICH' and individually as 'TRICH-1,' 'TRICH-2,' 'TRICH-3,' 'TRICH-4,' 'TRICH-S,' 'TRICH-6,' 'TRICH-7,' 'TRICH-8,' 'TRICH-9,' 'TRICH-10,' 'TRICH-11,' 'TRICH-12,' 'TRICH-13,' 'TRICH-14,' 'TRICH-15,' 'TRICH-16,' 'TRICH-17,' 'TRICH-18,' 'TRICH-19,' 'TRICH-20,' 'TRICH-21,' 'TRICH-22,' 'TRICH-23,' 'TRICH-24,' 'TRICH-25,' and 'TRICH-2f and methods for using these proteins and their encoding polynucleotides for the detection, diagnosis, and treatment of diseases and medical conditions. Embodiments also provide methods.for utilizing the purified transporters and ion channels and/or their encoding polynucleotides for facilitating the drug discovery process, including determination of efficacy, dosage, toxicity, and pharmacology.
Related embodiments provide methods for utilizing the purified transporters and ion channels and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical conditions.
An embodiment provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ )D N0:1-26, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ 1D
N0:1-26, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ D7 N0:1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ D7 NO:l-26. Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO:1-26:

Still another embodiment 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 >D N0:1-26, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ m N0:1-26, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ D7 N0:1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ )D N0:1-26. In another embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ )D N0:1-26. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ )D N0:27-52.
Still another embodiment 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 >D N0:1-26, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ m N0:1-26, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ )D N0:1-26.
Another embodiment provides a cell transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide.
Another embodiment provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ )D N0:1-26, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ >l7 NO:1-26, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ 117 N0:1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD N0:1-26.
'The method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
Yet another embodiment 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 ~ N0:1-26, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90%
identical to an amino acid sequence selected from the group consisting of SEQ 117 N0:1-26, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ )D N0:1-26, and d) an immunogenic fragment of a polypeptide having au amino acid sequence selected from the group consisting of SEQ D7 NO:1-26.
Still yet another embodiment provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID N0:27-52, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ll~ NO:27-52, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA
equivalent of a)-d). In other embodiments, the polynucleotide can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
1'et another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of.a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ m N0:27-52, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90%
identical or at least about 90%
identical to a polynucleotide sequence selected from the group consisting of SEQ ID N0:27-52, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). T'he 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. In a related embodiment, the method can include detecting the amount of the hybridization complex. In still other embodiments, the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
Still yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:27-52, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID
N0:27-52, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof. In a related embodiment, the method can include detecting the amount of the amplified target polynucleotide or fragment thereof.
Another embodiment provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ >D N0:1-26, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID N0:1-26, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-26, and a pharmaceutically acceptable excipient. In one embodiment, the composition can comprise an amino acid sequence selected from the group consisting of SEQ ID N0:1-26. Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional TRICH, comprising administering to a patient in need of such treatment the composition.
Yet another embodiment 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 117 N0:1-26, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ >D N0:1-26, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ
ID N0:1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ )D N0:1-26. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment 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.
Still yet another embodiment provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID N0:1-26, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90%
identical to an amino acid sequence selected from the group consisting of SEQ
)D NO:1-26, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-26. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample. Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment 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.
Another embodiment provides a method of screenitng for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID N0:1-26, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ D7 N0:1-26, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ
)D N0:1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the,group consisting of SEQ ~ NO:1-26. The method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.
Yet another embodiment provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a).a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ D7 N0:1-26, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID N0:1-26, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ
ID N0:1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-26. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
Still yet another embodiment provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ~ N0:27-52, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
Another embodiment 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
)D N0:27-52, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID
N0:27-52, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:27-52, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID N0:27-52, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA
equivalent of i)-iv).
Alternatively, the target polynucleotide can comprise a fragment of a polynucleotide selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for full length polynucleotide and polypeptide embodiments of the invention.
Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog, and the PROTEOIV>E database identification numbers and annotations of PROTEOME
database homologs, for polypeptide embodiments 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 embodiments, 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 embodiments, along with selected fragments of the polynucleotides.
Table 5 shows representative cDNA libraries for polynucleotide embodiments.
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 polynucleotides and polypeptides, along withapplicable descriptions, references, and threshold parameters.
Table ~ shows single nucleotide polymorphisms found in polynucleotide sequences of the invention, along with allele frequencies in different human populations.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleic acids, and methods are described, it is understood that embodiments of the invention are not limited to the particular machines, instruments, 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 invention.
As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host cell" includes a plurality of such host cells, and a reference to "an antibody' is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.
Although any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with various embodiments of 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
"TRICIT' refers to the amino acid sequences of substantially purified TRICH
obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the biological activity of TRICH. Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of TRICH either by directly interacting with TRICH or by acting on components of the biological pathway-in which TRICH
participates.
An "allelic variant" is an alternative form of the gene encoding TRICH.
Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
"Altered" nucleic acid sequences encoding TRICH include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as TRICH or a polypeptide with at least one functional characteristic of TRICH. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding TRICH, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide 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 one or more similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of TRICH is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, and positively charged amino acids may include lysine and arginine. Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine. Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" can refer to an oligopeptide, a peptide, a polypeptide, or a 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. Amplification may be carried out using polymerase chain reaction (PCR) technologies or other nucleic acid amplification 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, anticalins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of TRICH either by~ directly interacting with TRICH or by acting on components of the biological pathway in which TRICH participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding an epitopic determinant.
Antibodies that bind TRICH polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to .

immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (particular regions or three-dimensional structures on the protein). An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
The term "aptamer" refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target. Aptamers are derived from an in vitro evolutionary process (e.g., SELEX
(Systematic Evolution of Ligands by EXponential Enrichment), described in U.S.
Patent No.
5,270,163), which selects for target-specific aptamer sequences from large combinatorial libraries.
Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules. The nucleotide components of an aptamer may have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-NHZ), which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood. Aptamers may be conjugated to other molecules, e.g., a high molecular weight carn'er to slow clearance of the aptamer from the circulatory system.
Aptamers maybe specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker (Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13).
The term "intramer" refers to an aptamer which is expressed in vivo. For example, a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc.
Natl. Acad. Sci. USA
96:3606-3610).
The term "spiegelmer" refers to an aptamer which includes L-DNA, L-RNA, or other left-handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.
The term "antisense" refers to any composition capable of base-pairing with the "sense"
(coding) strand of a polynucleotide having a specific nucleic acid sequence.
Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, rnethylphosphonates, 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" and a "composition comprising a given polypeptide" can refer to any composition containing the given polynucleotide or polypeptide. The composition may comprise a dry formulation or an aqueous solution.
Compositions comprising polynucleotides 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 XL-PCR kit (Applied Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the 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 o least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. The table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.

Original Residue Conservative Substitution Ala Gly, Ser Arg His, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln, His Gly Ala ~ His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr S er, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Tle, Leu, Thr Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or polypeptide.
Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, aryl, hydroxyl, or amino group. A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A
derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
"Differential expression" refers to increased or upregulated; or decreased, downregulated, or 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 a polynucleotide encoding TRICH
which can be 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 about 5 to about 1000 contiguous nucleotides or amino acid residues. A fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, to may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule. For example, a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence. Clearly these lengths are exemplary, and any 15 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 117 NO:27-52 can comprise a region of unique polynucleotide sequence that specifically identifies SEQ ID N0:27-52, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ ID N0:27-52 can be employed 20 in one or more embodiments of methods of the invention,. for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID
N0:27-52 from related polynucleotides. The precise length. of a fragment of SEQ m N0:27-52 and the region of SEQ ll~
NO:27-52 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
25 A fragment of SEQ ID N0:1-26 is encoded by a fragment of SEQ ID N0:27-52. A
fragment of SEQ m N0:1-26 can comprise a region of unique amino acid sequence that specifically identifies SEQ 117 N0:1-26. For example, a fragment of SEQ ID N0:1-26 can be used as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:1-26.
The precise length of a fragment of SEQ ID NO:1-26 and the region of SEQ m NO:1-26 to which 30 the fragment corresponds can be determined based on the intended purpose for the fragment using one or more analytical methods described herein or otherwise known in the art.
A "full length" polynucleotide 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 one or more computer algorithms or programs known in the art or described herein. For example, percent identity can 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 which can be used is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol.
Biol. 215:403-410), which is available from several sources, including the NCBI, Bethesda, MD, and on the Internet at http://www.ncbi.nlm.nih.govBLAST/. The BLAST software suite includes various sequence analysis programs including "blastn," that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called "BLAST 2 Sequences" that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed below). BLAST
programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2Ø12 (April-21-2000) set at default parameters. Such default parameters may be, for example:
Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch: -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 D7 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 S0, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e ~.
sequence alignment program (described and referenced above). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows: I~tuple=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 paiiwise 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.' S0 Expect: 10 1o Word Size: 3 Filter: on Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ 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, afragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which. contain all of the elements required for chromosome replication, segregation and maintenance.
The term "humanized antibodya' refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing steps) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched. Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 ~tg/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out. Such wash temperatures are typically selected to be about S°C to 20°C lower than the thermal melting point (T"~ for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. An equation for calculating Tm and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring Harbor Press, Plaiuview 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, SS°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 acids 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 present in solution and another nucleic acid 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 polynucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of TRICH
which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal. The term "immunogenic fragment" also includes any polypeptide or oligopeptide fragment of TRICH which is useful in any of the antibody production methods disclosed herein or known in the art.
The term "microarra~' refers to an arrangement of a plurality of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide, polypeptide, antibody, 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 acids encoding TRICH, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acids. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. "Primers"
are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primex pairs can be used for amplification (and identification) of a nucleic acid, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
Methods for preparing and using probes and primers are described in the references, for example Sambrook, J. et al. (1989; Molecular Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY), Ausubel, F.M. et al. (1999; Short Protocols in Molecular Biolo , 4t'' ed., John Wiley & Sons, New York NY), and 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 genomc-wide scope. The Primer3 primer selection program (available to the public from the Whitehead Institute/MIT
Center for Genome Research, Cambridge MA) allows the user to input a "mispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.) The PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments. The oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a nucleic acid 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 maybe part of a viral vector, e.g., based on a, vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and other moieties known in the art.

An "RNA equivalent," in reference to a DNA molecule, is composed of the same linear sequence of nucleotides as the reference DNA molecule with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of containing TRICH, nucleic acids encoding TRICH, or fragments thereof may comprise a bodily fluid; an extract.from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding, composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A," the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60%
free, preferably at least about 75% free, and most preferably at least about 90% free from other components with which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
A "transcript image" or "expression profile" refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on tlae 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 trausiently 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. In another embodiment, the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002) Science 295:868-872). The erm genetic manipulation does not include classical cross breeding, or in vitro fertilization,, but rather is directed to the introduction of a recombinant DNA molecule. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. (1989), supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater 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 polynucleotides 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 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 lengthof one of the polypeptides..
THE INVENTION
Various embodiments of the invention include 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 embodiments of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Iucyte Project ID). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ
ID NO:) and an Incyte polypeptide sequence number (Iucyte 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.
Column 6 shows the Incyte ID numbers of physical, full length clones corresponding to the polypeptide and polynucleotide sequences of the invention. The full length clones encode polypeptides which have at least 95 % sequence identity to the polypeptide sequences shown in column 3.
Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME
database.
Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ZD NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention. Column 3 shows the GenBank identification number (GenBank ID NO:) of the nearest GenBank homolog and the PROTEOME database identification numbers (PROTEOME )D
NO:) of the nearest PROTEOME database homologs. Column 4 shows the probability scores for the matches between each polypeptide and its homolog(s). Column 5 shows the annotation of the GenBank and PROTEOME database homolog(s) along with relevant citations where applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and 2 show the polypeptide sequence identification number (SEQ 117 NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of the invention. Column 3 shows the number of amino acid residues in each polypeptide. Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS
program of the GCG sequence analysis software package (Genetics Computer Group, Madison WI).
Column 6 shows amino acid residues comprising signature sequences, domains, and motifs. Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the invention, and these properties establish that the claimed polypeptides are transporters and ion channels. For example, SEQ ID N0:1 is 49% identical, from residue S11 to residue K626, to human CTL1 protein (GenBank D7 g6996442) as determined by the Basic Local Alignment Search Tool (BLAST).
(See Table 2.) The BLAST probability score is 9.0e-168, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ m N0:1 also contains an eight transmembrane helices regions as determined by using a hidden Markov model for the prediction of txansmembrane helices. (See Table 3.) In an alternative example, SEQ 117 N0:3 is 57%
identical, from residue E10 to residue V115, to human SLC11A3 iron transporter (GenBank )D g8895485) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST
probability score is 4.7e-25, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. In an alternative example, SEQ >D N0:6 is 88% identical, from residue M1 to residue 5944, to rat potassium channel (GenBank D7 g2745729) 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 >l7 N0:6 also contains a PAC motif, a PAS domain, a cyclic nucleotide-binding domain, and an ion transport protein domain as determined by searching for statistically significant matches in the hidden Markov model (HIVI1VI) based PFAM database of conserved protein family domains. (See Table 3.) Data from BLAST_PRODOM and BLAST DOMO analyses provide further corroborative evidence that SEQ
ID NO:6 is a potassium chancel. In an alternative example, SEQ D7 N0:10 is 99%
identical, from residue M1 to residue I418, 95% identical, from residue 5420 to residue 5680, and 94% identical, from residue P665 to residue H894, to human Eag-related gene member 2 (GenBank ID
g11878259) 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 117 N0:10 is localized to the plasma membrane, has transporter and channel activity and is a voltage-gated potassium channel, as determined by BLAST analysis using the PROTEOME database. SEQ ll~ N0:10 also contains a PAC domain, cyclic nucleotide binding domain and ion transport domain as determined by searching for statistically significant matches in the hidden Markov model (HNINI) based PFAM database of conserved protein family domains. (See Table 3.) SEQ ID N0:10 contains five transmembrane-spanning regions as determined by T'LR analysis. Data from further BLAST analyses of the PRODOM and DOMO
databases provide additional corroborative evidence that SEQ lD N0:10 is a potassium channel. In an alternative example, SEQ ID N0:11 is 86% identical, from residue A94 to residue S785, to rat potassium channel (GenBank ID g2745729) 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. As determined by BLAST
analysis using the PROTEOME database, SEQ ID N0:11 is localized to the plasma membrane, is homologous to rat ether-a-go-go related 2, which is a slowly activating delayed rectifier potassium channel, and may facilitate the differentiation of pre-vertebral neurons (PROTEOME ll7 331276~Rn.10875); SEQ 177 N0:11 is also homologous to rat ether-a-go-go-related gene 3 which is an inward rectifier potassium channel that functions in potassium transport specifically in the nervous system (PROTEOME ll~ 331274~Rn.10874). SEQ ID N0:11 also contains a cyclic nucleotide-binding domain and an ion transport protein domain 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 additional BLAST
analyses provide further corroborative evidence that SEQ ID N0:11 is a potassium channel. In an alternative example, SEQ ID N0:14 is 38% identical, from residue Q13 to residue 51049, to Schizosaccharomyces pombe membrane ATPase (GenBank 1D g3451312) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 1.5e-189, which indicates the probability of obtaining the observed polypeptide sequencealignment by chance.
SEQ ID N0:14 is localized to the membrane, and is a member of the P-type, Ca2+-type, ATPase subfamily, as determined by BLAST analysis using the PROTEOME database. SEQ ID N0:14 also contains an E1-E2 ATPase domain as determined by searching for statistically significant matches in the hidden Markov model (I~V1M)-based PFAM database of conserved protein family domains.
(See Table 3.) Data from BLllVIPS, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ ID N0:14 is a membrane ATPase. In an alternative example, SEQ ID
N0:19 is 98%
identical, from residue M1 to residue L602, to human sodium-dependent high-affinity dicarboxylate transporter (GenBank ID 88132324) 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 D7 N0:19 also has homology to proteins that have transporter gene function and are sodium-dependent dicarboxylate transporters, as determined by BLAST analysis using the PROTEOME database. SEQ ID N0:19 also contains a sodium-dependent dicarboxylate transporter domain as determined by searching for statistically significant matches in the hidden Markov model (I~VIM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLllVIPS and BLAST analyses provide further corroborative evidence that SEQ ID N0:19 is a sodium-dependent dicarboxylate transporter. SEQ D7 N0:2, SEQ ll7 N0:4,-5, SEQ ID N0:7-9, SEQ ll7 NO:12-13, SEQ ID NO:15-18, and SEQ ID
NO:20-26 were analyzed and annotated in a sin-iilar manner. The algorithms and parameters for the analysis of SEQ JD N0:1-26 are described in Table 7.
As shown in Table 4, the full length polynucleotide embodiments were assembled using cDNA
sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences. Column 1 lists the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:), the corresponding Incyte polynucleotide consensus sequence number (Incyte ll~) for each polynucleotide of the invention, and the length of each polynucleotide sequence in basepairs.
Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA
and/or genomic sequences used to assemble the full length polynucleotide embodiments, and of fragments of the polynucleotides which are useful, for example, in hybridization or amplification technologies that identify SEQ ID NO:B-14 or that distinguish between SEQ ID N0:8-14 and related polynucleotides.
The polynucleotide fragments described in Column 2 of Table 4 may refer specifically, for example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from pooled cDNA
libraries. Alternatively, the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the full length polynucleotides. In addition, the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL
(The Sauger Centre, Cambridge, UK) database (i.e., those sequences including the designation "ENST"). Alternatively, the polynucleotide fragments described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences including the designation "NM" or "NT") or the NCBI RefSeq Protein Sequence Records (i.e., those sequences including the designation "NP"). Alternatively, the polynucleotide fragments described in column 2 may refer to assemblages of both cDNA and Genscan-predicted axons brought together by an "axon stitching" algorithm. For example, a polynucleotide sequence identified as FL I~:~~~LXXX NI N~ YI'YYI'_N3 N4 represents a "stitched" sequence in which ~'~~~XXX is the identification number of the cluster of sequences to which the algorithm was applied, and YYYYYis the number of the prediction generated by the algorithm, and N1,~,3,.,, if present, represent specific axons that may have been manually edited during analysis (See Example V).
Alternatively, the polynucleotide fragments in column 2 may refer to assemblages of axons brought together by an "axon-stretching" algorithm. For example, a polynucleotide sequence identified as FLX~:~_gA~9AAA~BBBBB_1 N is a "stretched" sequence, with 1~'~~~XXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the "axon-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 axons (See Example V). In instances where a RefSeq sequence was used as a protein homolog for the "axon-stretching" algorithm, a RefSeq identifier (denoted by "NM,"
"NP," or "NT") maybe 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 andlor examples of programs GNN, GFG,Exon prediction from genomic sequences using, ENST for example, GENSCAN (Stanford University, CA, USA) or FGENES
(Computer Genomics Group, The Sanger Centre, Cambridge, UK) GBI Hand-edited analysis of genomic sequences.

FL Stitched or stretched genomic sequences (see Example V).

INCY ~ Full length transcript and exon prediction from mapping of EST
sequences to the genome. Genomic location and EST
data are combined to predict the exons and resulting transcript.
In some cases, Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length polynucleotides 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 polynucleotides. The tissues and vectors which were used to construct the cDNA libraries shown in Table 5 are described in Table 6.
Table 8 shows single nucleotide polymorphisms (SNPs) found in polynucleotide sequences of the invention, along with,allele frequencies in different human populations.
Columns 1 and 2 show the polynucleotide sequence identification number (SEQ ID NO:) and the corresponding Incyte project identification number (PDT) for polynucleotides of the invention. Column 3 shows the Iucyte identification number for the EST in which the SNP was detected (EST ID), and column 4 shows the identification number for the SNP (SNP m). Column 5 shows the position within the EST sequence at which the SNP is located (EST SNP), and column 6 shows the position of the SNP within the full-length polynucleotide sequence (CB 1 SNP). Column 7 shows the allele found in the EST sequence.
Columns 8 and 9 show the two alleles found at the SNP site. Column 10 shows the amino acid encoded by the codon including the SNP site, based upon the allele found in the EST. Columns 11-14 show the frequency of allele 1 in four different human populations. An entry of n/d (not detected) indicates that the frequency of allele 1 in the population was too low to be detected, while n/a (not available) indicates that the allele frequency was not determined for the population.
The invention also encompasses TRICH variants. A preferred TRICH variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the TRICH amino acid sequence, and which contains at least one functional or structural characteristic of TRICH.
Various embodiments also encompass polynucleotides which encode TRICH. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID N0:27-52, which encodes TRICH. The polynucleotide sequences of SEQ D7 N0:27-52, 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 variants of a polynucleotide encoding TRICH. In particular, such a variant polynucleotide will have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a polynucleotide encoding TRICH. A
particular aspect of the invention encompasses a variant of a polynucleotide comprising a sequence selected from the group consisting of SEQ ll7 N0:27-52 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ IIJ N0:27-52. Any one of the polynucleotide variants described above can encode a polypeptide 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 encoding TRICH. A splice variant may have portions which have significant sequence identity to a polynucleotide 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 a polynucleotide 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 encoding TRICH.
For example, a polynucleotide comprising a sequence of SEQ ID N0:34 and a polynucleotide comprising a sequence of SEQ D7 N0:43 are splice variants of each other; a polynucleotide comprising a sequence of SEQ ID N0:46 and a polynucleotide comprising a sequence of SEQ ID
N0:52 are splice variants of each other; a polynucleotide comprising a sequence of SEQ ID N0:39 and a polynucleotide comprising a sequence of SEQ ID NO:50 are splice variants of each other; and a polynucleotide comprising a sequence of SEQ ID NO:32, a polynucleotide comprising a sequence of SEQ ID NO:36, and a polynucleotide comprising a sequence of SEQ TD NO:37 are splice variants of each other. Any one of the splice variants described above can encode a polypeptide which contains at least one functional or structural characteristic of TRICH.
It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of polynucleotide sequences encoding TRICH, some bearing minimal similarity to the polynucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring TRICH, and all such variations are to be considered as being specifically disclosed.
Although polynucleotides which encode TRICH and its variants are generally capable of hybridizing to polynucleotides encoding naturally occurring TRICH under appropriately selected conditions of stringency, it may be advantageous to produce polynucleotides 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 polynucleotides which encode TRICH and TRICH derivatives, or fragments thereof, entirely by synthetic chemistry.
After production, the synthetic polynucleotide 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 polynucleotide encoding TRICH or any fragment thereof.
Embodiments of the invention can also include polynucleotides that are capable of hybridizing to the claimed polynucleotides, and, in particular, to those having the sequences shown iu SEQ 177 NO:27-52 and fragments thereof, under various conditions of stringency (Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; IKimmel, A.R. (1987) Methods Enzymol.
152:507-511).
Hybridization conditions, including annealing and wash conditions, are described in "Definitions."
Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I; SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Iuvitrogen, Carlsbad CA). Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Applied Biosystems).
Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Amersham Biosciences), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art (Ausubel et al., supra, ch. 7; Meyers, R.A. (1995) Molecular Biology and Biotechnolo~y, Wiley VCH, New York NY, pp. 856-853).
The nucleic acids encoding TRICH may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstxeam 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 (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 (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 (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 (Parker, J.D. et al.
(1991) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested primers, and PROMOTERFIhTDER libraries (Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in fording intron/exon junctions. For all PCR-based methods, primers may be designed using commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
When screening for full length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. In addition, random-primed libraries, which often include sequences containing the 5' regions of genes, are preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.

Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirni 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, polynucleotides 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 polynucleotides which encode substantially the same or a functionally equivalent polypeptides may be produced and used to express TRICH.
The polynucleotides of the invention can be engineered using methods generally known in the art in order to alter TRICH-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA shuffling by~
random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent No.
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of TRICH, such as its biological or enzymatic activity or its ability to bind to ' other molecules or compounds. DNA shuffling is a process by which a library of gene variants is .
produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening. Thus, genetic diversity is created through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single.gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes iri the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
In another embodiment, polynucleotides encoding TRICH may be synthesized, in whole or in part, using one or more chemical methods well known in the art (Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; 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 known in the art. For example, peptide synthesis can be performed using various solution-phase or solid-phase techniques (Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH
Freeman, New York NY, pp. 55-60; Roberge, J.Y. et al. (1995) Science 269:202-204). Automated synthesis may be achieved using the ABI 431A peptide synthesizer (Applied Biosystems).
Additionally, the amino acid sequence of TRICH, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance liquid chromatography (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 (Creighton, supra, pp. 28-53).
2o In order to express a biologically active TRICH, the polynucleotides 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 polynucleotides encoding TRICH. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of polynucleotides encoding TRICH. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where a polynucleotide sequence 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 enhaucers appropriate for the particular host cell system used (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 polynucleotides encoding TRICH and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and itZ vivo genetic recombination (Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel et al., supra, ch. 1, 3, and 15).
A variety of expression vector/host systems may be utilized to contain and express polynucleotides 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 (Sambrook, supra;
Ausubel et al., supra; Van Heeke, G. and S.M. Schuster (1989) J, Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945;
Takamatsu, N. (1987) EMBO J. 6:307-311; The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw Hill, New York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad.
Sci. USA 81:3655-3659; 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 polynucleotides to the targeted organ, tissue, or cell population (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5:350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci.
USA 90:6340-6344; Buller, R.M. et al. (1985) Nature 317:813-815; McGregor, D.P. et al. (1994) Mol.
T_mmunol. 31:219-226; 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 polynucleotides encoding TRICH. For example, routine cloning, subcloning, and propagation of polynucleotides encoding TRICH can be achieved using a multifunctional E. eoli vector such as PBLUESCRIPT (Stratagem, La Jolla CA) or PSPORT1 plasmid (Invitrogen).
Ligation of polynucleotides 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 iri the cloned sequence (Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509). When large quantities of TRICH are needed, e.g. for the production of antibodies, vectors which direct high level expression of TRICH may be used. For example, vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of TRICH. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign polynucleotide sequences into the host genome for stable propagation (Ausubel et al., supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; Scorer, C.A.
et al. (1994) Bio/Technology 12:181-184).
Plant systems may also be used for expression of TRICH. Transcription of polynucleotides 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 (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Brogue, R. et al. (1984) Science 224:838-843; 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 (The McGraw Hill Yearbook of Science and TechnoloQV (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, polynucleotides 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 (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. HA.Cs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes (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, polynucleotides 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 rnay be allowed to grow for about 1 to 2 days'in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk' and apt cells, respectively (Wigler, M. et al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823). Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate; rieo confers resistance to the aminoglycosides neomycin and G-418; and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Wigler, M. et al.
(1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al.
(1981) J. Mol. Biol.
150:1-14). Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites (Hartman, S.C. and R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA
85:8047-8051). Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), ~3-glucuronidase and its substrate (3-glucuronide, or luciferase and its substrate luciferin may be used.
These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (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 polynucleotides 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 polynucleotide encoding TRICH and that express TRICH .may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR
amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.
Immunological methods for detecting and measuring the expression of TRICH
using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on TRICH is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art (Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN, Sect.
1V; Coligan, J.E. et al. (1997) Current Protocols in Itnmunolo~y, Greene Pub.
Associates and Wiley-Interscience, New York NY; Pound, J.D. (1998) Tmmunochemical Protocols, Humana Press, Totowa NJ).
A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding TRICH include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
Alternatively, polynucleotides 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 Biosciences, Promega (Madison WI), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with polynucleotides 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 polynucleotides or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" or "pro" form of the protein may also be used to specify protein targeting, folding, and/or activity.
Different host cells which have specific cellular machinery and characteristic mechanisms for l0 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 polynucleotides encoding TRICH may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems. For example, a chimeric TRICH
protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of TRICH activity. Heterologous protein and peptide 'moieties may also facilitate purification of fusion proteins using commercially available affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose 2o 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 hemagglutinir (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 TR.ICH 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 et al.
(supra, ch. 10 and 16). A
variety of commercially available kits may also be used to facilitate expression and purification of 3o fusion proteins.
In another embodiment, 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-metluonine.
TRICH, fragments of TRICH, or variants of TRICH may be used to screen for compounds that specifically bind to TRICH.. One or more test compounds may be screened for specific binding to TRICH. In various embodiments, 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened for specific binding to TRICH. Examples of test compounds can include antibodies, anticalins, oligonucleotides, proteins (e.g., ligands or receptors), or small molecules.
In related embodiments, variants of TRICH can be used to screen for binding of test compounds, such as antibodies, to TRICH, a variant of T1ZICH, or a combination of TRICH and/or one or more variants TRICH. In an embodiment, a variant of TRICH can be used to screen for compounds that bind to a variant of TRICH, but not to TRICH having the exact sequence of a sequence of SEQ LD N0:1-26. TRICH variants used to perform such screening can have a range of about 50% to about 99% sequence identity to T1ZICH, with various embodiments having 60%, 70%, 75%o, 80%, 85%, 90%, and 95% sequence identity.
In an embodiment, a compound identified in a screen for specific binding to TRICH can be 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 (Coligan, J.E.
et al. (1991) C~xrrent Protocols in hnmunolo~y 1(2):Chapter 5). In another embodiment, the compound thus identified can be a natural ligand of a receptor TRICH (Howard, A.D. et al. (2001) Trends Pharmacol. Sci.22:132-140; Wise, A. et al. (2002) Drug Discovery Today 7:235-246).
In other embodiments, a compound identified in a screen for specific binding to TRICH can be closely related to the natural receptor to which TRICH binds, at least a fragment of the receptor, or a fragment of the receptor including all or a portion of the ligand binding site or binding pocket. For example, the compound may be a receptor for T1ZICH which is capable of propagating a signal, or a decoy receptor for TRICH which is not capable of propagating a signal (Ashkenazi, A. and V.M.
Divit (1999) Curr. Opin. Cell Biol. 11:255-260; Mantovani, A. et al. (2001) Trends Tmmunol. 22:328-336). The compound can be rationally designed using known techniques. Examples of such techniques include those used to construct the compound etanercept (ENBREL;
Amgen Inc., Thousand Oaks CA), which is efficacious for treating rheumatoid arthritis in humans. Etanercept is an engineered p75 tumor necrosis factor (TNF) receptor dimer linked to the Fc portion of human IgGI
(Taylor, P.C. et al. (2001) Curr. Opin. Tmmunol. 13:611-616).

rr-mro rv.i In one embodiment, two or more antibodies having similar or, alternatively, different specificities can be screened for specific binding to TRICH, fragments of TRICH, or variants of TRICH.~ The binding specificity of the antibodies thus screened can thereby be selected to identify particular fragments or variants of TRICH. In one embodiment, an antibody can be selected such that its binding specificity allows for preferential identification of specific fragments or variants of TRICH.
In another embodiment, an antibody can be selected such that its binding specificity allows for preferential diagnosis of a specific disease or condition having increased, decreased, or otherwise abnormal production of TRICH.
In an embodiment, anticalins can be screened for specific binding to TRICH, fragments of TRICH, or variants of TRICH. Anticalins are ligand-binding proteins that have been constructed based on a lipocalin scaffold (Weiss, G.A. and H.B. Lowman (2000) Chem. Biol.
7:8177-8184;
Skerra, A. (2001) J. Biotechnol. 74:257-275). The protein architecture of lipocalins can include a beta-barrel having eight antiparallel beta-strands, which supports four loops at its open end. These loops form the natural ligand-binding site of the lipocalins, a site which can be re-engineered in vitro by amino acid substitutions to impart novel binding specificities. The amino acid substitutions can be made using methods known in the art or described herein, and can include conservative substitutions (e.g., substitutions that do not alter binding specificity) or substitutions that modestly, moderately, or significantly alter binding specificity.
In one embodiment, screening for compounds which specifically bind to, stimulate, or inhibit TRICH involves producing appropriate cells which express TRICH, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing TRICH or cell membrane fractions which contain TRICH are then contacted with a test compound and binding, stimulation, or inhibition of activity of either TRICH
or the compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label. For example, the assay may comprise the steps of combining at least one test compound with TRICH, either in solution or affixed to a solid support, and detecting the binding of TRICH to the compound. Alternatively, the assay may detect or measure binding of a test compound in the presence of a labeled competitor.
Additionally, the assay may be carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compounds) may be free in solution or affixed to a solid support.
An assay canbe used to assess the ability of a compound to bind to its natural ligand andlor to inhibit the binding of its natural ligand to its natural receptors. Examples of such assays include radio-labeling assays such as those described in U.S. Patent No. 5,914,236 and U.S.
Patent No. 6,372,724.
In a related embodiment, one or more amino acid substitutions can be introduced into a polypeptide compound (such as a receptor) to improve or alter its ability to bind to its natural ligands (Matthews, D.J. and J.A. Wells. (1994) Chem. Biol. 1:25-30). In another related embodiment, one or more amino acid substitutions can be introduced into a polypeptide compound (such as a ligand) to improve or alter its ability to bind to its natural receptors (Cunningham, B.C. and J.A. Wells (1991) Proc. Natl. Acad.
Sci. USA 88:3407-3411; Lowman, H.B. et al. (1991) J. Biol. Chem. 266:10982-10988).
TRICH, fragments of TRICH, or variants of TRICH 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). Fox 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) Clip. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains. Transgenic animals thus generated may be tested.with potential therapeutic or toxic agents.
Polynucleotides encoding TRICH may also be manipulated itt vitro in ES cells derived from human blastocysts. Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types. These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding TRICH can also be used to create "knockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease. With knockin technology, a region 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).
THERAPEUTTCS
Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of TRICH and transporters and ion channels. The expression of TRICH is closely associated with normal heart tissue, liver tumor tissue and diseased corpus callosum tissue. In addition, examples of tissues expressing TRICH can be found in Table 6 and can also be found in Example XI.
Therefore, TRICH appears to play a role in transport, neurological, muscle, immunological and cell proliferative disorders. In the treatment of disorders associated with increased TRICH expression or activity, it is desirable to decrease the expression or activity of TRICH. In the treatment of disorders associated with decreased TRICH expression or activity, it is desirable to increase the expression or activity of TRICH. , Therefore, in one embodiment, TRICH or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of TRICH. Examples of such disorders include, but are not limited to, a transport disorder such as akinesia, amyotrophic lateral sclerosis, ataxia telangiectasia, cystic fibrosis, Becker's muscular dystrophy, Bell's palsy; Charcot-Marie Tooth disease, diabetes mellitus, diabetes insipidus, diabetic neuropathy, Duchenne muscular dystrophy, hyperkalemic periodic paralysis, normokalemic periodic paralysis, Parkinson's disease, malignant hyperthermia, multidrug resistance, myasthenia gravis, myotonic dystrophy, catatonia, tardive dyskinesia, dystonias, peripheral neuropathy, cerebral neoplasms, prostate cancer, cardiac disorders associated with transport, e.g., angina, bradyarrythmia, tachyarrythmia, hypertension, Long QT syndrome, myocarditis, cardiomyopathy, nemaline myopathy, centronuclear myopathy, lipid myopathy, mitochondrial myopathy, thyrotoxic myopathy, ethanol myopathy, dermatomyositis, inclusion body myositis, infectious myositis, polymyositis, neurological disorders associated with transport, e.g., Alzheimer's disease, amnesia, bipolar disorder, dementia, depression, epilepsy, Tourette's disorder, paranoid psychoses, and schizophrenia, and other disorders associated with transport, e.g., neurofibromatosis, postherpetic neuralgia, trigeminal neuropathy, sarcoidosis, sickle cell anemia, Wilson's disease, cataracts, infertility, pulmonary artery stenosis, sensorineural autosomal deafness, hyperglycemia, hypoglycemia, Grave's disease, goiter, Cushing's disease, Addison's disease, glucose-galactose malabsorption syndrome, 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, atnyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, priors diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system including Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, hemiplegic migraine, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; a muscle disorder such as cardiomyopathy, myocarditis, Duchenne's muscular dystrophy, Becker's muscular dystrophy, myotonic dystrophy, central core disease, nemaline myopathy, centronuclear myopathy, lipid myopathy, mitochondrial myopathy, infectious myositis, polymyositis, dermatomyositis, inclusion body myositis, thyrotoxic myopathy, ethanol myopathy, angina, anaphylactic shock, arrhythmias, asthma, cardiovascular shock, Cushing's syndrome, hypertension, hypoglycemia, myocardial infarction, migraine, pheochromocytoma, and myopathies including encephalopathy, epilepsy, Kearns-Sayre syndrome, lactic acidosis, myoclonic disorder, ophthalmoplegia, acid maltase deficiency (AMD, also known as Pompe's disease), generalized myotonia, and myotonia congenita; an immunological disorder such as acquired immunodeficiency syndrome (A)DS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, seleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and heltninthic infections, and trauma; and a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.
In another embodiment, a vector capable of expressing TRICH or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of TRICH including, but not limited to, those described above.
In a further embodiment, a composition comprising a substantially purified TRICH in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of TRICH
including, but not limited to, those provided above.
In still auother embodiment, an agonist which modulates the activity of TRICH
may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of TRICH including, but not limited to, those listed above.
In a further embodiment, an antagonist of TRICH may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of TRICH.
Examples of such disorders include, but are not limited to, those transport, neurological, muscle, immunological and cell proliferative disorders described above. Iu. one aspect, an antibody which specifically binds TRICH
may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express TRICH.
In au additional embodiment, a vector expressing the complement of the polynucleotide encoding TRICH may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of TRICH including, but not limited to, those described above.
In other embodiments, any protein, agonist, antagonist, antibody, complementary sequence, or vector embodiments may be administered in combination with other appropriate therapeutic agents.
Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
An antagonist of TRICH may be produced using methods which are generally known in the art. In particular, purified TRICH may be used to produce. antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind TRICH.
Antibodies to TRICH may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are generally preferred for therapeutic use. Single chain antibodies (e.g., from camels or llamas) may be potent enzyme inhibitors and may have advantages in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S. (2001) J.
Biotechnol. 74:277-302).
For the production of antibodies, various hosts including goats, rabbits, rats, mice, camels, dromedaries, llamas, humans, and others may be immunized by injection with TRICH or with any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to TRICH have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein. Short stretches of TRICH amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
Monoclonal antibodies to TRICH may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV
hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al.
(1985) J. Tmmunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030;
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120).
In addition, techniques developed fox 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 (Morrison, S.L. et al. (1984) Proc. Natl. Acad.
Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608;
Takeda, S. et al. (1985) Nature 314:4.52-454). 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 (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 (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al.
(1991) Nature 349:293-299).
~o 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 (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 for TRICH.
The Ka determined for a preparation of monoclonal antibodies, which are monospecific for a particular TRICH epitope, represents a true measure of affinity. High-affinity antibody preparations with Ka ranging from about 109 to 1012 L/mole are preferred for use in immunoassays in which the TRICH-.
antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with Ka ranging from about 106 to 10' L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of TRICH, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL
Press, Washington DC;
Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
The titer and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For example, a polyclonal antibody preparation containing at least 1-2 mg specific antibody/mla preferably 5-10 mg specific antibody/ml, is generally employed in procedures requiring precipitation of TRICH-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available (Catty, supra; Coligan et al., supra).
In another embodiment of the invention, 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 (Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press, 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 (Slater, J.E. et al. (1998) J. Allergy Clip. Itnmunol. 102:469-475; Scanlon, K.J. et al. (1995) 9:1288-1296). Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (Miller, A.D. (1990) Blood 76:271; Ausubel et al., supra; Uckert, W.
and W. Walther (1994) Pharmacol. Ther. 63:323-347). Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art (Rossi, J.J.
(1995) Br. Med. Bull. 51:217-225; Boado, R.J. et al. (1998) J. Pharm. Sci.
87:1308-1315; Morris, M.C. et al. (1997) Nucleic Acids Res. 25:2730-2736).
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 (SC117)-X1 disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995) Science 270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familial 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 technologies for use with cells in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (ii) ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu.
Rev. Biochem.
62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J.-L. and H. Recipon (1998) Curr. Opin.
Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of TRICH include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Iuvitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), 2o and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
TRICH
may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or (3-actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci.
USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V. and H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen));
the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible promoter (Rossi, F.M.V.
and 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 LIPll7 TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in the art to deliver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters. In the alternative, transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by genetic defects with respect to TRICH expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding TRICH under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE) along with additional retrovirus cis-acting RNA sequences and coding sequences required for efficient vector propagation. Retrovirus vectors (e.g., PFB and PFBNEO) are commercially available (Stratagene) and are based on published data (Riviere, I. et al. (1995) Proc.
Natl. Aced. 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 (Range, 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;
Range, U. et al. (1998) Proc. Natl. Aced. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
In an embodiment, an adenovirus based gene therapy delivery system is used to deliver polynucleotides encoding TRICH to cells which have one or more genetic abnormalities with respect to the expression of TRICH. The construction and packaging of adenovirus based vectors are well known to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Trausplantation 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).
In another embodiment, 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. 5804,413 teaches the use of recombinant HSV.d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the control of the appropriate promoter for purposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSV vectors, see also Goins, W.F. et al. (1999; J. Virol. 73:519-532) and Xu, H. et al.
(1994; Dev. Biol. 163:152-161). 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 embodiment, an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding TRICH to target cells. The biology of the prototypic alphavirus, Semliki Forest Virus (SFV), has been studied extensively and gene transfer vectors have been based on the SFV genome (Garoff, H. and K.-J. Li (1998) C~rr. Opin. Biotechnol.
9:464-469). During alphavirus RNA replication, a subgenomic RNA is generated that normally encodes the viral capsid proteins. This subgenomic RNA replicates to higher levels than the full length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase). Similarly, inserting the coding sequence for TRICH into the alphavirus genome in place of the capsid-coding region results in the production of a large number of TRICH-coding RNAs and the synthesis of high levels of TRICH in vector transduced cells. While alphavirus infection is typically associated with cell lysis within a few days, the ability to establish a persistent infection in hamster normal kidney cells (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host range of alphaviruses will allow the introduction of TRICH into a variety of cell types. The specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction.
The methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA
transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art.
Oligonucleotides derived from the transcription initiation site, e.g., between about positions -10 and +10 from the start site, may also be employed to inhibit gene expression.
Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and T_m_m__unolog_lc Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-177). A
complementary sequence or antisense molecule may also be designed to block trauslation 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 RNA molecules encbding 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 may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo trauscription of DNA
molecules encoding TRICH. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA

polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontxaditional 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 3o may comprise, for example, an intact or permeabilized cell, or an in vitro cell-free or reconstituted biochemical system. Alterations in the expression of a polynucleotide encoding TRICH are assayed by any method commonly known in the art. Typically, the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding TRICH. The amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide. A screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a 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.
1o Commun. 268:8-13). A particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al.
(2000) U.S. Patent No.
6,022,691).
Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art (Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:462-466).
Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
An additional embodiment of the invention relates to the administration of a composition which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient.
Excipients may include, for example, sugars, starches, celluloses, gums, and proteins. Various formulations are commonly known and are thoroughly discussed in the latest edition of Remin~ton's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may consist of TRICH, antibodies to TRICH, and mimetics, agonists, antagonists, or inhibitors of TRICH.
The compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, infra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, ~s sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry powder form.
These compositions are generally aerosolized immediately prior to inhalation by the patient. In the case of small molecules (e.g. traditional low molecular weight organic drugs), aerosol delivery of fast-s 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, liposo~ne 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 xange depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
The exact dosage will be.determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half life and clearance rate of the particular formulation.
Normal dosage amounts may vary from about 0.1 ,ug to 100,000 fig, up to a total dose of about 1 gram, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind TRICH may be used for the diagnosis of disorders characterized by expression of TRICH, or in assays to monitor patients being treated with TRICH or agonists, antagonists, or inhibitors of TRICH.
Antibodies useful for diagnostic purposes may be prepared in the sarr~e manner as described above for therapeutics. Diagnostic assays for TRICH include methods which utilize the antibody and a label'to detect TRICH in human body fluids or in extracts of cells or tissues. The antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule. A wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
A variety of protocols for measuring TRICH, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of TRICH expression. Normal or standard values for TRICH expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibodies to TRICH under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of TRICH
expressed in so 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, polynucleotides encoding TRICH may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotides, complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of TRICH may be correlated with disease. The diagnostic assay. may be used to determine absence, presence, and excess expression of TRICH, and to monitor regulation of TRICH levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting polynucleotides, 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 S'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 m N0:27-52 or from genomic sequences including promoters, enhancers, and introns of the TRICH
gene.
Means for producing specific hybridization probes for polynucleotides encoding TRICH
include the cloning of polynucleotides encoding TRICH or TRICH derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32P or 35S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
Polynucleotides 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, tachyatTyrhinia, hypertension, Long QT syndrome, myocarditis, cardiomyopathy, nemaline myopathy, centronuclear myopathy, lipid myopathy, mitochondrial myopathy, thyrotoxic myopathy, ethanol myopathy, dermatomyositis, inclusion body myositis, infectious myositis, polymyositis, neurological . disorders associated with transport, e.g., Alzheimer's disease, amnesia, bipolar disorder, dementia, depression, epilepsy, Tourette's disorder, paranoid psychoses, and schizophrenia, and other disorders associated with transport, e.g., neurofibromatosis, postherpetic neuralgia, trigeminal neuropathy, sarcoidosis, sickle cell anemia, Wilson's disease, cataracts, infertility, pulmonary artery stenosis, sensorineural autosomal deafness, hyperglycemia, hypoglycemia, Grave's disease, goiter, Cushing's disease, Addison's disease, glucose-galactose malabsorption syndrome, 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, priors diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system including Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, hemiplegic migraine, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; a muscle disorder such as cardiomyopathy, myocarditis, Duchenne's muscular dystrophy, Becker's muscular dystrophy, myotonic dystrophy, central core disease, nemaline myopathy, centronuclear myopathy, lipid myopathy, mitochondrial myopathy, infectious myositis, polymyositis, dermatomyositis, inclusion body myositis, thyrotoxic myopathy, ethanol myopathy, angina, anaphylactic shock, arrhythmias, asthma, cardiovascular shock, Cushing's syndrome, hypertension, hypoglycemia, myocardial infarction, migraine, pheochromocytoma, and myopathies including encephalopathy, epilepsy, Kearns-Sayre syndrome, lactic acidosis, myoclonic disorder, ophthalmoplegia, 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-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 hehninthic infections, and trauma; and a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vets, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinorna, 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. Polynucleotides 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, polynucleotides encoding TRICH may be used in assays that detect the presence of associated disorders, particularly those mentioned above.
Polynucleotides complementary to 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 iri comparison to a control sample then the presence of altered levels of polynucleotides 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 1o combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding TRICH, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
With respect to cancer, the presence of an abnormal amount of transcript (either under- or 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.
Iu a particular aspect, oligonucleotide primers derived from polynucleotides encoding TRICH
may be used to detect single nucleotide polymorphisms (SNPs). SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans. Methods of SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP, oligonucleotide primers derived from polynucleotides encoding TRICH are used to amplify DNA using the polymerase chain reaction (PCR). The DNA
may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels. In fSCCP, the oligonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high-throughput equipment such as DNA sequencing machines.
Additionally, sequence database analysis methods, termed in silico SNP (isSNP), are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence. These computer-based methods filter out sequence variations due to laboratory preparation of DNA and sequencing errors using statistical models and automated analyses of DNA
sequence chromatograms. In the alternative, SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
SNPs may be used to study the genetic basis of human disease. For example, at least 16 common SNPs have been associated with non-insulin-dependent diabetes mellitus.
SNPs are also useful for examining differences in disease outcomes in monogenic disorders, such as cystic fibrosis, sickle cell anemia, or chronic granulomatous disease. For example, variants in the mannose binding lectin, MBL2, have been shown to be correlated with deleterious pulmonary outcomes in cystic fibrosis. SNPs also have utility in pharmacogenomics, the identification of genetic variants that influence a patient's response to a drug, such as life-threatening toxicity.
For example, a variation in N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drug isoniazid, while a variation in the core promoter of the ALOXS gene results in diminished clinical response to treatment with an anti-asthma drug that targets the 5-lipoxygenase pathway. Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as well as for tracing the origins of populations and their migrations (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512;
Kwok, P.-Y. and Z. Gu (1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. .(2001) Curr. Opin.
Neurobiol. 11:637-641).
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 (Melby, P.C. et al. (1993) J. Tmmunol. Methods 159:235-244;
Duplaa, C. et al. (1993) Anal. Biochem. 212:229- 236). The speed of quantitation of multiple samples maybe 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 polynucleotides 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 (Seilhamer et al., "Comparative Gene Transcript Analysis," U.S.
Patent No. 5,840,484;
hereby expressly incorporated by reference herein). Thus a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or cell type. In one embodiment, the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray. The resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues, cell lines, biopsies, or other biological samples. The transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals, as well as toxicological testing of industrial and naturally-occurring environmental compounds. All compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L.
Anderson (2000) Toxicol. Lett. 112-113:467-471). 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 an embodiment, the toxicity of a test compound can be 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 embodiment relates to the use of the polypeptides disclosed herein to analyze the s~

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
S 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 interest. 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 25' 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 maybe useful in the analysis of compounds which do not significantly affect the transcript image, but which ss alter the proteomic profile. In addition, the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more reliable and informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified. The amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample.
A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the to 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 (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;
Shalon, D. et al. (1995) 2o PCT application WO95/35505; Heller, R.A. et al. (1997) Proc. Natl. Acad.
Sci. USA 94:2150-2155;
Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662). Various types of microarrays are well known and thoroughly described in Schena, M., ed. (1999; DNA Microarrays: A
Practical Approach, Oxford University Press, London).
In another embodiment of the invention, nucleic acid sequences encoding TRICH
may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence.
Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (PACs), bacterial artificial chromosomes (BACs), bacterial P1 constructions, or single chromosome cDNA libraries (Harrington, J.J. et al.
(1997) Nat. Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; Trask, B.J. (1991) Trends Genet.
7:149-154). Once mapped, the nucleic acid sequences may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP) (Lander, E.S. and D. Botstein (1986) Proc. Natl.
Acad. Sci. LTSA 83:7353-7357).
Fluorescent iti situ hybridization (FISH) may be correlated with other physical and genetic map data (Heinz-Ulrich, et al. (1995) in Meyers, supf-a, 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 11q22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation (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 (Geysen, et al.
(1984) PCT application 4). In this method, large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with TRICH, or fragments thereof, and washed.
Bound TRICH is then detected by methods well known in the art. Purified TRICH
can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding TRICH specifically compete with a test compound for binding TRICH.
In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with TRICH.
In additional embodiments, the nucleotide sequences which encode TRICH may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
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.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above and below, 2o including U.S. Ser. No. 60/313,242, U.S. Ser. No. 60/324,782, U.S. Ser. No.
60/328,184, U.S. Ser.
No. 60/345,937, U.S. Ser. No. 60/335,698, U.S. Ser. No. 60/332,804, U.S. Ser.
No. 60/333,922, U.S.
Ser. No. 60/388,180, U.S. Ser. No. 60/375,637, and U.S. Ser. No. 60/377,444, 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). Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Invitrogen), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with 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 (Invitrogen), using the recommended procedures or similar methods known in the art (Ausubel et al., supf-a, ch. 5). Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes. For most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL
S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Biosciences) 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 (Invitrogen), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE (Incyte Genomics), or pINCY
(Incyte Genomics), or derivatives thereof. Recombinant plasmids were transformed into competent E. coli cells including XL1-Blue, XL1-BlueMRF, or SOLR from Stratagene or DHSa, DH10B, or ElectroMAX

from Invitrogen.
II. Isolation of cDNA Clones Plasmids obtained as described in Example I were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid, 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 3 84-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 Biosciences or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Amersham Biosciences);
the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art.
Reading frames within the cDNA sequences were identified using standard methods (Ausubel et al., supra, ch. 7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example V7JI.
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 nofvegicus, Mus rnusculus, Caeraor-ltabditis elegans, Sacchar~omyces cer~evisiae, Schizosacchar~omyces pombe, and Can.dida albicans (Incyte Genomics, Palo Alto CA); hidden Markov model (HMM)-based protein family databases such as PFAM, INCY, and TIGRFAM (Haft, D.H. et al. (2001) Nucleic Acids Res. 29:41-43); and FEVIM-based protein domain databases such as SMART (Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864;
Letunic, I. et al. (2002) Nucleic Acids Res. 30:242-244). (HMM is a probabilistic approach which analyzes consensus primary structures of gene families; see, for example, Eddy, S.R. (1996) Curr.
Opin. Struct. Biol.
6:361-365.) The queries were performed using programs based on BLAST, FASTA, BLIMPS, and IllVIMER. 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 1V 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 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, hidden Markov model (IITZM)-based protein family databases such as PFAM, INCY, and TIGRFAM; and I~~lM based protein domain databases such as SMART.
Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (MiraiBio, Alameda 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 Iucyte cDNA and full length sequences and provides applicable descriptions, references, and threshold parameters. The first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probability value, the greater the identity between two sequences).
The programs described above for the assembly and analysis of full length polynucleotide and polypeptide sequences were also used to identify polynucleotide sequence fragments from SEQ ID
N0:27-52. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies are described in Table 4, column 2.
IV. Identification and Editing of Coding Sequences from Genomic DNA

Putative transporters and ion channels were initially identified by running the Genscan gene identification program against public genomic sequence databases (e.g., gbpri and gbhtg). Genscan is a general-purpose gene identification program which analyzes genomic DNA
sequences from a variety of organisms (Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94;
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 chancels, 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 chancels. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri public databases. Where necessary, the Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct errors in the sequence predicted by Genscan, such as extra or omitted exons. BLAST analysis was also used to find any Incyte cDNA or public cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription. When Incyte cDNA coverage was available, this information was used to correct or con~trm 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 llI. Alternatively, full length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data "Stitched" Sequences Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example 1V. 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 progranuriing 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 III were queried against public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases using the BLAST program. The nearest GenBank protein homolog was then compared by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV. A chimeric protein was generated by using the resultant high-scoring segment pairs (HSPs) to map the translated sequences onto the GenBank proteinhomolog.
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 TRICII Encoding Polynucleotides The sequences which were used to assemble SEQ ID N0:27-52 were compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith-Waterman algorithm. Sequences from these databases that matched SEQ ID N0:27-52 were assembled into clusters of contiguous and overlapping sequences using 3o 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 au interval, in centiMorgans, is measured relative to the terminus of the chromosome's p-arm. (The centiMorgan (cM) is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.) The cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters. Human genome maps and other resources available to the public, such as the NCBI "GeneMap'99" World Wide Web site (http://www.ncbi.nlm.nih.govlgenemap~, can be employed to determine if previously identified disease genes map within or in proximity to the intervals indicated above.
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 (Sambrook, supra, ch. 7;
Ausubel et al., supt-a, ch. 4).
Analogous computer techniques applying BLAST were used to search for identical or related molecules in 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 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, polynucleotides 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 polynucleotides are produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment. One primer was synthesized to initiate 5' extension of the known fragment, and the other primer was synthesized to initiate 3' extension of the known fragment. The initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72°C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well known in the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mga+, (NH4)aS04, and 2-mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE
enzyme (Invitrogen), and Pfu DNA polymerase (Stratagene), with the following parameters for~primer pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 68 °C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 °C, 5 min;
Step 7: storage at 4 °C. In the alternative, the parameters for primer pair T7 and SK+ were as follows: Step 1: 94 °C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57 °C, 1 min; Step 4: 68 °C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 ~.1 PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1X TE
and 0.5 p1 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 ,u1 to 10 ,u1 aliquot of the reaction mixture was analyzed by electrophoresis on a 1 ~/o agarose gel to determine which reactions were successful in extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus~endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Biosciences). For shotgun sequencing, the digested nucleotides were separated on low concentration (0.6 to 0.8/0) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega). Extended clones were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Biosciences), treated with Pfu DNA polymerase (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 carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Biosciences) and Pfu DNA polymerase (Stxatagene) with the following parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 72°C, 2 min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA
recoveries were reamplified using the same conditions as described above. Samples were diluted with 20%
dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Biosciences) or the ABI PRISM BIGDYE
Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotides 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.
1o IX. Identification of Single Nucleotide Polymorphisms in TRICH Encoding Polynucleotides Common DNA sequence variants known as single nucleotide polymorphisms (SNPs) were identified in SEQ 117 NO:27-52 using the L1FESEQ database (Iucyte Genomics).
Sequences from the same gene were clustered together and assembled as described in Example III, allowing the identification of all sequence variants in the gene. An algorithm consisting of a series of filters was used to distinguish SNPs from other sequence variants. Preliminary filters removed the majority of basecall errors by requiring a minimum Phred quality score of 15, and removed sequence aligmnent errors and errors resulting from improper trimming of vector sequences, chimeras, and splice variants.
An automated procedure of advanced chromosome,analysis analysed the original chromatogram files in the vicinity of the putative SNP. Clone error filters used statistically generated algorithms to identify errors introduced during laboratory processing, such as those caused by reverse transcriptase, polymerase, or somatic mutation. Clustering error filters used statistically generated algorithms to identify errors resulting from clustering of close homologs or pseudogenes, or due to contamination by non-human sequences. A final set of filters removed duplicates and SNPs found in immunoglobulins or T-cell receptors.
Certain SNPs were selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at the SNP sites in four different human populations. The Caucasian population comprised 92 individuals (46 male, 46 female), including 83 from Utah, four French, three Venezualan, and two Amish individuals. The African population comprised 194 individuals (97 male, 97 female), all African Americans. The Hispanic population comprised 324 individuals (162 male, 162 female), all Mexican Hispanic. The Asian population comprised 126 individuals (64 male, 62 female) with a reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian.
Allele frequencies were first analyzed in the Caucasian population; in some cases those SNPs which showed no allelic variance in this population were not further tested in the other three populations.
X. Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ ID N0:27-52 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 /,tCi of [~y-3aP] adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled oligonucleotides are substantially purified using a superfine size exclusion dextran bead column (Amersham Biosciences). An aliquot containing 10' counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl 1I, 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.
XI. Microarrays The linkage or synthesis of array elements upon a microarray can be achieved utilizing photolithography, piezoelectric printing (ink jet printing; see, e.g., Baldeschweiler et al., 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, M., ed.
(1999) DNA Microarrays: A Practical Approach, Oxford University Press, London). 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 (Schena, M. et al. (1995) Science 270:467-470;

Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson (1998) Nat. Biotechnol.
16:27-31).
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers thereof may comprise the elements of the microarray. Fragments or oligomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR). The array elements are hybridized with polynucleotides in a biological sample. The polynucleotides in the 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/~,l oligo-(dT) primer (2lmer), 1X first strand buffer, 0.03 unitslpl RNase inhibitor, 500 ACM dATP, 500 ~,M dGTP, 500 p,M dTTP, 40 p,M
dCTP, 40 ~,M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences). The reverse transcription 2o reaction is performed in a 25 ml volume containing 200 ng poly(A)+ RNA with GEMBRIGHT kits (Incyte Genomics). Specific control poly(A)+ RNAs are synthesized by i~ 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 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, Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 ~,15X
SSC/0.2% SDS.
Microarray Preparation Sequences of the present invention are used to generate array elements. Each array element is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert. Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 fig.
Amplified array elements are then purified using SEPHACRYL-400 (Amersham Biosciences).
Purified array elements are immobilized on polymer-coated glass slides. Glass microscope slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with extensive distilled water washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in distilled water, and coated with 0.05% 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/~.1, is loaded into the open capillary printing element by a lugh-speed robotic apparatus. The apparatus then deposits about 5 n1 of array element sample per slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate buffered saline (PBS) (Tropix, lnc., Bedford MA) for 30 minutes at 60°
C followed by washes in 0.2%
SDS and distilled water as before.
Hybridization Hybridization reactions contain 9 ~Cl of sample mixture consisting of 0.2 ~,g each of Cy3 and 2o 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 cma 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 (0.1X SSC), and dried.
Detection Reporter-labeled hybridization complexes are detected with a microscope equipped with an lnnova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 llln 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 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 1BM-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 Genomics). Array elements that exhibited at least about a two-fold change in expression, a signal-to background ratio of at least 2.5, and an element spot size of at least 40% were identified as differentially expressed.
Expression For example, SEQ )D N0:30 showed differential expression in bone osteosarcoma tissues versus normal osteocytes, as determined by microarray analysis. Messenger RNA
from normal human osteoblasts was compared with mRNA from biopsy specimens, osteosarcoma tissues, primary cultures, or metastasized tissues. A normal osteoblast primary culture, NHOst 5488, was chosen as the reference in the initial experiments. One basic set of experiments is defined as the comparison of mRNA from biopsy specimen with that of debnitive surgical specimen from the same patient.
Extended study of this basic set includes mRNA from primary cell cultures of the definitive surgical specimen, muscle, or cartilage tissue from the same patient. Biopsy specimens, definitive surgical specimens, or lung metastatic tissues from different individuals were also included to reveal individual variability. °The expression of SEQ ID N0:30 was increased by at least two-fold in bone osteosarcoma tissues relative to normal osteocytes. Therefore, in various embodiments, SEQ m N0:30 can be used for one or more of the following: i) monitoring treatment of bone cancer, ii) diagnostic assays for bone cancer, and iii) developing therapeutics and/or other treatments for bone cancer.
In an alternative example, SEQ ID N0:33 showed differential expression in lung squamous carcinoma tissues versus normal lung tissues as determined by microarray analysis. In matched tissue experiments, the expression of SEQ 1D N0:33 was decreased by at least two-fold in lung squamous carcinoma tissues relative to grossly uninvolved normal lung tissues from the same donors. Therefore, in various embodiments, SEQ ID N0:33 can be used for one or more of the following: i) monitoring treatment of lung cancer, ii) diagnostic assays for lung cancer, and iii) developing therapeutics and/or other treatments for lung cancer.
SEQ ~ NO:33 also showed differential expression in ovarian adenocarcinoma tissues versus normal ovarian tissues as determined by microarray analysis. The expression of SEQ )D N0:33 was decreased by at least two-fold in ovarian adenocarcinoma tissues relative to grossly uninvolved normal ovarian tissues from the same donor. Therefore, in various embodiments, SEQ ID
N0:33 can be used for one or more of the following: i) monitoring treatment of ovarian cancer, ii) diagnostic assays for ovarian cancer, and iii) developing therapeutics and/or other treatments for ovarian cancer.
SEQ 117 N0:33 and SEQ ID NO:40 showed differential expression in association with immune and inflammatory responses as determined by microarray analysis. The expression of SEQ
ll~ N0:33 was increased by at least two-fold in human umbilical vein cells treated with PMA and ionomycin relative to untreated human umbilical vein cells. Human umbilical vein cells are derived from the endothelium of the human umbilical vein, and have been used as an experimental model for investigating the functional biology of human endothelial cells in vitro. PMA
is a broad activator of protein kinase C-dependent pathways and ionomycin is a calcium ionophore that permits the entry of calcium in the cell, hence increasing the cytosofic calcium concentration. The expression of SEQ m N0:40 was increased by at least 2.5-fold in vascular endothelial tissue treated with TNFa and IL-1(3 compared with untreated vascular endothelial tissue, as determined by microarray analysis. Human coronary artery endothelial cells and human coronary artery smooth muscle cells (BioWhittaker, Inc., San Diego CA) obtained from the same donor were cultured in tissue culture flasks in Endothelium Growth Medium or Smooth Muscle Growth Medium, respectively (BioWhittaker).
Cultures at 85%
confluency were either treated with recombinant human TNFoc and IL-1(3 (R&D
Systems, Minneapolis MN) at 10 ng/ml each for 24 hours at 37° C or were left untreated. Therefore, in various embodiments, SEQ ID NO:33 and SEQ 177 NO:40 can each be used for one or more of the following:
i) monitoring treatment of immune/inflammatory responses, ii) diagnostic assays for immune/inflammatory responses, and iii) developing therapeutics and/or other treatments for immune/inflammatory responses.
In an alternative example, SEQ )D N0:3 8 showed differential expression in breast carcinoma cell lines versus primary mammary epithelial cells as determined by microarray analysis. The breast carcinoma cell lines include MCF7, a breast adenocarcinoma cell line derived from the pleural effusion of a 69-year-old female; T-47D, a breast carcinoma cell line derived from a pleural effusion from a 54-year-old female with an infiltrating ductal carcinoma of the breast; Sk-BR-3, a breast adenocarcinoma cell line isolated from a malignant pleural effusion of a 43-year-old female; MDA-mb-231, a metastatic breast tumor cell line derived from the pleural effusion of a 51-year-old female with metastatic breast carcinoma; and MDA-mb-4355, a spindle shaped strain that evolved from a cell line isolated from the pleural effusion of a 31 year old female with metastatic, ductal adenocarcinoma of the breast. The primary mammary epithelial cell line HLV>EC was derived from normal human mammary tissue (Clonetics, San Diego, CA). All cell cultures were propagated in a chemically-defined medium, according to the supplier's recommendations and grown to 70-80% confluence prior to RNA isolation. The microarray experiments showed that the expression of SEQ
m N0:38 was decreased by at least two-fold iu all five breast carcinoma lines (MCF7, T-47D, Sk-BR-3, MDA-mb-231, and MDA-mb-4355) relative to primary mammary epithelial cells. Therefore, in various embodiments, SEQ m N0:3 8 can be used for one or more of the following: i) monitoring treatment of breast cancer, ii) diagnostic assays for breast cancer, and iii) developing therapeutics and/or other treatments for breast cancer.
SEQ )D NO:38 also showed differential expression in certain prostate carcinoma cell lines versus normal prostate epithelial cells as determined by microarray analysis.
The prostate carcinoma cell lines include CA-HPV-10, DU 145, LNCaP, PC-3, and MDAPCa2b. CA-HPV-7 was derived from cells from a 63 year old male with prostate adenocarcinoma and was transformed by transfection with HPV18 DNA. DU 145 was isolated from a metastatic site in the brain of a 69 year old male with widespread metastatic prostate carcinoma. DU 145 has no detectable sensitivity to hormones; forms colonies in semi-solid medium; is only weakly positive for acid phosphatase; and cells are negative for prostate specific antigen (PSA). LNCaP is a prostate carcinoma cell line isolated from a lymph node biopsy of a 50 year old male with metastatic prostate carcinoma. LNCaP
expresses PSA, produces prostate acid phosphatase, and expresses androgen receptors. PC-3, a prostate adenocarcinoma cell line, was isolated from a metastatic site in the bone of a 62 year old male with grade 1V prostate adenocarcinoma. MDAPCa2b is a prostate adenocarcinoma cell line isolated from a metastatic site in the bone of a 63 year old male. The MDAPCa2b cell line expresses PSA and androgen receptor and is androgen sensitive. The normal epithelial cell line, PrEC, is a primary prostate epithelial cell line isolated from a normal donor. The expression of SEQ ll~ N0:38 was decreased by at least two-fold in three out of five prostate carcinoma lines (DU 145, LNCaP, and PC-3) relative to cells from the normal prostate epithelial cell line, PrEC.
Therefore, in various embodiments, SEQ m NO:3 8 can be used for one or more of the following: i) monitoring treatment of prostate cancer, ii) diagnostic assays for prostate cancer, and iii) developing therapeutics and/or other treatments for prostate cancer.
2o In addition, SEQ ID NO:38 and SEQ ID N0:43 showed differential expression in toxicology studies as determined by microarray analysis. The expression of SEQ m N0:43 was increased by at least two-fold in C3A hepatoblastoma cells treated with 1-100 p,M
beclomethazone as compared with untreated C3A hepatoblastoma cells. The human C3A cell line is a clonal derivative of HepG2/C3 (hepatoma cell line, isolated from a 15-year-old male with liver tumor), which was selected for strong contact inhibition of growth. The use of a clonal population enhances the reproducibility of the cells.
C3A cells have many characteristics of primary human hepatocytes in culture:
i) expression of insulin receptor and insulin-like growth factor 1I receptor; ii) secretion of a high ratio of serum albumin compared with a-fetoprotein iii) conversion of ammonia to urea and glutamine;
iv) metabolism of aromatic amino acids; and v) proliferation in glucose-free and insulin-free medium. The C3A cell line 3o is now well established as an in vitro model of the mature human liver (Mickelson et al. (1995) Hepatology 22:866-875; Nagendra et al. (1997) Am J Physiol 272:6408-6416). C3A
cells were treated with 1-100 p,M beclomethazone for lhr, 3hr, 6hr and compared with untreated cells. In 10~

addition, the expression of SEQ 117 N0:34 was increased by at least two-fold in early confluent C3A
cells treated with progesterone, beclomethasone, medroxyprogesterone, budesonide, prednisone, dexamethasone, or betamethasone, for 1, 3, or 6 hours, as compared to untreated C3A cells. In addition, the expression of SEQ 117 N0:38 was decreased by at least two-fold in early confluent C3A
cells treated with progesterone, beclomethasone, medroxyprogesterone, budesonide, prednisone, dexamethasone, or betamethasone, for 1, 3, or 6 hours, as compared to untreated C3A cells. The effects of steroids on liver metabolism are important to the understanding of the pharmacodynamics of drugs. Therefore, in various embodiments, SEQ D7 N0:34, SEQ ID N0:38 and SEQ
T17 N0:43 can each be used for one or more of the following: i) monitoring treatment of liver toxicity, diseases and disorders, ii) diagnostic assays for liver toxicity, diseases and disorders, and iii) developing therapeutics and/or other treatments for liver toxicity, diseases and disorders.
In yet another example, the expression of SEQ ID N0:48 was differentially expressed in a specific region of human brain tissue as compared to pooled brain tissue control. Characterization of region-specific gene expression in the human brain provides a context and background for molecular neurobiology research in general. This knowledge may provide insight into the genetic basis of brain structure and function. The expression of SEQ 117 N0:48 was decreased by at least two-fold in normal human amygdala, entorhinal cortex, brain tissue as compared to the normal human pooled brain tissue used as a control. These experiments indicate that, SEQ 117 N0:48 exhibited significant differential expression patterns using microarray techniques, and further establishes its utility as a diagnostic marker or therapeutic agent which maybe useful in neurological disorders. Therefore, in various embodiments, SEQ 117 NO:48 can be used for one or more of the following: i) monitoring treatment of neurological disorders, ii) diagnostic assays for neurological disorders, and iii) developing therapeutics and/or other treatments for neurological disorders.
XII. Complementary Polynucleotides Sequences complementary to the TRICH-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring TRICH.
Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of TRICH. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence anal used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the TRICH-encoding transcript.
los XIII. Expression of TRICH
Expression and purification of TRICH is achieved using bacterial or virus-based expression systems. For expression of TRICH in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the tfp-lac (tac) hybrid promoter and the TS or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
Antibiotic resistant bacteria express TR1CH 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 Autogr~aplaica califo~nica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding TRTCH by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription. Recombinant baculovirus is used to infect Spodoptera, frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to baculovirus (Engelhard, E.K. et a1.
(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum.
Gene Ther. 7:1937-1945).
In most expression systems, TR1CH 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 Sclaistosoma japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Biosciences).
Following purification, the GST moiety can be proteolytically cleaved from TRICH at specifically engineered sites. FLAG, an 8-amino acid peptide, enables immunoaf~nity 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 (QIA.GEN).
Methods for protein expression and purification are discussed in Ausubel et al. (supt~a, ch. 10 and 16).
Purified TRICH obtained by these methods can be used directly in the assays shown in Examples XVII, XV)II, and XIX, where applicable.
XIV. 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 plasmid (Invitrogen, Carlsbad CA) and PCR3.1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter. 5-10 ,ug of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation. 1-2 ~g of an additional plasmid containing sequences encoding a marker proteinare 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.
XV. Production of TRICH Specific Antibodies TRICH substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize animals (e.g., rabbits, mice, etc.) and to produce antibodies using standard protocols.

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 (Ausubel et al., supf-a, ch. 11).
Typically, oligopeptides of about 15 residues in length are synthesized using an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to KLH (Sigma-Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity (Ausubel et al., supra). Rabbits are immunized with the oligopeptide-I~LH
complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide and anti-TRICH
activity by, for example, binding the peptide or TRICH to a substrate, blocking with 1 % BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
XVI. Purification of Naturally Occurring TRICH Using Specific Antibodies Naturally occurring or recombinant TRICH is substantially purified by immunoaffinity chromatography using antibodies specific for TRICH. An immunoaffinity column is constructed by covalently coupling anti-TRICH antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Biosciences). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing TRICH are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of TRICH (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/TRICH binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and TRICH is collected.
XVII. Identification of Molecules Which Interact with TRICH
TRICH, or biologically active fragments thereof, are labeled with lzsl Bolton-Hunter reagent (Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539). Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled TRICH, washed, and any wells with labeled TRICH complex are assayed. Data obtained using different concentrations of TRICH are used to calculate values for the number, affinity, and association of TRICH with the candidate molecules.
Alternatively, molecules interacting with TRICH are analyzed using the yeast two hybrid system as described in Fields, S. and O. Song (1989; Nature 340:245-246), or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
TRICH may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S.
Patent No. 6,057,101).
XVII. 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 1~I Bolton-Hunter reagent. (See, e.g., Bolton A.E. and W.M.
Hunter (1973) Biochem. J. 133:529-539.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled TRICH, washed, and any wells with labeled TRICH
complex are assayed. Data obtained using different concentrations of TRICH are used to calculate values for the number, affinity, and association of TRICH with the candidate molecules.
Alternatively, proteins that interact with TRICH 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 PATHCALL1NG process (C~raGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan, I~.
et al. (2000) U.S.
Patent No. 6,057,101).
Potential TRICH agonists or antagonists may be tested for activation or inhibition of TRICH
ion channel activity using the assays described in section XVIII.
XVIII. Demonstration of TRICH Activity Ion channel activity of TRICH is demonstrated using an electrophysiological assay for ion conductance. TRICH can be expressed by transforming a mammalian cell line such as COS7, HeLa or CHO with a eukaryotic expression vector encoding TRICH. Eukaryotic expression vectors are commercially available, and the techniques to introduce them into cells are well known to those skilled in the art. A second plasmid which expresses any one of a number of marker genes, such as 13-galactosidase, is co-transformed into the cells to allow rapid identification of those cells which have taken up and expressed the foreign DNA. The cells are incubated for 48-72 hours after transformation under conditions appropriate for the cell line to allow expression and accumulation of TRICH and 13-galactosidase.
Transformed cells expressing !3-galactosidase are stained blue when a suitable colorimetric substrate is added to the culture media under conditions that are well known in the art. Stained cells are tested for differences in membrane conductance by electrophysiological techniques that are well known in the art. Untransformed cells, and/or cells transformed with either vector sequences alone or 13-galactosidase sequences alone, are used as controls and tested in parallel.
Cells expressing TRICH
will have higher anion or canon conductance relative to control cells. The contribution of TRICH to conductance can be confirmed by incubating the cells using antibodies specific for TRICH. The antibodies will bind to the extracellular side of TRICH, thereby blocking the pore in the ion channel, and the associated conductance.
Alternatively, ion channel activity of TRICH is measured as current flow across a TRICH-containing Xeuopus laevis oocyte membrane using the two-electrode voltage-clamp technique (Ishi et al., supf~a; Jegla, T. and L. Salkoff (1997) J. Neurosci. 17:32-44). TRICH is subcloned into an appropriate Xenopus oocyte expression vector, such as pBF, and 0.5-5 ng of mRNA is injected into mature stage IV oocytes. Injected oocytes are incubated at 18 °C for 1-5 days. Inside-out macropatches are excised into an intracellular solution containing 116 mM I~-gluconate, 4 mM KCl, and 10 mM Hepes (pH 7.2). The intracellular solution is supplemented with varying concentrations of the TRICH mediator, such as CAMP, cGMP, or Ca+2 (in the form of CaCl2), where appropriate.
Electrode resistance is set at 2-5 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.
Transport activity of TRICH is assayed by measuring uptake of labeled substrates into Xenopus laevis oocytes. Oocytes at stages V and VI are injected with TRICH
mRNA (10 ng per oocyte) and incubated for 3 days at 18°C in OR2 medium (82.5mM NaCl, 2.5 mM KCl, 1mM CaCl2, 1mM MgCl2, 1mM NaaHP04, 5 mM Hepes, 3.8 mM NaOH , 50p,g/ml gentamycin, pH 7.8) to allow expression of TRICH. Oocytes are then transferred to standard uptake medium (100mM NaCl, 2 mM KCl, 1mM CaCl2, 1mM MgCl2, lO~mM HepeslTris pH 7.5). TJptake 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.
ATPase activity associated with TRICH can be measured by hydrolysis of radiolabeled ATP-[y-32P~, separation of the hydrolysis products by chromatographic methods, and quantitation of the recovered 3aP using a scintillation counter. The reaction mixture contains ATP-[~y-32P] and varying amounts of TRICH in a suitable buffer incubated at 37 °C for a suitable period of time. The reaction is terminated by acid precipitation with trichloroacetic acid and then neutralized with base, and an aliquot of the reaction mixture is subjected to membrane or filter paper-based chromatography to separate the reaction products. The amount of 32P liberated is counted in a scintillation counter. The amount of radioactivity recovered is proportional to the ATPase activity of TRICH in the assay.
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-paltmitic 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/M/cm at 361 nm).
A 700 p1 aliquot of 1 p.M TRICH in 10 mM Tris (pH 7.5), 2 mM EDTA, and 500 mM NaCl is placed in a 1 cm path length quartz cuvette and 1 p1 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.
XIX. Identification of TRICH Agonists and Antagonists TRICH is expressed in a eukaryotic cell line such as CHO (Chinese Hamster Ovary) or HEK
(Human Embryonic Kidney) 293. Ion channel activity of the transformed cells is measured in the presence and absence of candidate agonists or antagonists. Ion channel activity is assayed using patch clamp methods well known in the art or as described in Example XVIII.
Alternatively, ion channel activity is assayed using fluorescent techniques that measure ion flux across the cell membrane (Velicelebi, G. et al. (1999) Meth. Enzymol. 294:20-47; West, M.R.
and C.R. Molloy (1996) Anal. Biochem. 241:51-58). These assays may be adapted for high-throughput screening using microplates. Changes in internal ion concentration are measured using fluorescent dyes such as the Ca2+ indicator Fluo-4 AM, sodium-sensitive dyes such as SBFI and sodium green, or the Cl- indicator MQAE (all available from Molecular Probes) in combination with the FLIPR
fluorimetric plate reading system (Molecular Devices). In a more generic version of this assay, changes in membrane potential caused by ionic flux across the plasma membrane are measured using oxonyl dyes such as DiBAC4 (Molecular Probes). DiBAC4 equilibrates between the extracellular solution and cellular sites according to the cellular membrane potential. The dye's fluorescence intensity is 20-fold greater when bound to hydrophobic intracellular sites, allowing detection of 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 compositions, 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. It will be appreciated that the invention provides novel and useful proteins, and their encoding polynucleotides, which can be used in the drug discovery process, as well as methods for using these compositions for the detection, diagnosis, and treatment of diseases and conditions.
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.
Nor should the description of such embodiments be considered exhaustive or limit the invention to the precise forms disclosed. Furthermore, elements from one embodiment can be readily recombined with elements from one or more other embodiments. Such combinations can form a number of embodiments within the scope of the invention. It is intended that the scope of the invention be defined by the following claims and their equivalents.

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~n~n~n~nLn~n~nin~n~n~n~n~n~n~n r r~ ~ ~ r adz o <110> INCYTE GENOMICS, INC.
TANG, Y. Tom LAL, Preeti G.
YUE, Henry BAUGHN, Mariah R.
NGUYEN, Danniel B.
YAO, Monique G.
GREENE, Barrie D.
BOROWSKY, Mark L.
LEE, Sally EMERLING, Brooke M.
XU, Yuming BECHA, Shanya D.
GORVAD, Ann E.
AZIMZAI, Yalda YUE, Huibin ELLIOTT, Vicki S.
LEE, Ernestine A.
YANG, Junming LEHR-MASON, Patricia M.
RAMKUMAR, Jayalaacmi LEE, Soo Yeun FARIS, Mary TURNER, Christopher FURNESS, Michael BUCHBINDER, Jenny L.
WALIA, Narinder K.
LI, Joana X
FORSYTHE, Ian J.
GRIFFIN, Jennifer A.
GIETZEN, Kimberly J.
SWARNAKAR, Anita HAFALIA, April J.A.
LINDQUIST, Erika A.
JIANG, Xin JACKSON, Alan A.
WILSON, Amy D.
JIN, Pei KHARE, Reena MARQUIS, Joseph P.
<120> TRANSPORTERS AND ION CHANNELS
<130> PF-1148 PCT
<140> To Be Assigned <141> Herewith <150> 60/313,242 <151> 2001-08-17 <150> 60/324,782 <151> 2001-09-21 <150> 60/328,184 <151> 2001-10-02 <150> 60/345,937 <151> 2001-10-26 <150> 60/335,698 <151> 2001-11-01 <150> 60/332,804 <151> 2001-11-13 <150> 60/333,922 <151> 2001-11-27 <150> 60/375,637 <151> 2002-04-26 <150> 60/377,444 <151> 2002-05-03 <150> 60/388,180 <151> 2002-06-11 <160> 52 <170> PERL Program <210> 1 <211> 652 <212> PRT
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 1853191CD1 <400> 1 Met His Cys Leu Gly Ala Glu Tyr Leu Val Ser Ala Glu Gly Ala Pro Arg Gln Arg Glu Trp Arg Pro Gln Ile Tyr Arg Lys Cys Thr Asp Thr Ala Trp Leu Phe Leu Phe Phe Leu Phe Trp Thr Gly Leu Val Phe Ile Met Gly Tyr Ser Val Val Ala Gly Ala Ala Gly Arg Leu Leu Phe Gly Tyr Asp Ser Phe Gly Asn Met Cys Gly Lys Lys Asn Ser Pro Val Glu Gly Ala Pro Leu Ser Gly Gln Asp Met Thr Leu Lys Lys His Val Phe Phe Met Asn Ser Cys Asn Leu Glu Val Lys Gly Thr Gln Leu Asn Arg Met Ala Leu Cys Val Ser Asn Cys Pro Glu Glu Gln Leu Asp Ser Leu Glu Glu Val Gln Phe Phe Ala Asn Thr Ser Gly Ser Phe Leu Cys Val Tyr Ser Leu Asn Ser Phe Asn Tyr Thr His Ser Pro Lys Ala Asp Ser Leu Cys Pro Arg Leu Pro Val Pro Pro Ser Lys Ser Phe Pro Leu Phe Asn Arg Cys Val Pro Gln Thr Pro Glu Cys Tyr Ser Leu Phe Ala Ser Val Leu Ile Asn Asp Val Asp Thr Leu His Arg Ile Leu Ser Gly Ile Met Ser Gly Arg Asp Thr Ile Leu Gly Leu Cys Ile Leu Ala Leu Ala Leu Ser Leu Ala Met Met Phe Thr Phe Arg Phe Ile Thr Thr Leu Leu Val His Ile Phe Ile Ser Leu Val Ile Leu Gly Leu Leu Phe Val Cys Gly Val Leu Trp Trp Leu Tyr Tyr Asp Tyr Thr Asn Asp Leu Ser Ile Glu Leu Asp Thr Glu Arg Glu Asn Met Lys Cys Val Leu Gly Phe Ala Ile Val Ser Thr Gly Ile Thr Ala Val Leu Leu Val 290 . 295 300 Leu Ile Phe Val Leu Arg Lys Arg Ile Lys Leu Thr Val Glu Leu Phe Gln Ile Thr Asn Lys Ala Ile Ser Ser Ala Pro Phe Leu Leu Phe Gln Pro Leu Trp Thr Phe Ala Ile Leu Ile Phe Phe Trp Val Leu Trp Val Ala Val Leu Leu Ser Leu Gly Thr Ala Gly Ala Ala Gln Val Met Glu Gly Gly Gln Val Glu Tyr Lys Pro Leu Ser Gly Ile Arg Tyr Met Trp Ser Tyr His Leu Ile Gly Leu Ile Trp Thr Ser Glu Phe Ile Leu Ala Cys Gln Gln Met Thr Ile Ala Gly Ala Val Val Thr Cys Tyr Phe Asn Arg Ser Lys Asn Asp Pro Pro Asp His Pro Ile Leu Ser Ser Leu Ser Ile Leu Phe Phe Tyr His Gln Gly Thr Ile Val Lys Gly Ser Phe Leu Ile Ser Val Val Arg Ile Pro Arg Ile Ile Val Met Tyr Met Gln Asn Ala Leu Lys Glu Gln His Gly Ala Leu Ser Arg Tyr Leu Phe Arg Cys Cys Tyr Cys Cys Phe Trp Cys Leu Asp Lys Tyr Leu Leu His Leu Asn Gln Asn Ala Tyr Thr Thr Thr Ala Ile Asn Gly Thr Asp Phe Cys Thr Ser Ala Lys Asp Ala Phe Lys Ile Leu Ser Lys Asn Ser Ser His Phe Thr Ser Ile Asn Cys Phe Gly Asp Phe Ile Ile Phe Leu Gly Lys Val Leu Val Val Cys Phe Thr Val Phe Gly Gly Leu Met Ala Phe Asn Tyr Asn Arg Ala Phe Gln Val Trp Ala Val Pro Leu Leu Leu Val Ala Phe Phe Ala Tyr Leu Val Ala His Ser Phe Leu Ser Val Phe Glu Thr Val Leu Asp Ala Leu Phe Leu Cys Phe Ala Val Asp Leu Glu Thr Asn Asp Gly Ser Ser Glu Lys Pro Tyr Phe Met Asp G1n Glu Phe Leu Ser Phe Val Lys Arg Ser Asn Lys Leu Asn Asn Ala Arg Ala Gln Gln Asp Lys His Ser Leu Arg Asn Glu Glu Gly Thr Glu Leu Gln Ala Ile Val Arg <210> 2 <211> 345 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7497369CD1 <400> 2 Met Pro Gly Lys Arg Met Gly Tyr Arg Val Thr Glu Asn Tyr Ser Asp Val Leu Pro Leu Ser Leu Gly Val Gly Trp Gly Pro Trp Pro Leu Pro Leu Leu Pro Gly Gly Gly Gly Gly Ser Arg Gly His Pro His Gln Arg Cys Arg Phe Pro Ala Leu Phe Pro Glu Ser Pro Cys Trp Leu Leu Ala Thr Gly Gln Val Ala Arg Ala Arg Lys Ile Leu Trp Arg Phe Ala Glu Ala Ser Gly Val Gly Pro Gly Asp Ser Pro Leu Glu Glu Asn Ser Leu Ala Thr Glu Leu Thr Met Leu Ser Ala Arg Ser Pro Gln Pro Arg Tyr His Ser Pro Leu Gly Leu Leu Arg Thr Arg Val Thr Trp Arg Asn Gly Leu Ile Leu Gly Phe Ser Ser Leu Val Gly Gly Gly Ile Arg Ala Ser Phe Arg Arg Ser Leu Ala Pro Gln Val Pro Thr Phe Tyr Leu Pro Tyr Phe Leu Glu Ala Gly Leu Glu Ala Ala Ala Leu Val Phe Leu Leu Leu Thr Ala Asp Cys Cys Gly Arg Arg Pro Val Leu Leu Leu Gly Thr Met Val Thr Gly Leu Ala Ser Leu Leu Leu Leu Ala Gly Ala Gln Tyr Leu Pro Gly Trp Thr Val Leu Phe Leu Ser Val Leu Gly Leu Leu Ala Ser Arg Ala Val Ser Ala Leu Ser Ser Leu Phe Ala Ala Glu Val Phe Pro Thr Val Ile Arg Gly Ala Gly Leu Gly Leu Val Leu Gly Ala Gly Phe Leu Gly Gln Ala Ala Gly Pro Leu Asp Thr Leu His Gly Arg Gln Gly Phe Phe Leu Gln Gln Val Val Phe Ala Ser Leu Ala Val Leu Ala Leu Leu Cys Val Leu Leu Leu Pro Glu Ser Arg Ser Arg Gly Leu Pro Gln Ser Leu Gln Asp Ala Asp Arg Leu Arg Arg Ser Pro Leu Leu Arg Gly Arg Pro Arg Gln Asp His Leu Pro Leu Leu Pro Pro Ser Asn Ser Tyr Trp Ala Gly His Thr Pro Glu Gln His <210> 3 <211> 150 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1700438CD1 <400> 3 Met Gln Arg Leu Leu Cys Met Cys His Glu Gly Trp Glu Ala Tyr Cys Arg Gln Met Val Phe Leu Ala Gly Leu Cys Leu Val Phe Leu Tyr Met Thr Val Leu Gly Ser Gly Gly Ile Ile Thr Gly Tyr Ala Cys Thr Gln Gly Val Gly Asp Ser Leu Leu Ser Ile Leu Thr Ala Leu Ser Ala Leu Ser Gly Leu Met Gly Thr Val Leu Phe Thr Gln Leu Arg Gly His Tyr Gly Leu Val Thr Thr Gly Val Ile Ser Ser Gln Leu His Leu Gly Cys Leu Met Leu Cys Met Phe Ser Val Leu Ala Pro Gly Asn Ser Phe Asp Leu Ala Val Phe Ser Leu Pro Leu 110 115 ~ 120 Ser Lys Asn Pro Ser Asn Tyr Glu Leu Leu Val Gln Trp Met Glu Glu Gln Ser Arg Gly Met Ala Trp Phe Arg Phe Leu Ser Lys Gly <210> 4 <211> 537 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 535939CD1 <400> 4 Met Gly Asp Glu Asp Lys Arg Ile Thr Tyr Glu Asp Ser Glu Pro Ser Thr Gly Met Asn Tyr Thr Pro Ser Met His Gln Glu Ala Gln Glu Glu Thr Val Met Lys Leu Lys Gly Ile Asp Ala Asn Glu Pro Thr Glu Gly Ser Ile Leu Leu Lys Ser Ser Glu Lys Lys Leu Gln Glu Thr Pro Thr Glu Ala Asn His Val Gln Arg Leu Arg Gln Met Leu Ala Cys Pro Pro His Gly Leu Leu Asp Arg Val Ile Thr Asn Val Thr Ile Ile Val Leu Leu Trp Ala Val Val Trp Ser Ile Thr Gly Ser Glu Cys Leu Pro Gly Gly Asn Leu Phe Gly Ile Ile Ile Leu Phe Tyr Cys Ala Ile Ile Gly Gly Lys Leu Leu Gly Leu Ile Lys Leu Pro Thr Leu Pro Pro Leu Pro Ser Leu Leu Gly Met Leu Leu Ala Gly Phe Leu Ile Arg Asn Ile Pro Val Ile Asn Asp Asn Val Gln Ile Lys His Lys Trp Ser Ser Ser Leu Arg Ser Ile Ala Leu Ser Ile Ile Leu Val Arg Ala Gly Leu Gly Leu Asp Ser Lys Ala Leu Lys Lys Leu Lys Gly Val Cys Val Arg Leu Ser Met Gly Pro Cys Ile Val Glu Ala Cys Thr Ser Ala Leu Leu Ala His Tyr Leu Leu Gly Leu Pro Trp Gln Trp Gly Phe Ile Leu Gly Phe Val Leu Gly Ala Val Ser Pro Ala Val Val Val Pro Ser Met Leu Leu Leu Gln Gly Gly Gly Tyr Gly Val Glu Lys Gly Val Pro Thr Leu Leu Met Ala Ala Gly Ser Phe Asp Asp Ile Leu Ala Ile Thr Gly Phe Asn Thr Cys Leu Gly Ile Ala Phe Ser Thr Gly Ser Thr Val Phe Asn Val Leu Arg Gly Val Leu Glu Val Val Ile Gly Val Ala Thr Gly Ser Val Leu Gly Phe Phe Ile Gln Tyr Phe Pro Ser Arg Asp Gln Asp Lys Leu Val Cys Lys Arg Thr Phe Leu Val Leu Gly Leu Ser Val Leu Ala Val Phe Ser Ser Val His Phe Gly Phe Pro Gly Ser Gly Gly Leu Cys Thr Leu Val Met Ala Phe Leu Ala Gly Met Gly Trp Thr Ser Glu Lys Ala Glu Val Glu Lys Ile Ile Ala Val Ala Trp Asp Ile Phe Gln Pro Leu Leu Phe Gly Leu Ile Gly Ala Glu Val Ser Ile Ala Ser Leu Arg Pro Glu Thr Val Gly Leu Cys Val Ala Thr Val Gly Ile Ala Val Leu Ile Arg Ile Leu Thr Thr Phe Leu Met Val Cys Phe Ala Gly Phe Asn Leu Lys Glu Lys Ile Phe Ile Ser Phe Ala Trp Leu Pro Lys Ala Thr Val Gln Ala Ala Ile Gly Ser Val Ala Leu Asp Thr Ala Arg Ser His Gly Glu Lys Gln Leu Glu Asp Tyr Gly Met Asp Val Leu Thr Val Ala Phe Leu Ser Ile Leu Ile Thr Ala Pro Ile Gly Ser Leu Leu Ile Gly Leu Leu Gly Pro Arg Leu Leu Gln Lys Val Glu His Gln Asn Lys Asp Glu Glu Val Gln Gly Glu Thr Ser Val Gln Val <210> 5 <211> 1119 <212> PRT
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 55118067CD1 <400> 5 Met Thr Ala Ala Ala Ala Ser Asn Trp' Gly Leu Ile Thr Asn Ile Val Asn Ser Ile Val Gly Val Ser Val Leu Thr Met Pro Phe Cys Phe Lys Gln Cys Gly Ile Val Leu Gly Ala Leu Leu Leu Val Phe Cys Ser Trp Met Thr His Gln Ser Cys Met Phe Leu Val Lys Ser Ala Ser Leu Ser Lys Arg Arg Thr Tyr Ala Gly Leu Ala Phe His Ala Tyr Gly Lys Ala Gly Lys Met Leu Val Glu Thr Ser Met Ile Gly Leu Met Leu Gly Thr Cys Ile Ala Phe Tyr Val Val Ile Gly Asp Leu Gly Ser Asn Phe Phe Ala Arg Leu Phe Gly Phe Gln Val Gly Gly Thr Phe Arg Met Phe Leu Leu Phe Ala Val Ser Leu Cys Ile Val Leu Pro Leu Ser Leu Gln Arg Asn Met Met Ala Ser Ile Gln Ser Phe Ser Ala Met Ala Leu Leu Phe Tyr Thr Val Phe Met Phe Val Ile Val Leu Ser Ser Leu Lys His Gly Leu Phe Ser Gly Gln Trp Leu Arg Arg Val Ser Tyr Val Arg Trp Glu Gly Val Phe Arg Cys Ile Pro Ile Phe Gly Met Ser Phe Ala Cys Gln Ser Gln Val Leu Pro Thr Tyr Asp Ser Leu Asp Glu Pro Ser Val Lys Thr Met Ser Ser Ile Phe Ala Ser Ser Leu Asn Val Val Thr Thr Phe Tyr Val Met Val Gly Phe Phe Gly Tyr Val Ser Phe Thr Glu Ala Thr Ala Gly Asn Val Leu Met His Phe Pro Ser Asn Leu Val Thr Glu Met Leu Arg Val Gly Phe Met Met Ser Val Ala Val Gly Phe Pro Met Met Ile Leu Pro Cys Arg Gln Ala Leu Ser Thr Leu Leu Cys Glu Gln Gln Gln Lys Asp Gly Thr Phe Ala Ala Gly Gly Tyr Met Pro Pro Leu Arg Phe Lys Ala Leu Thr Leu Ser Val Val Phe Gly Thr Met Val Gly Gly Ile Leu Ile Pro Asn Val Glu Thr Ile Leu Gly Leu Thr Gly Ala Thr Met Gly Ser Leu Ile Cys Phe Ile Cys Pro Ala Leu Ile Tyr Lys Lys Ile His Lys Asn Ala Leu Ser Ser Gln Val Val Leu Trp Val Gly Leu Gly Val Leu Val Val Ser Thr Val Thr Thr Leu Ser Val Ser Glu Glu Val Pro Glu Asp Leu Ala Glu Glu Ala Pro Gly Gly Arg Leu Gly Glu Ala Glu Gly Leu Met Lys Val Glu Ala Ala Arg Leu Ser Ala Gln Asp Pro Val Val Ala Val Ala Glu Asp Gly Arg Glu Lys Pro Lys Leu Pro Lys Glu Arg Glu Glu Leu Glu Gln Ala Gln Ile Lys Gly Pro Val Asp Val Pro Gly Arg Glu Asp Gly Lys Glu Ala Pro Glu Glu Ala Gln Leu Asp Arg Pro Gly Gln Gly Ile Ala Val Pro Val Gly Glu Ala His Arg His Glu Pro Pro Val Pro His Asp Lys Val Val Val Asp Glu Gly Gln Asp Arg Glu Val Pro Glu Glu Asn Lys Pro Pro Ser Arg His Ala Gly Gly Lys Ala Pro Gly Val Gln Gly Gln Met Ala Pro Pro Leu Pro Asp Ser Glu Arg Glu Lys Gln Glu Pro Glu Gln Gly Glu Val Gly Lys Arg Pro Gly Gln Ala Gln Ala Leu Glu Glu Ala Gly Asp Leu Pro Glu Asp Pro Gln Lys Val Pro Glu Ala Asp Gly Gln Pro Ala Val Gln Pro Ala Lys Glu Asp Leu Gly Pro Gly Asp Arg Gly Leu His Pro Arg Pro Gln Ala Val Leu Ser Glu Gln Gln Asn Gly Leu Ala Val Gly Gly Gly Glu Lys Ala Lys Gly Gly Pro Pro Pro Gly Asn Ala Ala Gly Asp Thr Gly Gln Pro Ala Glu Asp Ser Asp His Gly Gly Lys Pro Pro Leu Pro Ala Glu Lys Pro Ala Pro Gly Pro Gly Leu Pro Pro Glu Pro Arg Glu Gln Arg Asp Val Glu Arg Ala Gly Gly Asn Gln Ala Ala Ser Gln Leu Glu Glu Ala Gly Arg Ala Glu Met Leu Asp His Ala Val Leu Leu Gln Val Ile Lys Glu Gln Gln Val Gln Gln Lys Arg Leu Leu Asp Gln Gln Glu Lys Leu Leu Ala Val Ile Glu Glu Gln His Lys Glu Ile His Gln Gln Arg Gln Glu Asp Glu Glu Asp Lys Pro Arg Gln Val Glu Val His Gln Glu Pro Gly Ala Ala Val Pro Arg Gly Gln Glu Ala Pro Glu Gly Lys Ala Arg Glu Thr Val Glu Asn Leu Pro Pro Leu Pro Leu Asp Pro Val Leu Arg Ala Pro Gly Gly Arg Pro Ala Pro Ser Gln Asp Leu Asn Gln Arg Ser Leu Glu His Ser Glu Gly Pro Val Gly Arg Asp Pro Ala Gly Pro Pro Asp Gly Gly Pro Asp Thr Glu Pro Arg Ala Ala Gln Ala Lys Leu Arg Asp Gly Gln Lys Asp Ala Ala Pro Arg Ala Ala Gly Thr Val Lys Glu Leu Pro Lys Gly Pro Glu Gln Val Pro Val Pro Asp Pro Ala Arg Glu Ala Gly Gly Pro Glu Glu Arg Leu Ala Glu Glu Phe Pro Gly Gln Ser Gln Asp Val Thr Gly Gly Ser Gln Asp Arg Lys Lys Pro Gly Lys Glu Val Ala Ala Thr Gly Thr Ser Ile Leu Lys Glu Ala Asn Trp Leu Val Ala Gly Pro Gly Ala Glu Thr Gly Asp Pro Arg Met Lys Pro Lys Gln Val Ser Arg Asp Leu Gly Leu Ala Ala Asp Leu Pro Gly Gly Ala Glu Gly Ala Ala Ala Gln Pro Gln Ala Val Leu Arg Gln Pro Glu Leu Arg Val Ile Ser Asp Gly Glu Gln Gly Gly Gln Gln Gly His Arg Leu Asp His Gly Gly His Leu Glu Met Arg Lys Ala Arg Gly Gly Asp His Val Pro Val Ser His Glu Gln Pro Arg Gly Gly Glu Asp Ala Ala Val Gln Glu Pro Arg Gln Arg Pro Glu Pro Glu Leu Gly Leu Lys Arg Ala Val Pro Gly Gly Gln Arg Pro Asp Asn Ala Lys Pro Asn Arg Asp Leu Lys Leu Gln Ala Gly Ser Asp Leu Arg Arg Arg Arg Arg Asp Leu Gly Pro His Ala Glu Gly Gln Leu Ala Pro Arg Asp Gly Val Ile Ile Gly Leu Asn Pro Leu Pro Asp Val Gln Val Asn Asp Leu Arg Gly Ala Leu Asp Ala Gln Leu Arg Gln Ala Ala Gly Gly Ala Leu Gln Val Val His Ser Arg Gln Leu Arg Gln Ala Pro Gly Pro Pro Glu Glu Ser <210> 6 <211> 947 <212> PRT
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 7502087CD1 <400> 6 Met Pro Val Arg Arg Gly His Val Ala Pro Gln Asn Thr Tyr Leu Asp Thr Ile Ile Arg Lys Phe Glu Gly Gln Ser Arg Lys Phe Leu Ile Ala Asn Ala Gln Met Glu Asn Cys Ala Ile Ile Tyr Cys Asn Asp Gly Phe Cys Glu Leu Phe Gly Tyr Ser Arg Val Glu Val Met Gln Gln Pro Cys Thr Cys Asp Phe Leu Thr Gly Pro Asn Thr Pro Ser Ser Ala Val Ser Arg Leu Ala Gln Ala Leu Leu Gly Ala Glu Glu Cys Lys Val Asp Ile Leu Tyr Tyr Arg Lys Asp Ala Ser Ser Phe Arg Cys Leu Val Asp Val Val Pro Val Lys Asn Glu Asp Gly Ala Val Ile Met Phe Ile Leu Asn Phe Glu Asp Leu Ala Gln Leu Leu Ala Lys Cys Ser Ser Arg Ser Leu Ser Gln Arg Leu Leu Ser Gln Ser Phe Leu Gly Ser Glu Gly Ser His Gly Arg Pro Gly Gly Pro Gly Pro Gly Thr Gly Arg Gly Lys Tyr Arg Thr Ile Ser Gln Ile Pro Gln Phe Thr Leu Asn Phe Val Glu Phe Asn Leu Glu Lys His Arg Ser Ser Ser Thr Thr Glu Ile Glu Ile Ile Ala Pro His Lys Val Val Glu Arg Thr Gln Asn Val Thr Glu Lys Val Thr Gln Val Leu Ser Leu Gly Ala Asp Val Leu Pro Glu Tyr Lys Leu Gln Ala Pro Arg Ile His Arg Trp Thr Ile Leu His Tyr Ser Pro Phe Lys Ala Val Trp Asp Trp Leu Ile Leu Leu Leu Val Ile Tyr Thr Ala Val Phe Thr Pro Tyr Ser Ala Ala Phe Leu Leu Ser Asp Gln Asp Glu Ser Arg Arg Gly Ala Cys Ser Tyr Thr Cys Ser Pro Leu Thr Val Val Asp Leu Ile Val Asp Ile Met Phe Val Val Asp Ile Val Ile Asn Phe Arg Thr Thr Tyr Val Asn Thr Asn Asp Glu Val 320 325 , 330 Val Ser His Pro Arg Arg Ile Ala Val His Tyr Phe Lys Gly Trp Phe Leu Ile Asp Met Val Ala Ala Ile Pro Phe Asp Leu Leu Ile Phe Arg Thr Gly Ser Asp Glu Thr Thr Thr Leu Ile Gly Leu Leu Lys Thr Ala Arg Leu Leu Arg Leu Val Arg Val Ala Arg Lys Leu Asp Cys Tyr Ser Glu Tyr Gly Ala Ala Val Leu Phe Leu Leu Met Cys Thr Phe Ala Leu Ile Ala His Trp Leu Ala Cys Ile Trp Tyr Ala Ile Gly Asn Val Glu Arg Pro Tyr Leu Glu His Lys Ile Gly Trp Leu Asp Ser Leu Gly Val Gln Leu Gly Lys Arg Tyr Asn Gly Ser Asp Pro Ala Ser Gly Pro Ser Val Gln Asp Lys Tyr Val Thr Ala Leu Tyr Phe Thr Phe Ser Ser Leu Thr Ser Val Gly Phe Gly Asn Val Ser Pro Asn Thr Asn Ser Glu Lys Val Phe Ser Ile Cys Val Met Leu Ile Gly Ser Leu Met Tyr Ala Ser Ile Phe Gly Asn Val Ser Ala Ile Ile Gln Arg Leu Tyr Ser Gly Thr Ala Arg Tyr His Thr Gln Met Leu Arg Val Lys Glu Phe Ile Arg Phe His Gln Ile Pro Asn Pro Leu Arg Gln Arg Leu Glu Glu Tyr Phe Gln His Ala Trp Ser Tyr Thr Asn Gly Ile Asp Met Asn Ala Val Leu Lys Gly Phe Pro Glu Cys Leu Gln Ala Asp Ile Cys Leu His Leu His Arg Ala Leu Leu Gln His Cys Pro Ala Phe Ser Gly Ala Gly Lys Gly Cys Leu Arg Ala Leu Ala Val Lys Phe Lys Thr Thr His Ala Pro Pro Gly Asp Thr Leu Val His Leu Gly Asp Val Leu Ser Thr Leu Tyr Phe Ile Ser Arg Gly Ser Ile Glu Ile Leu Arg Asp~Asp Val Val Val Ala Ile Leu Gly Lys Asn Asp Ile Phe Gly Glu Pro Val Ser Leu His Ala Gln Pro Gly Lys Ser Ser Ala Asp Val Arg Ala Leu Thr Tyr Cys Asp Leu His Lys Ile Gln Arg Ala Asp Leu Leu Glu Val Leu Asp Met Tyr Pro Ala Phe Ala Glu Ser Phe Trp Ser Lys Leu Glu Val Thr Phe Asn Leu Arg Asp Ala Pro Gly Ser Gln Asp His Gln Gly Phe Phe Leu Ser Asp Asn Gln Ser Asp Ala Ala Pro Pro Leu Ser Ile Ser Asp Ala Ser Gly Leu Trp Pro Glu Leu Leu Gln Glu Met Pro Pro Arg His Ser Pro Gln Ser Pro Gln Glu Asp Pro Asp Cys Trp Pro Leu Lys Leu Gly Ser Arg Leu Glu Gln Leu Gln Ala Gln Met Asn Arg Leu Glu Ser Arg Val Ser Ser Asp Leu Ser Arg Ile Leu Gln Leu Leu Gln Lys Pro Met Pro Gln Gly His Ala Ser Tyr Ile Leu Glu Ala Pro Ala Ser Asn Asp Leu Ala Leu Val Pro Ile Ala Ser Glu Thr Thr Ser Pro Gly Pro Arg Leu Pro Gln Gly Phe Leu Pro Pro Ala Gln Thr Pro Ser Tyr Gly Asp Leu Asp Asp Cys Ser Pro Lys His Arg Asn Ser Ser Pro Arg Met Pro His Leu Ala Val Ala Met Asp Lys Thr Leu Ala Pro Ser Ser Glu Gln Glu Gln Pro Glu Gly Leu Trp Pro Pro Leu Ala Ser Pro Leu His Pro Leu Glu Val Gln Gly Leu Ile Cys Gly Pro Cys Phe Ser Ser Leu Pro Glu His Leu Gly Ser Val Pro Lys Gln Leu Asp Phe Gln Arg His Gly Ser Asp Pro Gly Phe Ala Gly Ser Trp Gly His <210> 7 <211> 80 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7500819CD1 <400> 7 Met Glu Leu Val Leu Val Phe Leu Cys Ser Leu Leu Ala Pro Met Val Leu Ala Ser Ala Ala Glu Lys Glu Lys Glu Met Asp Pro Phe His Tyr Asp Tyr Gln Thr Leu Arg Ile Gly Gly Leu Val Phe Ala Val Val Leu Phe Ser Val Gly Ile Leu Leu Ile Leu Ser Arg Arg Cys Lys Cys Ser Phe Tyr Ser Ala Pro Gly Glu Cys Val Pro Cys Ile Ser Ser Gln Gln <210> 8 <211> 531 <212> PRT
<213> Homo Sapiens <220>
<221> misc_~eature <223> Incyte ID No: 7503413CD1 <400> 8 Met Ala Ser Ala Leu Ser Tyr Val Ser Lys Phe Lys Ser Phe Val Ile Leu Phe Val Thr Pro Leu Leu Leu Leu Pro Leu Val Ile Leu Met Pro Ala Lys Phe Val Arg Cys Ala Tyr Val Ile Ile Leu Met Ala Ile Tyr Trp Cys Thr Glu Val Ile Pro Leu Ala Val Thr Ser Leu Met Pro Val Leu Leu Phe Pro Leu Phe Gln Ile Leu Asp Ser 65. 70 75 Arg Gln Val Cys Val Gln Tyr Met Lys Asp Thr Asn Met Leu Phe Leu Gly Gly Leu Ile Val Ala Val Ala Val Glu Arg Trp Asn Leu His Lys Arg Ile Ala Leu Arg Thr Leu Leu Trp Val Gly Ala Lys Pro Ala Arg Leu Met Leu Gly Phe Met Gly Val Thr Ala Leu Leu 125 130 ~ 135 Ser Met Trp Ile Ser Asn Thr Ala Thr Thr Ala Met Met Val Pro Ile Val Glu Ala Ile Leu Gln Gln Met Glu Ala Thr Ser Ala Ala Thr Glu Ala Gly Leu Glu Leu Val Asp Lys Gly Lys Ala Lys Glu Leu Pro Gly Ser Gln Val Ile Phe Glu Gly Pro Thr Leu Gly Gln Gln Glu Asp Gln Glu Arg Lys Arg Leu Cys Lys Ala Met Thr Leu Cys Ile Cys Tyr Ala Ala Ser Ile Gly Gly Thr Ala Thr Leu Thr Gly Thr Gly Pro Asn Val Val Leu Leu Gly Gln Met Asn Glu Leu Phe Pro Asp Ser Lys Asp Leu Val Asn Phe Ala Ser Trp Phe Ala Phe Ala Phe Pro Asn Met Leu Val Met Leu Leu Phe Ala Trp Leu Trp Leu Gln Phe Val Tyr Met Arg Phe Asn Phe Lys Lys Ser Trp Gly Cys Gly Leu Glu Ser Lys Lys Asn Glu Lys Ala Ala Leu Lys Val Leu Gln Glu Glu Tyr Arg Lys Leu Gly Pro Leu Ser Phe Ala Glu Ile Asn Val Leu Ile Cys Phe Phe Leu Leu Val Ile Leu Trp Phe Ser Arg Asp Pro Gly Phe Met Pro Gly Trp Leu Thr Val Ala Trp Val Glu Glu Arg Lys Thr Pro Phe Tyr Pro Pro Pro Leu Leu Asp Trp Lys Val Thr Gln Glu Lys Val Pro Trp Gly Ile Val Leu 365 370 ~ 375 Leu Leu Gly Gly Gly Phe Ala Leu Ala Lys Gly Ser Glu Ala Ser Gly Leu Ser Val Trp Met Gly Lys Gln Met Glu Pro Leu His Ala Val Pro Pro Ala Ala Ile Thr Leu Ile Leu Ser Leu Leu Val Ala Val Phe Thr Glu Cys Thr Ser Asn Val Ala Thr Thr Thr Leu Phe Leu Pro Ile Phe Ala Ser Met Ser Arg Ser Ile Gly Leu Asn Pro Leu Tyr Ile Met Leu Pro Cys Thr Leu Ser Ala Ser Phe Ala Phe Met Leu Pro Val Ala Thr Pro Pro Asn Ala Ile Val Phe Thr Tyr Gly His Leu Lys Val Ala Asp Met Val Lys Thr Gly Val Ile Met Asn Ile Ile Gly Val Phe Cys Val Phe Leu Ala Val Asn Thr Trp Gly Arg Ala Ile Phe Asp Leu Asp His Phe Pro Asp Trp Ala Asn Val Thr His Ile Glu Thr <210> 9 <211> 510 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7500007CD1 <400> 9 Met Asp Ser Arg Val Ser Gly Thr Thr Ser Asn Gly Glu Thr Lys Pro Val Tyr Pro Val Met Glu Lys Lys Glu Glu Asp Gly Thr Leu Glu Arg Gly His Trp Asn Asn Lys Met Glu Phe Val Leu Ser Val Ala Gly Glu Ile Ile Gly Leu Gly Asn Val Trp Arg Phe Pro Tyr Leu Cys Tyr Lys Asn Gly Gly Glu His Cys Met Glu Phe Gln Lys Thr Asn Gly Ser Leu Asn Gly Thr Ser Glu Asn Ala Thr Ser Pro Val Ile Glu Phe Trp Glu Arg Arg Val Leu Lys Ile Ser Asp Gly Ile Gln His Leu Gly Ala Leu Arg Trp Glu Leu Ala Leu Cys Leu Leu Leu Ala Trp Val Ile Cys Tyr Phe Cys Ile Trp Lys Gly Val Lys Ser Thr Gly Lys Val Val Tyr Phe Thr Ala Thr Phe Pro Tyr Leu Met Leu Val Val Leu Leu Ile Arg Gly Val Thr Leu Pro Gly Ala Ala Gln Gly Ile Gln Phe Tyr Leu Tyr Pro Asn Leu Thr Arg Leu Trp Asp Pro Gln Val Trp Met Asp Ala Gly Thr Gln Ile Phe Phe Ser Phe Ala Ile Cys Leu Gly Cys Leu Thr Ala Leu Gly Ser Tyr Asn Lys Tyr His Asn Asn Cys Tyr Arg Asp Cys Ile Ala Leu Cys Phe Leu Asn Ser Gly Thr Ser Phe Val Ala Gly Phe Ala Ile Phe Ser Ile Leu Gly Phe Met Ser Gln Glu Gln Gly Val Pro Ile Ser Glu Val Ala Glu Ser Gly Pro Gly Leu Ala Phe Ile Ala Tyr Pro Arg Ala Val Val Met Leu Pro Phe Ser Pro Leu Trp Ala Cys Cys Phe Phe Phe Met Val Val Leu Leu Gly Leu Asp Ser Gln Phe Val Cys Val Glu Ser Leu Val Thr Ala Leu Val Asp Met Tyr Pro His Val Phe Arg Lys Lys Asn Arg Arg Glu Val Leu Ile Leu Gly Val Ser Val Val Ser Phe Leu Val Gly Leu Ile Met Leu Thr Glu Gly Gly Met Tyr Val Phe Gln Leu Phe Asp Tyr Tyr Ala Ala Ser Gly Met Cys Leu Leu Phe Val Ala Ile Phe Glu Ser Leu Cys Val Ala Trp Val Tyr Gly Ala Lys Arg Phe Tyr Asp Asn Ile Glu Asp Met Ile Gly Tyr Arg Pro Trp Pro Leu Ile Lys Tyr Cys Trp Leu Phe Leu Thr Pro Ala Val Cys Thr Ala Thr Phe Leu Phe Ser Leu Ile Lys Tyr Thr Pro Leu Thr Tyr Asn Lys Lys Tyr Thr Tyr Pro Trp Trp Gly Asp Ala Leu Gly Trp Leu Leu Ala Leu Ser Ser Met Val Cys Ile Pro Ala Trp Ser Leu Tyr Arg Leu Gly Thr Leu Lys 455 460 4&5 Gly Pro Phe Arg Glu Arg Ile Arg Gln Leu Met Cys Pro Ala Glu Asp Leu Pro Gln Arg Asn Pro Ala Gly Pro Ser Ala Pro Ala Thr Pro Arg Thr Ser Leu Leu Arg Leu Thr Glu Leu Glu Ser His Cys <210> 10 <211> 894 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7500025CD1 <400> 10 Met Ser Val Arg Arg Gly His Val Ala Pro Gln Asn Thr Tyr Leu Asp Thr Ile Ile Arg Lys Phe Glu Gly Gln Ser Arg Lys Phe Leu Ile Ala Asn Ala Gln Met Glu Asn Cys Ala Ile Ile Tyr Cys Asn Asp Gly Phe Cys Glu Leu Phe Gly Tyr Ser Arg Val Glu Val Met Gln Gln Pro Cys Thr Cys Asp Phe Leu Thr Gly Pro Asn Thr Pro Ser Ser Ala Val Ser Arg Leu Ala Gln Ala Leu Leu Gly Ala Glu Glu Cys Lys Val Asp Ile Leu Tyr Tyr Arg Lys Asp Ala Ser Ser Phe Arg Cys Leu Val Asp Val Val Pro Val Lys Asn Glu Asp Gly Ala Val Ile Met Ser Ile Leu Asn Phe Glu Asp Leu Ala Gln Leu Leu Ala Lys Cys Ser Ser Arg Ser Leu Ser Gln Arg Leu Leu Ser Gln Ser Phe Leu Gly Ser Glu Gly Ser His Gly Arg Pro Gly Gly Pro Gly Pro Gly Thr Gly Arg Gly Lys Tyr Arg Thr Ile Ser Gln 170 ~ 175 180 Ile Pro Gln Phe Thr Leu Asn Phe Val Glu Phe Asn Leu Glu Lys His Arg Ser Ser Ser Thr Thr Glu Ile Glu Ile Ile Ala Pro His Lys Val Val Glu Arg Thr Gln Asn Val Thr Glu Lys Val Thr Gln Val Leu Ser Leu Gly Ala Asp Val Leu Pro Glu Tyr Lys Leu Gln Ala Pro Arg Ile His Arg Trp Thr Ile Leu His Tyr Ser Pro Phe Lys Ala Val Trp Asp Trp Leu Ile Pro Leu Leu Val Ile Tyr Thr Ala Val Phe Thr Pro Tyr Ser Ala Ala Phe Leu Leu Ser Asp Gln Asp Glu Ser Arg Arg Gly Ala Cys Ser Tyr Thr Cys Ser Pro Leu Thr Val Val Asp Leu Ile Val Asp Ile Met Phe Val Val Asp Ile Val Ile Asn Phe Arg Thr Thr Tyr Val Asn Thr Asn Asp Glu Val Val Ser His Pro Arg Arg Ile Ala Val His Tyr Phe Lys Gly Trp Phe Leu Ile Asp Met Val Ala Ala Ile Pro Phe Asp Leu Leu Ile Phe Arg Thr Gly Ser Asp Glu Thr Thr Thr Leu Ile Gly Leu Leu Lys Thr Ala Arg Leu Leu Arg Leu Val Arg Val Ala Arg Lys Leu Asp Arg Tyr Ser Glu Tyr Gly Ala Ala Val Leu Phe Leu Leu Met Cys Thr Phe Ala Leu Ile Ala His Trp Leu Ala Cys Ile Cys Ser Leu Thr Ser Val Gly Phe Gly Asn Val Ser Pro Asn Thr Asn Ser Glu Lys Val Phe Ser Ile Cys Val Met Leu Ile Gly Ser Leu Met Tyr Ala Ser Ile Phe Gly Asn Val Ser Ala Ile Ile Gln Arg Leu Tyr Ser Gly Thr Ala Arg Tyr His Thr Gln Met Leu Arg Val Lys Glu Phe Ile Arg Phe His Gln Ile Pro~Asn Pro Leu Arg Gln Arg Leu Glu Glu Tyr Phe Gln His Ala Trp Ser Tyr Thr Asn Gly Ile Asp Met Asn Ala Val Leu Lys Gly Phe Pro Glu Cys Leu Gln Ala Asp Ile Cys Leu His Leu His Arg Ala Leu Leu Gln His Cys Pro Ala Phe Ser Gly Ala Gly Lys Gly Cys Leu Arg Ala Leu Ala Val Lys Phe Lys Thr Thr His Ala Pro Pro Gly Asp Thr Leu Val His Leu Gly Asp Val Leu Ser Thr Leu Tyr Phe Ile Ser Arg Gly Ser Ile Glu Ile Leu Arg Asp Asp Val Val Val Ala Ile Leu Gly Lys Asn Asp Ile Phe Gly Glu Pro Val Ser Leu His Ala Gln Pro Gly Lys Ser Ser Ala Asp Val Arg Ala Leu Thr Tyr Cys Asp Leu His Lys Ile Gln Arg Ala Asp Leu Leu Glu Val Leu Asp Met Tyr Pro Ala Phe Ala Glu Ser Phe Trp Ser Lys Leu Glu Val Thr Phe Asn Leu Arg Asp Ala Pro Gly Ser Gln Asp His Gln Gly Phe Phe Leu Ser Asp Asn Gln Ser Asp Ala Ala Pro Pro Leu Ser Ile Ser Asp Ala Ser Gly Leu Trp Pro Glu Leu Leu Gln Glu Met Pro Pro Arg His Ser Pro Gln Ser Pro Gln Glu Asp Pro Asp Cys Trp Pro Leu Lys Leu Gly Ser Arg Leu Glu Gln Leu Gln Ala Gln Met Asn Arg Leu Glu Ser Arg Val Ser Ser Asp Leu Ser Arg Ile Leu Gln Leu Leu Gln Lys Pro Met Pro Gln Gly His Ala Ser Tyr Ile Leu Glu Ala Pro Ala Ser Asn Asp Leu Ala Leu Val Pro Ile Ala Ser Glu Thr Thr Ser Pro Gly Pro Arg Leu Pro Gln Gly Phe Leu Pro Pro Ala Gln Thr Pro Ser Tyr Gly Asp Leu Asp Asp Cys Ser Pro Lys His Arg Asn Ser Ser Pro Arg Met Pro His Leu Ala Val Ala Met Asp Lys Thr Leu Ala Pro Ser Ser Glu Gln Glu Gln Pro Glu Gly Leu Trp Pro Pro Leu Ala Ser Pro Leu His Pro Leu Glu Val Gln Gly Leu Ile Cys Gly Pro Cys Phe Ser Ser Leu Pro Glu His Leu Gly Ser Val Pro Lys Gln Leu Asp Phe Gln Arg His Gly Ser Asp Pro Gly Phe Ala Gly Ser Trp Gly His <210> 11 <211> 788 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7502736CD1 <400> 11 Met Ala Gly Gln Ala Asp Gln Gly Gln Ala Gln Ala Gly Ala Ser Thr Gly Pro Ser Ala Arg Ser His Ser Ser Arg Ser Thr Ser Trp Ser Ser Thr Trp Arg Ser Thr Ala Pro Ala Pro Pro Arg Arg Leu Arg Ser Ser Arg Pro Ile Arg Trp Trp Ser Gly His Arg Thr Ser Leu Arg Arg Ser Pro Arg Ser Cys Pro Trp Ala Arg Met Cys Cys Arg Ser Thr Ser Cys Arg Arg Arg Ala Ser Thr Ala Gly Pro Ser Cys Thr Thr Ala Pro Phe Lys Ala Val Trp Asp Trp Leu Ile Leu Leu Leu Val Ile Tyr Thr Ala Val Phe Thr Pro Tyr Ser Ala Ala Phe Leu Leu Ser Asp Gln Asp Glu Ser Arg Arg Gly Ala Cys Ser Tyr Thr Cys Ser Pro Leu Thr Val Val Asp Leu Ile Val Asp Ile Met Phe Val Val Asp Ile Val Ile Asn Phe Arg Thr Thr Tyr Val Asn Thr Asn Asp Glu Val Val Ser His Pro Arg Arg Ile Ala Val His Tyr Phe Lys Gly Trp Phe Leu Ile Asp Met Val Ala Ala Ile Pro Phe Asp Leu Leu Ile Phe Arg Thr Gly Ser Asp Glu Thr Thr Thr Leu Ile Gly Leu Leu Lys Thr Ala Arg Leu Leu Arg Leu Val Arg Val Ala Arg Lys Leu Asp Arg Tyr Ser Glu Tyr Gly Ala Ala Val Leu Phe Leu Leu Met Cys Thr Phe Ala Leu Ile Ala His Trp Leu Ala Cys Ile Trp Tyr Ala Ile Gly Asn Val Glu Arg Pro Tyr Leu Glu His Lys Ile Gly Trp Leu Asp Ser Leu Gly Val Gln Leu Gly Lys Arg Tyr Asn Gly Ser Asp Pro Ala Ser Gly Pro Ser Val Gln Asp Lys Tyr Val Thr Ala Leu Tyr Phe Thr Phe Ser Ser Leu Thr Ser Val Gly Phe Gly Asn Val Ser Pro Asn Thr Asn Ser Glu Lys Val Phe Ser Ile Cys Val Met Leu Ile Gly Ser Leu Met Tyr Ala Ser Ile Phe Gly Asn Val Ser Ala Ile Ile Gln Arg Leu Tyr Ser Gly Thr Ala Arg Tyr His Thr Gln Met Leu Arg Val Lys Glu Phe Ile Arg Phe His Gln Ile Pro Asn Pro Leu Arg Gln Arg Leu Glu Glu Tyr Phe Gln His Ala Trp Ser Tyr Thr Asn Gly Ile Asp Met Asn Ala Val Leu Lys Gly Phe Pro Glu Cys Leu Gln Ala Asp Ile Cys Leu His Leu His Arg Ala Leu Leu Gln His Cys Pro Ala Phe Ser Gly Ala Gly Lys Gly Cys Leu Arg Ala Leu Ala Val Lys Phe Lys Thr Thr His Ala Pro Pro Gly Asp Thr Leu Val His Leu Gly Asp Val Leu Ser Thr Leu Tyr Phe Ile Ser Arg Gly Ser Ile Glu Ile Leu Arg Asp Asp Val Val Val Ala Ile Leu Gly Lys Asn Asp Ile Phe Gly Glu Pro Val Ser Leu His Ala Gln Pro Gly Lys Ser Ser Ala Asp Val Arg Ala Leu Thr Tyr Cys Asp Leu His Lys Ile Gln Arg Ala Asp Leu Leu Glu Val Leu Asp Met Tyr Pro Ala Phe Ala Glu Ser Phe Trp Ser Lys Leu Glu Val Thr Phe Asn Leu Arg Asp Ala Pro Gly Ser Gln Asp His Gln Gly Phe Phe Leu Ser Asp Asn Gln Ser Asp Ala Ala Pro Pro Leu Ser Ile Ser Asp Ala Ser Gly Leu Trp Pro Glu Leu Leu Gln Glu Met Pro Pro Arg His Ser Pro Gln Ser Pro Gln Glu Asp Pro Asp Cys Trp Pro Leu Lys Leu Gly Ser Arg Leu Glu Gln Leu Gln Ala Gln Met Asn Arg Leu Glu Ser Arg Val Ser Ser Asp Leu Ser Arg Ile Leu Gln Leu Leu Gln Lys Pro Met Pro Gln Gly His Ala Ser Tyr Ile Leu Glu Ala Pro Ala Ser Asn Asp Leu Ala Leu Val Pro Ile Ala Ser Glu Thr Thr Ser Pro Gly Pro Arg Leu Pro Gln Gly Phe Leu Pro Pro Ala Gln Thr Pro Ser Tyr Gly Asp Leu Asp Asp Cys Ser Pro Lys His Arg Asn Ser Ser Pro Arg Met Pro His Leu Ala Val Ala Met Asp Lys Thr Leu Ala Pro Ser Ser Glu Gln Glu Gln Pro Glu Gly Leu Trp Pro Pro Leu Ala Ser Pro Leu His Pro Leu Glu Val Gln Gly Leu Ile Cys Gly Pro Cys Phe Ser Ser Leu Pro Glu His Leu Gly Ser Val Pro Lys Gln Leu Asp Phe Gln Arg His Gly Ser Asp Pro Gly Phe Ala Gly Ser Trp Gly His <210> 12 <211> 501 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503570CD1 <400> 12 Met Ser Gln Asp Thr Glu Val Asp Met Lys Glu Val Glu Leu Asn Glu Leu Glu Pro Glu Lys Gln Pro Met Asn Ala Ala Ser Gly Ala Ala Met Ser Leu Ala Gly Ala Glu Lys Asn Gly Leu Val Lys Ile Lys Val Ala Glu Asp Glu Ala Glu Ala Ala Ala Ala Ala Lys Phe Thr Gly Leu Ser Lys Glu Glu Leu Leu Lys Val Ala Gly Ser Pro Gly Trp'Val Arg Thr Arg Trp Ala Leu Leu Leu Leu Phe Trp Leu Gly Trp Leu Gly Met Leu Ala Gly Ala Val Val Ile Ile Val Arg Ala Pro Arg Cys Arg Glu Leu Pro Ala Gln Lys Trp Trp His Thr Gly Ala Leu Tyr Arg Ile Gly Asp Leu Gln Ala Phe Gln Gly His Gly Ala Gly Asn Leu Ala Gly Leu Lys Gly Arg Leu Asp Tyr Leu 140 14.5 150 Ser Ser Leu Lys Val Lys Gly Leu Val Leu Gly Pro Ile His Lys Asn Gln Lys Asp Asp Val Ala Gln Thr Asp Leu Leu Gln Ile Asp Pro Asn Phe Gly Ser Lys Glu Asp Phe Asp Ser Leu Leu Gln Ser Ala Lys Lys Lys Ser Ile Arg Val Ile Leu Asp Leu Thr Pro Asn Tyr Arg Gly Glu Asn Ser Trp Phe Ser Thr Gln Val Asp Thr Val Ala Thr Lys Val Lys Asp Ala Leu Glu Phe Trp Leu Gln Ala Gly Val Asp Gly Phe Gln Val Arg Asp Ile Glu Asn Leu Lys Asp Ala Ser Ser Phe Leu Ala Glu Trp Gln Asn Ile Thr Lys Gly Phe Ser Glu Asp Arg Leu Leu Ile Ala Gly Thr Asn Ser Ser Asp Leu Gln Gln Ile Leu Ser Leu Leu Glu Ser Asn Lys Asp Leu Leu Leu Thr Ser Ser Tyr Leu Ser Asp Ser Gly Ser Thr Gly Glu His Thr Lys Ser Leu Val Thr Gln Tyr Leu Asn Ala Thr Gly Asn Arg Trp Cys Ser Trp Ser Leu Ser Gln Ala Arg Leu Leu Thr Ser Phe Leu Pro Ala Gln Leu Leu Arg Leu Tyr Gln Leu Met Leu Phe Thr Leu Pro Gly Thr Pro Val Phe Ser Tyr Gly Asp Glu Ile Gly Leu Asp Ala Ala Ala Leu Pro Gly Gln Gly Gln Ser Glu Asp Pro Gly Ser Leu Leu Ser Leu Phe Arg Arg Leu Ser Asp Gln Arg Ser Lys Glu Arg Ser Leu Leu His Gly Asp Phe His Ala Phe Ser Ala Gly Pro Gly Leu Phe Ser Tyr Ile Arg His Trp Asp Gln Asn Glu Arg Phe Leu Val Val Leu Asn Phe Gly Asp Val Gly Leu Ser Ala Gly Leu Gln Ala Ser Asp Leu Pro Ala Ser Ala Ser Leu Pro Ala Lys Ala Asp Leu Leu Leu Ser Thr Gln Pro Gly Arg Glu Glu Gly Ser Pro Leu Glu Leu Glu Arg Leu Lys Leu Glu Pro His Glu Gly Leu Leu Leu Arg Phe Pro Tyr Ala Ala <210> 13 <211> 721 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7504008CD1 <400> 13 Met Gly Leu Ala Asp Ala Ser Gly Pro Arg Asp Thr Gln Ala Leu Leu Ser Ala Thr Gln Ala Met Asp Leu Arg Arg Arg Asp Tyr His Met Glu Arg Pro Leu Leu Asn Gln Glu His Leu Glu Glu Leu Gly Arg Trp Gly Ser Ala Pro Arg Thr His Gln Trp Arg Thr Trp Leu Gln Cys Ser Arg Ala Arg Ala Tyr Ala Leu Leu Leu Gln His Leu Pro Val Leu Val Trp Leu Pro Arg Tyr Pro Val Arg Asp Trp Leu Leu Gly Asp Leu Leu Ser Gly Leu Ser Val Ala Ile Met Gln Leu Pro Gln Gly Leu Ala Tyr Ala Leu Leu Ala Gly Leu Pro Pro Val Phe Gly Leu Tyr Ser Ser Phe Tyr Pro Val Phe Ile Tyr Phe Leu Phe Gly Thr Ser Arg His Ile Ser Val Gly Thr Phe Ala Val Met Ser Val Met Val Gly Ser Val Thr Glu Ser Leu Ala Pro Gln Ala Leu Asn Asp Ser Met Ile Asn Glu Thr Ala Arg Asp Ala Ala Arg Val Gln Val Ala Ser Thr Leu Ser Val Leu Val Gly Leu Phe Gln Val Gly Leu Gly Leu Ile His Phe Gly Phe Val Val Thr Tyr Leu Ser Glu Pro Leu Val Arg Gly Tyr Thr Thr Ala Ala Ala Val Gln Val Phe Val Ser Gln Leu Lys Tyr Val Phe Gly Leu His Leu Ser Ser His Ser Gly Pro Leu Ser Leu Ile Tyr Thr Val Leu Glu Val Cys Trp Lys Leu Pro Gln Ser Lys Leu Ile Gly Ala Thr Gly Ile Ser Tyr Gly Met Gly Leu Lys His Arg Phe Glu Val Asp Val Val Gly Asn Ile Pro Ala Gly Leu Val Pro Pro Val Ala Pro Asn Thr Gln Leu Phe Ser Lys Leu Val Gly Ser Ala Phe Thr Ile Ala Val Val Gly Phe Ala Ile Ala Ile Ser Leu Gly Lys Ile Phe Ala Leu Arg His Gly Tyr Arg Val Asp Ser Asn Gln Glu Leu Val Ala Leu Gly Leu Ser Asn Leu Ile Gly Gly Ile Phe Gln Cys Phe Pro Val Ser Cys Ser Met Ser Arg Ser Leu Val Gln Glu Ser Thr Gly Gly Asn Ser Gln Val Ala Gly Ala Ile Ser Ser Leu Phe Ile Leu Leu Ile Ile Val Lys Leu Gly Glu Leu Phe His Asp Leu Pro Lys Ala Val Leu Ala Ala Ile Ile Ile Val Asn Leu Lys Gly Met Leu Arg Gln Leu Ser Asp Met Arg Ser Leu Trp Lys Ala Asn Arg Ala Asp Leu Leu Ile Trp Leu Val Thr Phe Thr Ala Thr Ile Leu Leu Asn Leu Asp Leu Gly Leu Val Val Ala Val Ile Phe Ser Leu Leu Leu Val Val Val Arg Thr Gln Met Pro His Tyr Ser Val Leu Gly Gln Val Pro Asp Thr Asp Ile Tyr Arg Asp Val Ala Glu Tyr Ser Glu Ala Lys Glu Val Arg Gly Val Lys Val Phe Arg Ser Ser Ala Thr Val Tyr Phe Ala Asn Ala Glu Phe Tyr Ser Asp Ala Leu Lys Gln Arg Cys Gly Val Asp Val Asp Phe Leu Ile Ser Gln Lys Lys Lys Leu Leu Lys Lys Gln Glu Gln Leu Lys Leu Lys Gln Leu Gln Lys Glu Glu Lys Leu Arg Lys Gln Ala Ala Ser Pro Lys Gly Ala Ser Val Ser Ile Asn Val Asn Thr Ser Leu Glu Asp Met Arg Ser Asn Asn Val Glu Asp Cys Lys Met Met Gln Val Ser Ser Gly Asp Lys Met Glu Asp Ala Thr Ala Asn Gly Gln Glu Asp Ser Lys Ala Pro Asp Gly Ser Thr Leu Lys Ala Leu Gly Leu Pro Gln Pro Asp Phe His Ser Leu Ile Leu Asp Leu Gly Ala Leu Ser Phe Val Asp Thr Val Cys Leu Lys Ser Leu Lys Asn Ile Phe His Asp Phe Arg Glu Ile Glu Val Glu Val Tyr Met Ala Ala Cys His Ser Pro Val Val Ser Gln Leu Glu Ala Gly His Phe Phe Asp Ala Ser Ile Thr Lys Lys His Leu Phe Ala Ser Val His Asp Ala Val Thr Phe Ala Leu Gln His Pro Arg Pro Val Pro Asp Ser Pro Val Ser Val Thr Arg Leu <210> 14 <211> 1226 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503559CD1 <400> 14 Met Asp Arg Glu Glu Arg Lys Thr Ile Asn Gln Gly Gln Glu Asp Glu Met Glu Ile Tyr Gly Tyr Asn Leu Ser Arg Trp Lys Leu Ala Ile Val Ser Leu Gly Val Ile Cys Ser Gly Gly Phe Leu Leu Leu Leu Leu Tyr Trp Met Pro Glu Trp Arg Val Lys Ala Thr Cys Val Arg Ala Ala Ile Lys Asp Cys Glu Val Val Leu Leu Arg Thr Thr Asp Glu Phe Lys Met Trp Phe Cys Ala Lys Ile Arg Val Leu Ser Leu Glu Thr Tyr Pro Val Ser Ser Pro Lys Ser Met Ser Asn Lys Leu Ser Asn Gly His Ala Val Cys Leu Ile Glu Asn Pro Thr Glu 110 115 ~ 120 Glu Asn Arg His Arg Ile Ser Lys Tyr Ser Gln Thr Glu Ser Gln Gln Ile Arg Tyr Phe Thr His His Ser Val Lys Tyr Phe Trp Asn Asp Thr Ile His Asn Phe Asp Phe Leu Lys Gly Leu Asp Glu Gly Val Ser Cys Thr Ser Ile Tyr Glu Lys His Ser Ala Gly Leu Thr Lys Gly Met His Ala Tyr Arg Lys Leu Leu Tyr Gly Val Asn Glu Ile Ala Val Lys Val Pro Ser Val Phe Lys Leu Leu Ile Lys Glu Val Leu Asn Pro Phe Tyr Ile Phe Gln Leu Phe Ser Val Ile Leu Trp Ser Thr Asp Glu Tyr Tyr Tyr Tyr Ala Leu Ala Ile Val Val Met Ser Ile Val Ser Ile Val Ser Ser Leu Tyr Ser Ile Arg Lys 245 , 250 255 Gln Tyr Val Met Leu His Asp Met Val Ala Thr His Ser Thr Val Arg Val Ser Val Cys Arg Val Asn Glu Glu Ile Glu Glu Ile Phe Ser Thr Asp Leu Val Pro Gly Asp Val Met Val Ile Pro Leu Asn Gly Thr Ile Met Pro Cys Asp Ala Val Leu Ile Asn Gly Thr Cys Ile Val Asn Glu Ser Met Leu Thr Gly Glu Ser Val Pro Val Thr Lys Thr Asn Leu Pro Asn Pro Ser Val Asp Val Lys Gly Ile Gly Asp Glu Leu Tyr Asn Pro Glu Thr His Lys Arg His Thr Leu Phe Cys Gly Thr Thr Val Ile Gln Thr Arg Phe Tyr Thr Gly Glu Leu Val Lys Ala Ile Val Val Arg Thr Gly Phe Ser Thr Ser Lys Gly Gln Leu Val Arg Ser Ile Leu Tyr Pro Lys Pro Thr Asp Phe Lys Leu Tyr Arg Asp Ala Tyr Leu Phe Leu Leu Cys Leu Val Ala Val Ala Gly Ile Gly Phe Ile Tyr Thr Ile Ile Asn Ser Ile Leu Asn Glu Val Gln Val Gly Val Ile Ile Ile Glu Ser Leu Asp Ile Ile Val Val Val Arg Thr Gln Met Pro His Tyr Ser Val Leu G

Thr Ile Thr Val Pro Pro Ala Leu Pro Ala Ala Met Thr Ala Gly Ile Val Tyr Ala Gln Arg Arg Leu Lys Lys Ile Gly Ile Phe Cys Ile Ser Pro Gln Arg Ile Asn Ile Cys Gly Gln Leu Asn Leu Val Cys Phe Asp Lys Thr Gly Thr Leu Thr Glu Asp Gly Leu Asp Leu Trp Gly Ile Gln Arg Val Glu Asn Ala Arg Phe Leu Ser Pro Glu Glu Asn Val Cys Asn Glu Met Leu Val Lys Ser Gln Phe Val Ala Cys Met Ala Thr Cys His Ser Leu Thr Lys Ile Glu Gly Val Leu Ser Gly Asp Pro Leu Asp Leu Lys Met Phe Glu Ala Ile Gly Trp Ile Leu Glu Glu Ala Thr Glu Glu Glu Thr Ala Leu His Asn Arg Ile Met Pro Thr Val Val Arg Pro Pro Lys Gln Leu Leu Pro Glu Ser Thr Pro Ala Gly Asn Gln Glu Met Glu Leu Phe Glu Leu Pro Ala Thr Tyr Glu Ile Gly Ile Val Arg Gln Phe Pro Phe Ser Ser Ala Leu Gln Arg Met Ser Val Val Ala Arg Val Leu Gly Asp Arg Lys Met Asp Ala Tyr Met Lys Gly Ala Pro Glu Ala Ile Ala Gly Leu Cys Lys Pro Glu Thr Val Pro Val Asp Phe Gln Asn Val Leu Glu Asp Phe Thr Lys Gln Gly Phe Arg Val Ile Ala Leu Ala His Arg Lys Leu Glu Ser Lys Leu Thr Trp His Lys Val Gln Asn Ile Ser Arg Asp Ala Ile Glu Asn Asn Met Asp Phe Met Gly Leu Ile Ile Met Gln Asn Lys Leu Lys Gln Glu Thr Pro Ala Val Leu Glu Asp Leu His Lys Ala Asn Ile Arg Thr Val Met Val Thr Gly Asp Ser Met Leu Thr Ala Val Ser Val Ala Arg Asp Cys Gly Met Ile Leu Pro Gln Asp Lys Val Ile Ile Ala Glu Ala Leu Pro Pro Lys Asp Gly Lys Val Ala Lys Ile Asn Trp His Tyr Ala Asp Ser Leu Thr Gln Cys Ser His Pro Ser Ala Ile Asp Pro Glu Ala Ile Pro Val Lys Leu Val His Asp Ser Leu Glu Asp Leu Gln Met Thr Arg Tyr His Phe Ala Met Asn Gly Lys Ser Phe Ser Val Ile Leu Glu His Phe Gln Asp Leu Val Pro Lys Leu Met Leu His Gly Thr Val Phe Ala Arg Met Ala Pro Asp Gln Lys Thr Gln Leu Ile Glu Ala Leu Gln Asn Val Asp Tyr Phe Val Gly Met Cys Gly Asp Gly Ala Asn Asp Cys Gly Ala Leu Lys Arg Ala His Gly Gly Ile Ser Leu Ser Glu Leu Glu Ala Ser Val Ala Ser Pro Phe Thr Ser Lys Thr 905 910 ~ 915 Pro Ser Ile Ser Cys Val Pro Asn Leu Ile Arg Glu Gly Arg Ala Ala Leu Ile Thr Ser Phe Cys Val Phe Lys Phe Met Ala Leu Tyr Ser Ile Ile Gln Tyr Phe Ser Val Thr Leu Leu Tyr Ser Ile Leu Ser Asn Leu Gly Asp Phe Gln Phe Leu Phe Ile Asp Leu Ala Ile Ile Leu Val Val Va1 Phe Thr Met Ser Leu Asn Pro Ala Trp Lys Glu Leu Val Ala Gln Arg Pro Pro Ser Gly Leu Ile Ser Gly Ala Leu Leu Phe Ser Val Leu Ser Gln Ile Ile Ile Cys Ile Gly Phe Gln Ser Leu Gly Phe Phe Trp Val Lys Gln Gln Pro Trp Tyr Glu Val Trp His Pro Lys Ser Asp Ala Cys Asn Thr Thr Gly Ser Gly Phe Trp Asn Ser Ser His Val Asp Asn Glu Thr Glu Leu Asp Glu His Asn Ile Gln Asn Tyr Glu Asn Thr Thr Val Phe Phe Ile Ser Ser Phe Gln Tyr Leu Ile Val Ala Ile Ala Phe Ser Lys Gly Lys Pro Phe Arg Gln Pro Cys Tyr Lys Asn Tyr Phe Phe Val Phe Ser Val Ile Phe Leu Tyr Ile Phe Ile Leu Phe Ile Met Leu Tyr Pro Val Ala Ser Va1 Asp Gln Val Leu Gln Ile Val Cys Val Pro Tyr Gln Trp Arg Val Thr Met Leu Ile Ile Val Leu Val Asn Ala Phe Val Ser Ile Thr Val Glu Glu Ser Val Asp Arg Trp Gly Lys Cys Cys Leu Pro Trp Ala Leu Gly Cys Arg Lys Lys Thr Pro Lys Ala Lys Tyr Met Tyr Leu Ala Gln Glu Leu Leu Val Asp Pro Glu Trp Pro Pro Lys Pro Gln Thr Thr Thr Glu Ala Lys Ala Leu Val Lys 1205 1210 ' 1215 Glu Asn Gly Ser Cys Gln Ile Ile Thr Ile Thr <210> 15 <211> 638 <212> PRT
<213> Homo sapiens <220>
<221> misc feature <223> Incyte ID No: 6243872CD1 <400> 15 Met Phe Val Gly Val Ala Arg His Ser Gly Ser Gln Asp Glu Val Ser Arg Gly Val Glu Pro Leu Glu Ala Ala Arg Ala Gln Pro Ala Lys Asp Arg Arg Ala Lys Gly Thr Pro Lys Ser Ser Lys Pro Gly Lys Lys His Arg Tyr Leu Arg Leu Leu Pro Glu Ala Leu Ile Arg Phe Gly Gly Phe Arg Lys Arg Lys Lys Ala Lys Ser Ser Val Ser Lys Lys Pro Gly Glu Val Asp Asp Ser Leu Glu Gln Pro Cys Gly Leu Gly Cys Leu Val Ser Thr Cys Cys Glu Cys Cys Asn Asn Ile Arg Cys Phe Met Ile Phe Tyr Cys Ile Leu Leu Ile Cys Gln Gly Val Val Phe Gly Leu Ile Asp Val Ser Ile Gly Asp Phe Gln Lys Glu Tyr Gln Leu Lys Thr Ile Glu Lys Leu Ala Leu Glu Lys Ser Tyr Asp Ile Ser Ser Gly Leu Thr Val Gln Gly Ile Ala Gly Met Pro Leu Tyr Ile Leu Gly Ile Thr Phe Ile Asp Glu Asn Val Ala Thr His Ser Ala Gly Ile Tyr Leu Gly Ile Ala Glu Cys Thr Ser Met Ile Gly Tyr Ala Leu Gly Tyr Val Leu Gly Ala Pro Leu Val Lys Val Pro Glu Asn Thr Thr Ser Ala Thr Asn Thr Thr Val Asn Asn Gly Ser Pro Glu Trp Leu Trp Thr Trp Trp Ile Asn Phe Leu Phe Ala Ala Val Val Ala Trp Cys Thr Leu Ile Pro Leu Ser Cys Phe Pro Asn Asn Met Pro Gly Ser Thr Arg Ile Lys Ala Arg Lys Arg Lys Gln Leu His Phe Phe Asp Ser Arg Leu Lys Asp Leu Lys Leu Gly Ile Asn Ile Lys Asp Leu Cys Ala Ala Leu Trp Ile Leu Met Lys Asn Pro Val Leu Ile Cys Leu Ala Leu Ser Lys Ala Thr Glu Tyr Leu Val Ile Ile Gly Ala Ser Glu Phe Leu Pro Ile Tyr Leu Glu Asn Gln Phe Ile Leu Thr Pro Thr Val Ala Thr Thr Leu Ala Gly Leu Val Leu Ile Pro Gly Gly Ala Leu Gly Gln Leu Leu Gly Gly Val Ile Val Ser Thr Leu Glu Met Ser Cys Lys Ala Leu Met Arg Phe Ile Met Val Thr Ser Val Ile Ser Leu Ile Leu Leu Val Phe Ile Ile Phe Val Arg Cys Asn Pro Val Gln Phe Ala Gly Ile Asn Glu Asp Tyr Asp Gly Thr Gly Lys Leu Gly Asn Leu Thr Ala Pro Cys Asn Glu Lys Cys Arg Cys Ser Ser Ser Ile Tyr Ser Ser Ile Cys Gly Arg Asp Asp Ile Glu Tyr Phe Ser Pro Cys Phe Ala Gly Ile Val Ser Cys Leu Gln Tyr Ser Gln Met Tyr Tyr Asn Cys Ser Cys Ile Lys Glu Gly Leu Ile Thr Ala Asp Ala Glu Gly Asp Phe Ile Asp Ala Arg Pro Gly Lys Cys Asp Ala Lys Cys Tyr Lys Leu Pro Leu Phe Ile Ala Phe Ile Phe Ser Thr Leu Ile Phe Ser Gly Phe Ser Gly Val Pro Ile Val Leu Ala Met Thr Arg Val Val Pro Asp Lys Leu Arg Ser Leu Ala Leu Gly Val Ser Tyr Val Ile Leu Arg Ile Phe Gly Thr Ile Pro Gly Pro Ser Ile Phe Lys Met Ser Gly Glu Thr Ser Cys Ile Leu Arg Asp Val Asn Lys Cys Gly His Thr Gly Arg Cys Trp Ile Tyr Asn Lys Thr Lys Met Ala Phe Leu Leu Val Gly Ile Cys Phe Leu Cys Lys Leu Cys Thr Ile Ile Phe Thr Thr Ile Ala Phe Phe Ile Tyr Lys Arg Arg Leu Asn Glu Asn Thr Asp Phe Pro Asp Val Thr Val Lys Asn Pro Lys Val Lys Lys Lys Glu Glu Thr Asp Leu <210> 16 <211> 507 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 90011608CD1 <400> 16 Met Thr Ala Ser Thr Pro Glu Ala Thr Pro Asn Met Glu Leu Lys Ala Pro Ala Ala Gly Gly Leu Asn Ala Gly Pro Val Pro Pro Ala Ala Leu Ser Thr Gln Arg Leu Arg Asn Glu Asp Tyr His Asp Tyr Ser Ser Thr Asp Val Ser Pro Glu Glu Ser Pro Ser Glu Gly Leu Asn Asn Leu Ser Ser Pro Gly Ser Tyr Gln Arg Phe Gly Gln Ser Asn Ser Thr Thr Trp Phe Gln Thr Leu Ile His Leu Leu Lys Gly Asn Ile Gly Thr Gly Leu Leu Gly Leu Pro Leu Ala Val Lys Asn Ala Gly Ile Val Met Gly Pro Ile Ser Leu Leu Ile Ile Gly Ile Val Ala Val His Cys Met Gly Ile Leu Val Lys Cys Ala His His Phe Cys Arg Arg Leu Asn Lys Ser Phe Val Asp Tyr Gly Asp Thr 140 145 ' 150 Val Met Tyr Gly Leu Glu Ser Ser Pro Cys Ser Trp Leu Arg Asn His Ala His Trp Gly Arg Arg Val Val Asp Phe Phe Leu Ile Val Thr Gln Leu Gly Phe Cys Cys Val Tyr Phe Val Phe Leu Ala Asp Asn Phe Lys Gln Val Ile Glu Ala Ala Asn Gly Thr Thr Asn Asn Cys His Asn Asn Glu Thr Val Ile Leu Thr Pro Thr Met Asp Ser Arg Leu Tyr Met Leu Ser Phe Leu Pro Phe Leu Val Leu Leu Val Phe Ile Arg Asn Leu Arg Ala Leu Ser Ile Phe Ser Leu Leu Ala Asn Ile Thr Met Leu Val Ser Leu Val Met Ile Tyr Gln Phe Ile Val Gln Arg Ile Pro Asp Pro Ser His Leu Pro Leu Val Ala Pro Trp Lys Thr Tyr Pro Leu Phe Phe Gly Thr Ala Ile Phe Ser Phe Glu Gly Ile Gly Met Val Leu Pro Leu Glu Asn Lys Met Lys Asp Pro Arg Lys Phe Pro Leu Ile Leu Tyr Leu Gly Met Val Ile Val Thr Ile Leu Tyr Ile Ser Leu Gly Cys Leu Gly Tyr Leu Gln Phe Gly Ala Asn Ile Gln Gly Ser Ile Thr Leu Asn Leu Pro Asn Cys Trp Leu Tyr Gln Ser Val Lys Leu Leu Tyr Ser Ile Gly Ile Phe Phe Thr Tyr Ala Leu Gln Phe Tyr Val Pro Ala Glu Ile Ile Ile Pro Phe Phe Val Ser Arg Ala Pro Glu His Cys Glu Leu Val Val Asp Leu Phe Val Arg Thr Val Leu Val Cys Leu Thr Cys Ile Leu Ala Ile Leu Ile Pro Arg Leu Asp Leu Val Ile Ser Leu Val Gly Ser Val Ser Ser Ser Ala Leu Ala Leu Ile Ile Pro Pro Leu Leu Glu Val Thr Thr Phe Tyr Ser Glu Gly Met Ser Pro Leu Thr Ile Phe Lys Asp Ala Leu Ile Ser Ile Leu Gly Phe Val Gly Phe Val Val Gly Thr Tyr Glu Ala Leu Tyr Glu Leu Ile Gln Pro Ser Asn Ala Pro Ile Phe Ile Asn Ser Thr Cys Ala Phe Ile <210> 17 <211> 568 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 90024583CD1 <400> 17 Met Ala Ser Ala Leu Ser Tyr Val Ser Lys Phe Lys Ser Phe Val 1 5 ~ 10 15 Ile Leu Phe Val Thr Pro Leu Leu Leu Leu Pro Leu Val Ile Leu Met Pro Ala Lys Phe Val Arg Cys Ala Tyr Val Ile Ile Leu Met Ala Ile Tyr Trp Cys Thr Glu Val Ile Pro Leu Ala Val Thr Ser Leu Met Pro Val Leu Leu Phe Pro Leu Phe Gln Ile Leu Asp Ser Arg Gln Val Cys Val Gln Tyr Met Lys Asp Thr Asn Met Leu Phe Leu Gly Gly Leu Ile Val Ala Val Ala Val Glu Arg Trp Asn Leu His Lys Arg Ile Ala Leu Arg Thr Leu Leu Trp Val Gly Ala Lys Pro Ala Arg Leu Met Leu Gly Phe Met Gly Val Thr Ala Pro Leu Ser Met Trp Ile Ser Asn Thr Ala Thr Thr Ala Met Met Val Pro Ile Val Glu Ala Ile Leu Gln Gln Met Glu Ala Thr Ser Ala Ala Thr Glu Ala Gly Leu Glu Leu Val Asp Lys Gly Lys Ala Lys Glu Leu Pro Gly Ser Gln Val Ile Phe Glu Gly Pro Thr Leu Gly Gln Gln Glu Asp Gln Glu Arg Lys Arg Leu Cys Lys Ala Met Thr Leu Cys Ile Cys Tyr Ala Ala Ser Ile Gly Gly Thr Ala Thr Leu Thr Gly Thr Gly Pro Asn Val Val Leu Leu Gly Gln Met Asn Glu Leu Phe Pro Asp Ser Lys Asp Leu Val Asn Phe Ala Ser Trp Phe Ala Phe Ala Phe Pro Asn Met Leu Val Met Leu Leu Phe Ala Trp Leu Trp Leu Gln Phe Val Tyr Met Arg Phe Asn Phe Lys Lys Ser Trp Gly Cys Gly Leu Glu Ser Lys Lys Asn Glu Lys Ala Ala Leu Lys Val Leu Gln Glu Glu Tyr Arg Lys Leu Gly Pro Leu Ser Phe Ala Glu Ile Asn Val Leu Ile Cys Phe Phe Leu Leu Val Ile Leu Trp Phe Ser Arg Asp Pro Gly Phe Met Pro Gly Trp Leu Thr Val Ala Trp Val Glu Gly Glu Thr Lys Tyr Val Ser Asp Ala Thr Val Ala Ile Phe Val Ala Thr Leu Leu Phe Ile Val Pro Ser Gln Lys Pro Lys Phe Asn Phe Arg Ser Gln Thr Glu Glu Glu Arg Lys Thr Pro Phe Tyr Pro Pro Pro Leu Leu Asp Trp Lys Val Thr Gln Glu Lys Val Pro Trp Gly Ile Val Leu Leu Leu Gly Gly Gly Phe Ala Leu Ala Lys Gly Ser Glu Ala Ser Gly Leu Ser Val Trp Met Gly Lys Gln Met Glu Pro Leu His Ala Val Pro Pro Ala Ala Ile Thr Leu Ile Leu Ser Leu Leu Val Ala Val Phe Thr Glu Cys Thr Ser Asn Val Ala Thr Thr Thr Leu Phe Leu Pro Ile Phe Ala Ser Met Ser Arg Ser Asn Gly Leu Asn Pro Leu Tyr Ile Met Leu Pro Cys Thr Leu Ser Ala Ser Phe Ala Phe Met Leu Pro Val Ala Thr Pro Pro Asn Ala Ile Val Phe Thr Tyr Gly His Leu Lys Val Ala Asp Met Val Lys Thr Gly Val Ile Met Asn Ile Ile Gly Val Phe Cys Val Phe Leu Ala Val Asn Thr Trp Gly Arg Ala Ile Phe Asp Leu Asp His Phe Pro Asp Trp Ala Asn Val Thr His Ile Glu Thr <210> 18 <211> 595 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 90113658CD1 <400> 18 Met Ile Leu Ile Lys Gln Lys Arg Leu Phe Pro Cys Trp Ile Pro Ala Leu Phe Ile Gly Phe Ser Gln Phe Ser Asp Ser Phe Leu Leu Asp Gln Pro Asn Phe Trp Cys Arg Gly Ala Gly Lys Gly Thr Glu Leu Ala Gly Val Thr Thr Thr Gly Arg Gly Gly Asp Met Gly Asn Trp Thr Ser Leu Pro Thr Thr Pro Phe Ala Thr Ala Pro Trp Glu Ala Ala Gly Asn Arg Ser Asn Ser Ser Gly Ala Asp Gly Gly Asp Thr Pro Pro Leu Pro Ser Pro Pro Asp Lys Gly Asp Asn Ala Ser Asn Cys Asp Cys Arg Ala Trp Asp Tyr Gly Ile Arg Ala Gly Leu Val Gln Asn Val Val Ser Lys Trp Asp Leu Val Cys Asp Asn Ala Trp Lys Val His Ile Ala Lys Phe Ser Leu Leu Val Gly Leu Ile Phe Gly Tyr Leu Ile Thr Gly Cys Ile Ala Asp Trp Val Gly Arg Arg Pro Val Leu Leu Phe Ser Ile Ile Phe Ile Leu Ile Phe Gly Leu Thr Val Ala Leu Ser Val Asn Val Thr Met Phe Ser Thr Leu Arg Phe Phe Glu Gly Phe Cys Leu Ala Gly Ile Ile Leu Thr Leu Tyr Ala Leu Arg Ile Glu Leu Cys Pro Pro Gly Lys Arg Phe Met Ile Thr Met Val Ala Ser Phe Val Ala Met Ala Gly Gln Phe Leu Met Pro Gly Leu Ala Ala Leu Cys Arg Asp Trp Gln Val Leu Gln Ala Leu Ile Ile Cys Pro Phe Leu Leu Met Leu Leu Tyr Trp Ser Ile Phe Pro Glu Ser Leu Arg Trp Leu Met Ala Thr Gln Gln Phe Glu Ser Ala Lys Arg Leu Ile Leu His Phe Thr Gln Lys Asn Arg Met Asn Pro Glu Gly Asp Ile Lys Gly Val Ile Pro Glu Leu Glu Lys Glu Leu Ser Arg Arg Pro Lys Lys Val Cys Ile Val Lys Val Val Gly Thr Arg Asn Leu Trp Lys Asn Ile Val Val Leu Cys Val Asn Ser Leu Thr Gly Tyr Gly Ile His His Cys Phe Ala Arg Ser Met Met Gly His Glu Val Lys Val Pro Leu Leu Glu Asn Phe Tyr Ala Asp Tyr Tyr Thr Thr Ala Ser Ile Ala Leu Val Ser Cys Leu Ala Met Cys Val Val Val Arg Phe Leu Gly Arg Arg Gly Gly Leu Leu Leu Phe Met Ile.Leu Thr Ala Leu Ala Ser Leu Leu Gln Leu Gly Leu Leu Asn Leu Ile Gly Lys Tyr Ser Gln His Pro Asp Ser Gly Met Ser Asp Ser Val Lys Asp Lys Phe Ser Ile Ala Phe Ser Ile Val Gly Met Phe Ala Ser His Ala Val Gly Ser Leu Ser Val Phe Phe Cys Ala.Glu Ile Thr Pro Thr Val Ile Arg Cys Gly Gly Leu Gly Leu Val Leu Ala Ser Ala Gly Phe Gly Met Leu Thr Ala Pro Ile Ile Glu Leu His Asn Gln Lys Gly Tyr Phe Leu His His Ile Ile Phe Ala Cys Cys Thr Leu Ile Cys Ile Ile Cys Ile Leu Leu Leu Pro Glu Ser Arg Asp Gln Asn Leu Pro Glu Asn Ile Ser Asn Gly Glu His Tyr Thr Arg Gln Pro Leu Leu Pro His Lys Lys Gly Glu Gln Pro Leu Leu Leu Thr Asn Ala Glu Leu Lys Asp Tyr Ser Gly Leu His Asp Ala Ala Ala Ala Gly Asp Thr Leu Pro Glu Gly Ala Thr Ala Asn Gly Met Lys Ala Met <210> 19 <211> 602 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3942766CD1 <400> 19 Met Ala Ala Leu Ala Ala Ala Ala Lys Lys Val Trp Ser Ala Arg Arg Leu Leu Val Leu Leu Phe Thr Pro Leu Ala Leu Leu Pro Val Val Phe Ala Leu Pro Pro Lys Glu Gly Arg Cys Leu Phe Val Ile Leu Leu Met Ala Val Tyr Trp Cys Thr Glu Ala Leu Pro Leu Ser Val Thr Ala Leu Leu Pro Ile Val Leu Phe Pro Phe Met Gly Ile Leu Pro Ser Asn Lys Val Cys Pro Gln Tyr Phe Leu Asp Thr Asn Phe Leu Phe Leu Ser Gly Leu Ile Met Ala Ser Ala Ile Glu Glu Trp Asn Leu His Arg Arg Ile Ala Leu Lys Ile Leu Met Leu Val Gly Val Gln Pro Ala Arg Leu Ile Leu Gly Met Met Val Thr Thr Ser Phe Leu Ser Met Trp Leu Ser Asn Thr Ala Ser Thr Ala Met Met Leu Pro Ile Ala Asn Ala Ile Leu Lys Ser Leu Phe Gly Gln Lys Glu Val Arg Lys Asp Pro Ser Gln Glu Ser Glu Glu Asn Thr Ala Ala Val Arg Arg Asn Gly Leu His Thr Val Pro Thr Glu Met Gln Phe Leu Ala Ser Thr Glu Ala Lys Asp His Pro Gly Glu Thr Glu Val Pro Leu Asp Leu Pro Ala Asp Ser Arg Lys Glu Asp Glu Tyr Arg Arg Asn Ile Trp Lys Gly Phe Leu I1e Ser Ile Pro Tyr Ser Ala Ser Ile Gly Gly Thr Ala Thr Leu Thr Gly Thr Ala Pro Asn Leu Ile Leu Leu Gly Gln Leu Lys Ser Phe Phe Pro Gln Cys Asp Val Val Asn Phe Gly Ser Trp Phe Ile Phe Ala Phe Pro Leu Met Leu Leu Phe Leu Leu Ala Gly Trp Leu Trp Ile Ser Phe Leu Tyr Gly Gly Leu Ser Phe Arg Gly Trp Arg Lys Asn Lys Ser Glu Ile Arg Thr Asn Ala Glu Asp Arg Ala Arg Ala Val Ile Arg Glu Glu Tyr Gln Asn Leu Gly Pro Ile Lys Phe Ala Glu Gln Ala Val Phe Ile Leu Phe Cys Met Phe Ala Ile Leu Leu Phe Thr Arg Asp Pro Lys Phe Ile Pro Gly Trp Ala Ser Leu Phe Asn Pro Gly Phe Leu Ser Asp Ala Val Thr Gly Val Ala Ile Val Thr Ile Leu Phe Phe Phe Pro Ser Gln Arg Pro Ser Leu Lys Trp Trp Phe Asp Phe Lys Ala Pro Asn Thr Glu Thr Glu Pro Leu Leu Thr Trp Lys Lys Ala Gln Glu Thr Val Pro Trp Asn Ile Ile Leu Leu Leu Gly Gly Gly Phe Ala Met Ala Lys Gly Cys Glu Glu Ser Gly Leu Ser Val Trp Ile Gly Gly Gln Leu His Pro Leu Glu Asn Val Pro Pro Ala Leu Ala Val Leu Leu Ile Thr Val Val Ile Ala Phe Phe Thr Glu Phe Ala Ser Asn Thr Ala Thr Ile Ile Ile Phe Leu Pro Val Leu Ala Glu Leu Ala Ile Arg Leu Arg Val His Pro Leu Tyr Leu Met Ile Pro Gly Thr Val Gly Cys Ser Phe Ala Phe Met Leu Pro Val Ser Thr Pro Pro Asn Ser Ile Ala Phe Ala Ser Gly His Leu Leu Val Lys Asp Met Val Arg Thr Gly Leu Leu Met Asn Leu Met Gly Val Leu Leu Leu Ser Leu Ala Met Asn Thr Trp Ala Gln Thr Ile Phe Gln Leu Gly Thr Phe Pro Asp Trp Ala Asp Met Tyr Ser Val Asn Val Thr Ala Leu Pro Pro Thr~Leu Ala Asn Asp Thr Phe Arg Thr Leu <210> 20 <211> 372 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7501987CD1 <400> 20 Met Val Pro Ala Gly Trp Val Arg Gly Leu Glu Leu Ser Leu Trp Gly Gly Asp Pro Val Val Pro Trp Ser Cys Arg Phe Cys Ser Gln Gln Asp Asp Gly Gln Asp Arg Glu Arg Leu Thr Tyr Phe Gln Asn Leu Pro Glu Ser Leu Thr Ser Leu Leu Val Leu Leu Thr Thr Ala Asn Asn Pro Asp Val Met Ile Pro Ala Tyr Ser Lys Asn Arg Ala Tyr Ala Ile Phe Phe Ile Val Phe Thr Val Ile Gly Ser Leu Phe Leu Met Asn Leu Leu Thr Ala Ile Ile Tyr Ser Gln Phe Arg Gly 95 100 . 105 Tyr Leu Met Lys Ser Leu Gln Thr Ser Leu Phe Arg Arg Arg Leu Gly Thr Arg Ala Ala Phe Glu Val Leu Ser Ser Met Val Gly Glu Gly Gly Ala Phe Pro Gln Ala Val Gly Val Lys Pro Gln Asn Leu Leu Gln Val Leu Gln Lys Val Gln Leu Asp Ser Ser His Lys Gln Ala Met Met Glu Lys Val Arg Ser Tyr Gly Ser Val Leu Leu Ser Ala Glu Glu Phe Gln Lys Leu Phe Asn Glu Leu Asp Arg Ser Val Val Lys Glu His Pro Pro Arg Pro Glu Tyr Gln Ser Pro Phe Leu Gln Ser Ala Gln Phe Leu Phe Gly His Tyr Tyr Phe Asp Tyr Leu Gly Asn Leu Ile Ala Leu Ala Asn Leu Val Ser Ile Cys Val Phe Leu Val Leu Asp Ala Asp Val Leu Pro Ala Glu Arg Asp Asp Phe Ile Leu Gly Ile Leu Asn Cys Val Phe Ile Val Tyr Tyr Leu Leu Glu Leu Leu Leu Lys Val Phe Ala Leu Gly Leu Arg Gly Tyr Leu Ser Tyr Pro Ser Asn Val Phe Asp Gly Leu Leu Thr Val Val Leu Leu Glu Ala Gly Asp Gly Gly Pro Ala Val Ala Val Gly His Asp Pro His Ala Glu His Ala His Arg Val Pro Leu Pro Ala Tyr His Pro Gln His Glu Ala Asp Gly Arg Gly Gly Gln Tyr Arg Pro Gly Pro Gly Ala Glu His Ala Cys Val Trp Arg Asp Pro Gly Gly Gly Leu Leu Arg Ile Cys His His Trp Asp Gln Leu Val <210> 21 <211> 165 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503223CD1 <400> 21 Met Thr Leu Leu Pro Gly Asp Asn Ser Asp Tyr Asp Tyr Ser Ala Leu Ser Cys Thr Ser Asp Ala Ser Phe His Pro Ala Phe Leu Pro Gln Arg Gln Ala Ile Lys Gly Ala Phe Tyr Arg Arg Ala Gln Arg Leu Arg Pro Gln Asp Glu Pro Arg Gln Gly Cys Gln Pro Glu Asp Arg Arg Arg Arg Ile Ile Ile Asn Val Gly Gly Ile Lys Tyr Ser Leu Pro Trp Thr Thr Leu Asp Glu Phe Pro Leu Thr Arg Leu Gly Gln Leu Lys Ala Cys Thr Asn Phe Asp Asp Ile Leu Asn Val Cys Asp Asp Tyr Asp Val Thr Cys Asn Glu Phe Phe Phe Asp Arg Asn Pro Gly Ala Phe Gly Thr Ile Leu Thr Phe Leu Arg Ala Gly Lys Leu Arg Leu Leu Arg Glu Met Cys Ala Leu Ser Phe Gln Asp Ser Asp Ile Leu Phe Gly Ser Ala Ser Ser Asp Thr Arg Asp Asn Asn <210> 22 <211> 497 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503566CD1 <400> 22 Met Leu Arg Thr Ile Leu Asp Ala Pro Gln Arg Leu Leu Lys Glu Gly Arg Ala Ser Arg Gln Leu Val Leu Val Val Val Phe Val Ala Leu Leu Leu Asp Asn Met Leu Phe Thr Val Val Val Pro Ile Val Pro Thr Phe Leu Tyr Asp Met Glu Phe Lys Glu Val Asn Ser Ser Leu His Leu Gly His Ala Gly Ser Ser Pro His Ala Leu Ala Ser Pro Ala Phe Ser Thr Ile Phe Ser Phe Phe Asn Asn Asn Thr Val Ala Val Glu Glu Ser Val Pro Ser Gly Ile Ala Trp Met Asn Asp Thr Ala Ser Thr Ile Pro Pro Pro Ala Thr G1u Ala Ile Ser Ala His Lys Asn Asn Cys Leu Gln Gly Thr Gly Phe Leu Glu Glu Glu Thr Thr Arg Val Gly Val Leu Phe Ala Ser Lys Ala Val Met Gln Leu Leu Val Asn Pro Phe Val Gly Pro Leu Thr Asn Arg Ile Gly Tyr His Ile Pro Met Phe Ala Gly Phe Val Ile Met Phe Leu Ser Thr Val Ser Leu Gly Met Leu Ala Ser Val Tyr Thr Asp Asp His Glu Arg Gly Arg Ala Met Gly Thr Ala Leu Gly Gly Leu Ala Leu Gly Leu Leu Val Gly Ala Pro Phe Gly Ser Val Met Tyr Glu Phe Val Gly Lys Ser Ala Pro Phe Leu Ile Leu Ala Phe Leu Ala Leu Leu Asp Gly Ala Leu Gln Leu Cys Ile Leu Gln Pro Ser Lys Val Ser Pro Glu Ser Ala Lys Gly Thr Pro Leu Phe Met Leu Leu Lys Asp Pro Tyr Ile Leu Val Ala Ala Gly Ser Ile Cys Phe Ala Asn Met Gly Val Ala Ile Leu Glu Pro Thr Leu Pro Ile Trp Met Met Gln Thr Met Cys Ser Pro Lys Trp Gln Leu Gly Leu Ala Phe Leu Pro Ala Ser Val Ser Tyr Leu Ile Gly Thr Asn Leu Phe Gly Val Leu Ala Asn Lys Met Gly Arg Trp Leu Cys Ser. Leu Ile Gly Met Leu Val Val Gly Thr Ser Leu Leu Cys Val Pro Leu Ala His Asn Ile Phe Gly Leu Ile Gly Pro Asn Ala Gly Leu Gly Leu Ala Ile Gly Met Val Asp Ser Ser Met Met Pro Ile Met Gly His Leu Val Asp Leu Arg His Thr Ser Val Tyr Gly Ser Val Tyr Ala Ile Ala Asp Val Ala Phe Cys Met Gly Phe Ala Ile Gly Pro Ser Thr Gly Gly Ala Ile Val Lys Ala Ile Gly Phe Pro Trp Leu Met Val Ile Thr Gly Val Ile Asn Ile Val Tyr Ala Pro Leu Cys Tyr Tyr Leu Arg Ser Pro Pro Ala Lys Glu Glu Lys Leu Ala Ile Leu Ser Gln Asp Cys Pro Met Glu Thr Arg Met Tyr Ala Thr Gln Lys Pro Thr Lys Glu Phe Pro Leu Gly Glu Asp Ser Asp Glu Glu Pro Asp His Glu Glu <210> 23 <211> 67 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7505122CD1 <400> 23 Met Gln Lys Val Thr Leu Gly Leu Leu Val Phe Leu Ala Gly Phe Pro Val Leu Asp Ala Asn Asp Leu Glu Asp Lys Asn Ser Pro Phe Tyr Tyr Asp Trp His Ser Leu Gln Val Gly Gly Leu Ile Cys Ala Gly Val Leu Cys Met Ala Gly Pro His Leu Thr Ser Arg Lys Arg Val Ser Leu Phe Asn Phe Phe <210> 24 <211> 152 <212> PRT
~213> Homo sapiens ~220>
<221> misc_feature <223> Incyte ID No: 7511620CD1 <400> 24 Met Gly Leu Ala Asp Ala Ser Gly Pro Arg Asp Thr Gln Ala Leu Leu Ser Ala Thr Gln Ala Met Asp Leu Arg Arg Arg Asp Tyr His Met Glu Arg Pro Leu Leu Asn Gln Glu His Leu Glu Glu Leu Gly Arg Trp Gly Ser Ala Pro Arg Thr His Gln Trp Arg Thr Trp Leu Gln Cys Ser Arg Ala Arg Ala Tyr Ala Leu Leu Leu Gln His Leu Pro Val Leu Val Trp Leu Pro Arg Tyr Pro Val Arg Asp Trp Leu Leu Gly Asp Leu Leu~Ser Gly Leu Ser Val Ala Ile Met Gln Leu Pro Gln Gly Leu Ala Tyr Ala Leu Leu Ala Gly Leu Pro Pro Val Phe Gly Leu Tyr Ser Ser Phe Tyr Pro Val Phe Ile Tyr Phe Leu Phe Gly Thr Ser Arg His Ile Ser Val Gly Leu Glu Arg Leu His Asp Gln <210> 25 <211> 467 <212> PRT
<213> Homo Sapiens <220>
<221> misc feature ~223> Incyte ID No: 7506995CD1 <400> 25 Met Val Pro Ala Gly Trp Val Arg Gly Leu Glu Leu Ser Leu Trp Gly Gly Asp Pro Val Val Pro Trp Ser Cys Arg Phe Cys Ser Gln Gln Asp Asp Gly Gln Asp Arg Glu Arg Leu Thr Tyr Phe Gln Asn Leu Pro Glu Ser Leu Thr Ser Leu Leu Val Leu Leu Thr Thr Ala Asn Asn Pro Asp Val Met Ile Pro Ala Tyr Ser Lys Asn Arg Ala Tyr Ala Ile Phe Phe Ile Val Phe Thr Val Ile Gly Ser Leu Phe Leu Met Asn Leu Leu Thr Ala Ile Ile Tyr Ser Gln Phe Arg Gly Tyr Leu Met Lys Ser Leu Gln Thr Ser Leu Phe Arg Arg Arg Leu Gly Thr Arg Ala Ala Phe Glu Val Leu Ser Ser Met Val Gly Glu Gly Gly Ala Phe Pro Gln Ala Thr Arg Arg Gly Pro Ser Thr Ser Leu Arg Phe Cys Arg Ala Pro Ser Ser Ser Ser Ala Thr Thr Thr Leu Thr Thr Trp Gly Thr Ser Ser Pro Trp Gln Thr Trp Cys Pro Phe Ala Cys Ser Trp Cys Trp Met Gln Met Cys Cys Leu Leu Ser Val Met Thr Ser Ser Trp Gly Phe Ser Thr Ala Ser Ser Leu Cys Thr Thr Cys Trp Arg Cys Cys Ser Arg Ser Leu Pro Trp Ala Cys Glu Gly Thr Cys Pro Thr Pro Ala Thr Cys Leu Thr Gly Ser Ser Pro Leu Ser Cys Trp Arg Pro Glu Met Val Gly Leu Leu Ser Leu Trp Asp Met Thr Arg Met Leu Asn Met Leu Ile Val Phe Arg Phe Leu Arg Ile Ile Pro Ser Met Lys Pro Met Ala Val Val Ala Ser Thr Val Leu Gly Leu Val Gln Asn Met Arg Ala Phe Gly Gly Ile Leu Val Val Val Tyr Tyr Val Phe Ala Ile Ile Gly Ile Asn Leu Phe Arg Gly Val Ile Val Ala Leu Pro Gly Asn Ser Ser Leu Ala Pro Ala Asn Gly Ser Ala Pro Cys Gly Ser Phe Glu Gln Leu Glu Tyr Trp Ala Asn Asn Phe Asp Asp Phe Ala Ala Ala Leu Val Thr Leu Trp Asn Leu Met Val Val Asn Asn Trp Gln Val Phe Leu Asp Ala Tyr Arg Arg Tyr Ser Gly Pro Trp Ser Lys Ile Tyr Phe Val Leu Trp Trp Leu Val Ser Ser Val Ile Trp Val Asn Leu Phe Leu Ala Leu Ile Leu Glu Asn Phe Leu His Lys Trp Asp Pro Arg Ser His Leu Gln Pro Leu Ala Gly Thr Pro Glu Ala Thr Tyr Gln Met Thr Val Glu Leu Leu Phe Arg Asp Ile Leu Glu Glu Pro Gly Glu Asp Glu Leu Thr Glu Arg Leu Ser Gln His Pro His Leu Trp Leu Cys Arg <210> 26 <211> 490 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7506996CD1 <400> 26 Met Val Pro Ala Gly Trp Val Arg Gly Leu Glu Leu Ser Leu Trp 1 5 . 10 15 Gly Gly Asp Pro Val Val Pro Trp Ser Cys Arg Phe Cys Ser Gln Gln Asp Asp Gly Gln Asp Arg Glu Arg Leu Thr Tyr Phe Gln Asn Leu Pro Glu Ser Leu Thr Ser Leu Leu Val Leu Leu Thr Thr Ala Asn Asn Pro Asp Val Met Ile Pro Ala Tyr Ser Lys Asn Arg Ala Tyr Ala Ile Phe Phe Ile Val Phe Thr Val Ile Gly Ser Leu Phe Leu Met Asn Leu Leu Thr Ala Ile Ile Tyr Ser Gln Phe Arg Gly Tyr Leu Met Lys Ser Leu Gln Thr Ser Leu Phe Arg Arg Arg Leu Gly Thr Arg Ala Ala Phe Glu Val Leu Ser Ser Met Val Gly Glu Gly Gly Ala Phe Pro Gln Ala Val Gly Val Lys Pro Gln Asn Leu Leu Gln Val Leu Gln Lys Val Gln Leu Asp Ser Ser His Lys Gln Ala Met Met Glu Lys Val Arg Ser Tyr Gly Ser Val Leu Leu Ser Ala Glu Glu Phe Gln Lys Leu Phe Asn Glu Leu Asp Arg Ser Val Val Lys Glu His Pro Pro Arg Pro Glu Tyr Gln Ser Pro Phe Leu Gln Ser Ala Gln Phe Leu Phe Gly His Tyr Tyr Phe Asp Tyr Leu Gly Asn Leu Ile Ala Leu Ala Asn Leu Val Ser Ile Cys Val Phe Leu Val Leu Asp Ala Asp Val Leu Pro Ala Glu Arg Asp Asp Phe Ile Leu Gly Ile Leu Asn Cys Val Phe Ile Val Tyr Tyr Leu Leu Glu Leu Leu Leu Lys Val Phe Ala Leu Gly Leu Arg Gly Tyr Leu Ser Tyr Pro Ser Asn Val Phe Asp Gly Leu Leu Thr Val Val Leu Leu Pro Met Ala Val Val Ala Ser Thr Val Leu Gly Leu Val Gln Asn Met Arg Ala Phe Gly Gly Ile Leu Val Val Val Tyr Tyr Val Phe Ala Ile Ile Gly Ile Asn Leu Phe Arg Gly Val Ile Val Ala Leu Pro Gly Asn Ser Ser Leu Ala Pro Ala Asn Gly Ser Ala Pro Cys Gly Ser Phe Glu Gln Leu Glu Tyr Trp Ala Asn Asn Phe Asp Asp Phe Ala Ala Ala Leu Val Thr Leu Trp Asn Leu Met Val Val Asn Asn Trp Gln Val Phe Leu Asp Ala Tyr Arg Arg Tyr Ser Gly Pro Trp Ser Lys Ile Tyr Phe Val Leu Trp Trp Leu Val Ser Ser Val Ile Trp Val Asn Leu Phe Leu Ala Leu Ile Leu Glu Asn Phe Leu His Lys Trp Asp Pro Arg Ser His Leu Gln Pro Leu Ala Gly Thr Pro Glu Ala Thr Tyr Gln Met Thr Val Glu Leu Leu Phe Arg Asp Ile Leu Glu Glu Pro Gly Glu Asp Glu Leu Thr Glu Arg Leu Ser Gln His Pro His Leu Trp Leu Cys Arg <210> 27 <211> 2343 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1853191CB1 <400> 27 cgcgtacagg aggcggcggc ggctcccagt caccggcccc cgccggcgag cgcacgatgc 60 actgcctggg cgccgagtac ctggtttctg cagaaggagc ccctaggcaa agggagtggc 120 gaccccagat ttataggaaa tgcacagata cggcatggtt attcctgttc tttctctttt 180 ggactggttt ggtgtttatc atgggctact cggtggtggc tggagccgcg ggaagactcc 240 tctttggcta tgacagcttt ggcaacatgt gtggcaagaa gaactccccc gtggaagggg 300 cccctctttc agggcaggac atgaccctaa aaaaacacgt gttctttatg aattcctgca 360 acctggaagt caaaggtacg cagctcaacc gcatggccct ctgtgtatcc aactgccctg 420 aagagcagct tgactccctg gaagaggtcc agttctttgc aaacaccagt gggtccttcc 480 tgtgtgttta tagtttgaat tccttcaact atacccacag tccaaaagca gactcactgt 540 gtcccaggct accagttcct ccaagcaagt catttccctt atttaaccga tgtgtccctc 600 aaacacctga gtgctactcc ctatttgcat ctgttttgat aaatgatgtt gacaccctcc 660 accgaattct aagtggaatc atgtcgggaa gagatacaat ccttggcctg tgtatcctcg 720 cattagcctt gtctttggcc atgatgttta ccttcagatt catcaccacc cttctggttc 780 acattttcat ttcattggtt attttgggat tgttgtttgt ctgcggtgtt ttatggtggc 840 tgtattatga ctataccaac gacctcagca tagaattgga cacagaaagg gaaaatatga 900 agtgcgtgct ggggtttgct atcgtatcca caggcatcac ggcagtgctg ctcgtcttga 960 tttttgttct cagaaagaga ataaaattga cagttgagct tttccaaatc acaaataaag 1020 ccatcagcag tgctcccttc ctgctgttcc agccactgtg gacatttgcc atcctcattt 1080 tcttctgggt cctctgggtg gctgtgctgc tgagcctggg aactgcagga gctgcccagg 1140 ttatggaagg cggccaagtg gaatataagc ccctttcggg cattcggtac atgtggtcgt 1200 accatttaat tggcctcatc tggactagtg aattcatcct tgcgtgccag caaatgacta 1260 tagctggggc agtggttact tgttatttca acagaagtaa aaatgatcct cctgatcatc 1320 ccatcctttc gtctctctcc attctcttct tctaccatca aggaaccatt gtgaaagggt 1380 catttttaat ctctgtggtg aggattccga gaatcattgt catgtacatg caaaacgcac 1440 tgaaagaaca gcatggtgca ttgtccaggt acctgttccg atgctgctac tgctgtttct 1500 ggtgtcttga caaatacctg ctccatctca accagaatgc atatactaca actgctatta 1560 atgggacaga tttctgtaca tcagcaaaag atgcattcaa aatcttgtcc aagaactcaa 1620 gtcactttac atctattaac tgctttggag acttcataat ttttctagga aaggtgttag 1680 tggtgtgttt cactgttttt ggaggactca tggcttttaa ctacaatcgg gcattccagg 1740 tgtgggcagt ccctctgtta ttggtagctt tttttgccta cttagtagcc catagttttt 1800 tatctgtgtt tgaaactgtg ctggatgcac ttttcctgtg ttttgctgtt gatctggaaa 1860 caaatgatgg atcgtcagaa aagccctact ttatggatca agaatttctg agtttcgtaa 1920 aaaggagcaa caaattaaac aatgcaaggg cacagcagga caagcactca ttaaggaatg 1980 aggagggaac agaactccag gccattgtga gatagatacc catttaggta tctgtacctg 2040 gaaaacattt ccttctaaga gccatttaca gaatagaaga tgagaccact agagaaaagt 2100 tagtgaattt ttttttaaaa gacctaataa accctattct tcctcattgt ctttgtcatt 2160 attgtttgac caggtaacaa tactggaact atattagttt accttttttt gtacaaatta 2220 ggacagaaaa actcttctaa aaccatgttt atatgcatca acttacaaag tacactatgt 2280 aagaactgag gtaagtttgt aagtgcacaa ctaataaata aaccttttta agataaggaa 2340 aaa 2343 <210> 28 <211> 3145 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7497369CB1 <400> 28 ctggtgtccc cggggtccct cggatagagg tggaaacagg tcggacaaga gctggccgtg 60 ggggaagagc aatggtgtgc ggaagtgggg agccctatct agggacaagc agaaaactta 120 gggtggcaga tccccccgac cctgtgcagg ggctgccgga gaccagagag gctgctgtgt 180 gacctggacc aagccccttg ccctctcatg cctcagtttc cccttctctc cacggaggga 240 ttgagcagga catggagggc cccttccagt agccattctg ggactccctg cacctgtccc 300 caggggaggg tcctgagctg gcctccacgt ggctgctgtt ccccatagtg gaaccttgtg 360 tgtggagacg gctggaaggt cccgctggag caggtgagcc acctcctggg ctggctgctg 420 ggctgtgtca tcctgggagc aggctgtgac cggtgaggcc ccttcccttt tcctgctgcc 480 cagcagccct cccctccgcc cactctcagg gttcagctag acctggtgct gccaggatga 540 aggttggacc ctgggttatc ctgaccctgt ccaagcatgt cccccgctgc ccaccaggtt 600 tggacgccgg gcagtttttg tggcctccct ggtgctgacc acaggcctgg gggccagtga 660 ggccctggct gccagcttcc ctaccctgct ggtcctgcgc ctactccacg ggggcacatt 720 ggcaggggcc ctcctcgccc tgtatctggc tcgtgagtac cctgggggct gctgtcactg 780 ggggaggggc agtgggtggc ccgcaggcct ctgaggctcc cttgccgagg gccccgagct 840 gcagggacag tgagcagtga gtcccttggg catcccgctc ctgggcaggt caccaatagg 900 tccccgcagt tcccaatgga actgttccag tcctccccga ggcctccact tcaacctgtc 960 tgtgtctgcc caggcctgga gttgtgtgac cctccccacc gcctggcctt ctccatgggg 1020 gctggccttt tctcggtggt gggcaccctg ctgctgcccg gcctggctgc gcttgtgcag 1080 gactggcgtc ttctgcaggg gctgggcgcc ctgatgagtg gactcttgct gctcttttgg 1140 gggtaagtgt ggtgggagct gggccagcag ccctcatcgg ggctgactat gtagcttccc 1200 tctgcaccga cgtatcccat gccaggtaaa cgcatgggtt acagggtcac agagaactac 1260 agtgatgtcc tgcccctgag cctgggggtg gggtgggggc cctggccttt gcctctcctt 1320 ccaggaggag gtggagggag ccgtgggcat cctcaccaac gctgcaggtt cccggccctg 1380 ttccccgagt ctccctgctg gctgctggcc acaggtcagg tagctcgagc caggaagatc 1440 ctgtggcgct ttgcagaagc cagtggcgtg ggccccgggg acagtccctt ggaggagaac 1500 tccctggcta cagagctgac catgctgtct gcacggagcc cccagccccg gtaccactcc 1560 ccactggggc ttctgcgtac ccgagtcacc tggagaaacg ggcttatctt gggcttcagc 1620 tcgctggttg gtggaggcat cagagctagc ttccgccgca gcctggcacc tcaggtgccg 1680 accttctacc tgccctactt cctggaggcc ggcctggagg cggcagcctt ggtcttcctg 1740 ctcctgacgg cagattgctg tggacgccgc cccgtgctgc tgctgggcac catggtcaca 1800 ggcctggcat ccctgctgct cctcgctggg gcccagtatc tgccaggctg gactgtgctg 1860 ttcctctctg tcctggggct cctggcctcc cgggctgtgt ccgcactcag cagcctcttc 1920 gcggccgagg tcttccccac ggtgatcagg ggggccgggc tgggcctggt gctgggggcc 1980 gggttcctgg gccaggcagc cggccccctg gacaccctgc acggccggca gggcttcttc 2040 ctgcaacaag tcgtcttcgc ctcccttgct gtccttgccc tgctgtgtgt cctgctgctg 2100 cctgagagcc gaagccgggg gctgccccag tcactgcagg acgccgaccg cctgcgccgc 2160 tccccactcc tgcggggccg cccccgccag gaccacctgc ctctgctgcc gccctccaac 2220 tcctactggg ccggccacac ccccgagcag cactagtcct gcctggtggc cctgggagcc 2280 aggatgggac caaagtcaag gcctggggca tggctgagta ccccagacgt ctggtccagg 2340 gcagacacat tcctctcaga agcccgtgtc tcagtgcagg tggagccgtg gggacagcgt 2400 gaaggtgtct ccagccaggc cccaggcact gggaggccct gggtctcccc ccagccacac 2460 ccagtaggtg tggaggataa aggcttctgt ggaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2520 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2580 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa acaaaacaaa caacaaaaac aaaaaacaaa 2640 aaaaacacac aaaaaaaaaa aaagaaaaga agaagatcac agcgacgata taagcgtgaa 2700 tcacagagag agacaggggg acagagcgtg aactgcacac cgcagcgatg gtacgcgcag 2760 ccagctgagc gagggcgacc gtggcgcgca gcccagccga ccagcgcggt acgcgagaga 2820 gagagacgag ctagactgcc accgaaacgc agccaacaac aagagggaac acatagaagt 2880 aggtgataca gcgcacacag acaaggacga agaagaagac agcagggagg tagagcaata 2940 aacaaaaaca taaaagaaac gaaaaaaata acaaaaaaaa aaaaaaacta taaactaata 3000 caatagataa aacacattaa caatctaacc aataaaaaat aaagcgccat aaaacatatt 3060 aagcaatata taagataaaa tcgcacacaa ttaactaatc cctacatact acagaatcca 3.120 acaaacatat aaacaataca tatat 3145 <210> 29 <211> 763 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1700438CB1 <400> 29 ttctcgctct gtcaccaggc tggagtgcag tggcgcgatc tcgactcact gcaagctccg 60 cctcccggat tcacgccatt gtcccgcctc agcctctcga gtagctggga atacaggcgc 120 ccaccaccat gcccggctga tgttttgtat ttttagtaga gacggggttt caccgtgtta 180 gccaggatgg cctcgatctc ctgacctcgt gatccgtctg cctcggcctc ccaaagtgct 240 gggattatag gcatgagcca ctgcgcccgg cctccttttc acctttaaaa catgcaaagg 300 ctgctctgca tgtgccatga gggatgggaa gcctactgca ggcagatggt cttcttagct 360 ggcttgtgct tggtcttcct ctacatgaca gttctggggt ctggcggcat catcactggc 420 tatgcctgta cccagggggt tggagactcc ctgcttagca tcctcacggc cctttcagct 480 ctctctggcc tgatgggcac cgttctcttc acccagctta gggggcacta tggcttggtc 540 acaactggag tcatatctag ccagctccat ttaggctgtc taatgctctg catgttttct 600 gtcttggctc ctggaaattc ttttgatctg gctgttttct cacttccatt aagtaaaaat 660 ccttcaaact atgagttatt ggtccagtgg atggaagaac agtccagagg aatggcttgg 720 ttcaggtttc tttcaaaggg ataaacatgg gtctggttct ctt 763 <210> 30 <211> 2720 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 535939CB1 <400> 30 tgtgaattat aggcagctgt gatgtcgcgt gcgcgcgttc gttagctcgg tcgttcggtg 60 cgagggaaag atgcggcgaa tcaggaaccc cagagcgcgg tggctagacc gggctccgcc 120 gcctccccca cagccccttt cctaatcgtt cagacggagc ctggtcgact tcgccggaga 180 ctgccagatc tcgttcctct tccctgtgtc atcttcttaa ttataaataa tgggggatga 240 agataaaaga attacatatg aagattcaga accatccaca ggaatgaatt acacgccctc 300 catgcatcaa gaagcacagg aggagacagt tatgaagctc aaaggtatag atgcaaatga 360 accaacagaa ggaagtattc ttttgaaaag cagtgaaaaa aagctacaag aaacaccaac 420 tgaagcaaat cacgtacaaa gactgagaca aatgctggct tgccctccac atggtttact 480 ggacagggtc ataacaaatg ttaccatcat tgttcttctg tgggctgtag tttggtcaat 540 tactggcagt gaatgtcttc ctggaggaaa cctatttgga attataatcc tattctattg 600 tgccatcatt ggtggtaaac ttttggggct tattaagtta cctacattgc ctccactgcc 660 ttctcttctt ggcatgctgc ttgcagggtt tctcatcaga aatatcccag tcatcaacga 720 taatgtgcag atcaagcaca agtggtcttc ctctttgaga agcatagccc tgtctatcat 780 tctggttcgt gctggccttg gtctggattc aaaggccctg aagaagttaa agggcgtttg 840 tgtaagactg tccatgggtc cctgtattgt ggaggcgtgc acatctgctc ttcttgccca 900 ttacctgctg ggtttaccat ggcaatgggg atttatactg ggttttgttt taggtgctgt 960 atctccagct gttgtggtgc cttcaatgct ccttttgcag ggaggaggct atggtgttga 1020 gaagggtgtc ccaaccttgc tcatggcagc tggcagcttc gatgacattc tggccatcac 1080 tggcttcaac acatgcttgg gcatagcctt ttccacaggc tctactgtct ttaatgtcct 1140 cagaggagtt ttggaggtgg taattggtgt ggcaactgga tctgttcttg gatttttcat 1200 tcagtacttt ccaagccgtg accaggacaa acttgtgtgt aagagaacat tccttgtgtt 1260 ggggttgtct gtgctagctg tgttcagcag tgtgcatttt ggtttccctg gatcaggagg 1320 actgtgcacg ttggtcatgg ctttccttgc aggcatggga tggaccagcg aaaaggcaga 1380 ggttgaaaag ataattgcag ttgcctggga catttttcag ccccttcttt ttggactaat 1440 tggagcagag gtatctattg catctctcag accagaaact gtaggccttt gtgttgccac 1500 cgtaggcatt gcagtattga tacgaatttt gactacattt ctgatggtgt gttttgctgg 1560 ttttaactta aaagaaaaga tatttatttc ttttgcatgg cttccaaagg ccacagttca 1620 ggctgcaata ggatctgtgg ctttggacac agcaaggtca catggagaga aacaattaga 1680 agactatgga atggatgtgt tgacagtggc atttttgtcc atcctcatca cagccccaat 1740 tggaagtctg cttattggtt tactgggccc caggcttctg cagaaagttg aacatcaaaa 1800 taaagatgaa gaagttcaag gagagacttc tgtgcaagtt tagaggtgaa aagagagagt 1860 gctgaacata atgtttagaa agctgctact tttttcaaga tgcatattga aatatgtaat 1920 gtttaagctt aaaatgtaat agaaccaaaa gtgtagctgt ttctttaaac agcattttta 1980 gcccttgctc tttccatgtg ggtggtaatg atctatatca ccaaccttaa tctctctgcc 2040 ttttttttca aacacccctt catcatccat cttaatttgc ataaggacat atctacttta 2100 atgtactacc acagtttaca gttaatgtgg gaaagaccag cttcagtatc ctcttcagct 2160 aggattgccc taacttttaa ctttcacagt ttcctgattc atatttgccc aggctctgat 2220 gccttgaatt ggttttggct ctcttttttg gatctgtttt tgttgttaaa catcataatg 2280 cagtctctca ttaattttta ccatcattta ccctgataat ctgcctcttc tccatttctc 2340 cttcccttac tacctttctt tgaattactg taactgattg gtcccaccaa aattttaaag 2400 tacatgaagt atcttcattg gttcatcctc ttgccccctc cagatgtcaa aaaactttat 2460 cctgccccct agctgaccac ccaggttcct ttatttcagt ggcccatgtg agtctacctt 2520 tctttggcta t cccctaagga gtgccctaat ccagcccttt ttttgtttct tatgacccat atctttaggc 2580 tcttcccatt tctaggtggg agataggtaa gtttcaaatc tatgccagtc ttatgaatat 2640 tacattaggg taatgtgcta taatgaagaa ataaaaaata cagtgcttaa aagaaaataa 2700 aattctattt ctgtctaatg 2720 <210> 31 <211> 4464 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 55118067CB1 <400> 31 ggcagctgag ttgggctgag gtgtccctag ctggctctgc ggctcttccg ggtctgggct 60 cggagattca caggcggccc gcgaggccga gcgagggacg catggccctg aggcggccgc 120 agggcttggc ggggtccgga ggttgacctc gcccccgcag ccggccttcg aggctgcctc 180 ctccaggcag cctctggggc ccgcgcccgc gcctgctcag gctcccgtgt tcaggctgcc 240 catcccctcc ccaccggcgt cccggacgtt gggacctgtg accgtggcct cgggctgggc 300 ttccaaagcc ggccgcagcc cggcgacccc cgaggcctct cgccccgggc ccctagacct 360 ctcactatga ccgcggccgc cgcctccaac tgggggctga tcacgaacat cgtgaacagc 420 atcgtagggg tcagtgtcct caccatgccc ttctgcttca aacagtgcgg catcgtcctg 480 ggggcgctgc tcttggtctt ctgctcatgg atgacgcacc agtcgtgcat gttcttggtg 540 aagtcggcca gcctgagcaa gcggaggacc tacgccggcc tggcattcca cgcctacggg 600 aaggcaggca agatgctggt ggagaccagc atgatcgggc tgatgctggg cacctgcatc 660 gccttctacg tcgtgatcgg cgacttgggg tccaacttct ttgcccggct gttcgggttt 720 caggtgggcg gcaccttccg catgttcctg ctgttcgccg tgtcgctgtg catcgtgctc 780 ccgctcagcc tgcagcggaa catgatggcc tccatccagt ccttcagcgc catggccctc 840 ctcttctaca ccgtgttcat gttcgtgatc gtgctctcct ctctcaagca cggcctcttc 900 agtgggcagt ggctgcggcg ggtcagctac gtccgctggg agggcgtctt ccgctgcatc 960 cccatcttcg gcatgtcctt cgcctgccag tcccaggtgc tgcccaccta cgacagcctg 1020 gatgagccgt cagtgaaaac catgagctcc atatttgctt cctcccttaa tgtggtcacc 1080 accttctacg tcatggtggg gtttttcggc tacgtcagct tcaccgaggc cacggccggc 1140 aacgtgctca tgcactttcc ctccaacctg gtgacggaga tgctccgtgt gggcttcatg 1200 atgtcagtgg ctgtgggctt ccccatgatg atcctgccat gcaggcaggc cctgagcacg 1260 ctgctgtgtg agcagcagca aaaagatggc acctttgcag cagggggcta catgccccct 1320 ctccggttta aagcacttac cctctctgtg gtgtttggaa ccatggttgg tggcatcctt 1380 atccccaacg tggagaccat cctgggcctc acaggagcga ccatgggaag cctcatctgc 1440 ttcatctgcc cggcgctgat ctacaagaaa atccacaaga acgcactttc ctcccaggtg 1500 gtgctgtggg tcggcctggg cgtcctggtg gtgagcactg tcaccacact gtctgtgagc 1560 gaggaggtcc ccgaggactt ggcagaggaa gcccctggcg gccggcttgg agaggccgag 1620 ggtttgatga aggtggaggc agcgcggctc tcagcccagg atccggttgt ggccgtggct 1680 gaggatggcc gggagaagcc gaagctgccg aaggagagag aggagctgga gcaggcccag 1740 atcaaggggc ccgtggatgt gcctggacgg gaagatggca aggaggcacc ggaggaggca 1800 cagctcgatc gccctgggca agggattgct gtgcctgtgg gcgaggccca ccgccacgag 1860 cctcctgttc ctcacgacaa ggtggtggta gatgaaggcc aagaccgaga ggtgccagaa 1920 gagaacaaac ctccatccag acacgcgggc ggaaaggctc caggggtcca gggccagatg 1980 gcgccgcctc tgcccgactc agaaagagag aaacaagagc cggagcaggg agaggttggg 2040 aagaggcctg gacaggccca ggccttggag gaggcgggtg atcttcctga agatccccag 2100 aaagttccag aagcagatgg tcagccagct gtccagcctg caaaggagga cctggggcca 2160 ggagacaggg gcctgcatcc tcggccccag gcagtgctgt ctgagcagca gaacggcctg 2220 gcggtgggtg gaggggaaaa ggccaagggg ggaccgccgc caggcaacgc cgccggggac 2280 acagggcagc ccgcagagga cagcgaccac ggtgggaagc ctcccctccc agcggagaag 2340 ccggctccag ggcctgggct gccgcccgag cctcgcgagc agagggacgt ggagcgagcg 2400 ggtggaaacc aggcggccag ccagctggag gaagctggca gggcggagat gctggaccac 2460 gccgtcctgc ttcaggtgat caaagaacag caggtgcagc aaaagcgctt gctggaccag 2520 caggagaagc tgctggcggt gatcgaggag cagcacaagg agatccacca gcagaggcag 2580 gaggacgagg aggataaacc caggcaggtg gaggtgcatc aagagcccgg ggcagcggtg 2640 cccagaggcc aggaggcccc tgaaggcaag gccagggaga cggtggagaa tctgcctccc 2700 ctgcctttgg accctgtcct cagagctcct gggggccgcc ctgctccatc ccaggacctt 2760 aaccagcgct ccctggagca ctctgagggg cctgtgggca gagaccctgc tggccctcct 2820 gacggcggcc ctgacacaga gcctcgggca gcccaggcca agctgagaga tggccagaag 2880 gatgccgccc ccagggcagc tggcactgtg aaggagctcc ccaagggccc ggagcaggtg 2940 cccgtgccag accccgccag ggaagccggg ggcccagagg agcgcctcgc agaggaattc 3000 cctgggcaaa gtcaggacgt tactggcggt tcccaagaca ggaaaaaacc tgggaaggag 3060 gtggcagcca ctggcaccag cattctgaag gaagccaact ggctcgtggc agggccagga 3120 gcagagacgg gggaccctcg catgaagccc aagcaagtga gccgagacct gggccttgca 3180 gcggacctgc ccggtggggc ggaaggagca gctgcacagc cccaggctgt gttacgccag 3240 ccggaactgc gggtcatctc tgatggcgag cagggtggac agcagggcca ccggctggac 3300 catggcggtc acctggagat gagaaaggcc cgcggggggg accatgtgcc tgtgtcccac 3360 gagcagccga gaggcgggga ggacgctgct gtccaggagc ccaggcagag gccagagcca 3420 gagctggggc tcaaacgagc tgtcccgggg ggccagaggc cggacaatgc caagcccaac 3480 cgggacctga aactgcaggc tggctccgac ctccggaggc gacggcggga ccttggccct 3540 catgcagagg gtcagctggc cccgagggat ggggtcatca ttggccttaa ccccctgcct 3600 gatgtccagg tgaacgacct ccgtggcgcc ctggatgccc agctccgcca ggctgcgggg 3660 ggagctctgc aggtggtcca cagccggcag cttagacagg cgcctgggcc tccagaggag 3720 tcctagcacc tgctggccat gagggccacg ccagccactg ccctcctcgg ccagcagcag 3780 gtctgtctca gccgcatccc agccaaactc tggaggtcac actcgcctct ccccagggtt 3840 tcatgtctga ggccctcacc aagtgtgagt gacagtataa aagattcact gtggcatcgt 3900 ttccagaatg ttcttgctgt cgttctgttg cagctcttag tctgaggtcc tctgacctct 3960 agactctgag ctcactccag cctgtgagga gaaacggcct ccgctgcgag ctggctggtg 4020 cactcccagg ctcaggctgg ggagctgctg cgtctgtggt caggcctcct gctcctgcca 4080 gggagcacgc gtggtcttcg ggttgagctc ggccgtgcgt ggaggtgcgc atggctgctc 4140 atggtcccaa cacaggctac tgtgagagcc agcatccaac cccacgcttg cagtgactca 4200 gaatgataat tattatgact gtttatcgat gcttcccaca gtgtggtaga aagtcttgaa 4260 taaacacttt tgccttcacc cagcctcggt ggatgctgtt tggtgtccag gaagcacagg 4320 gagcaggggc cagacaagcc gggtgtccag gggcactggc cggtgccgcc ctttgcactc 4380 ctcatggttg gcccagccct ccattgtctg tgtttttcaa aatccaattg tggctttttt 4440 taaaaagtaa aaaacaccac tgtg 4464 <210> 32 <211> 3135 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7502087CB1 <220>
<221> unsure <222> 2961 <223> a, t, c, g, or other <400> 32 ggcggagacg ccgggagcca gtggcgcctg tggctccggg caggggccgc ggccgaaaga 60 tgccggtccg caggggccac gtcgctcccc aaaacactta cctggacacc atcatccgca 120 agttcgaggg ccaaagtcgg aagttcctga ttgccaatgc tcagatggag aactgcgcca 180 tcatttactg caacgacggc ttctgcgaac tcttcggcta ctcccgagtg gaggtgatgc 240 agcaaccctg cacctgcgac ttcctcacag gccccaacac accaagcagc gccgtgtccc 300 gcctagcgca ggccctgctg ggggctgagg agtgcaaggt ggacatcctc tactaccgca 360 aggatgcctc cagcttccgc tgcctggtag atgtggtgcc cgtgaagaac gaggacgggg 420 ctgtcatcat gttcattctc aacttcgagg acctggccca gctcctggcc aagtgcagca 480 gccgcagctt gtcccagcgc ctgttgtccc agagcttcct gggctccgag ggctctcatg 540 gcaggccagg cggaccaggg ccaggcacag gcaggggcaa gtacaggacc atcagccaga 600 tcccacagtt cacgctcaac ttcgtggagt tcaacttgga gaagcaccgc tccagctcca 660 ccacggagat tgagatcatc gcgccccata aggtggtgga gcggacacag aacgtcactg 720 agaaggtcac ccaggtcctg tccctgggcg cggatgtgct gccggagtac aagctgcagg 780 cgccgcgcat ccaccgctgg accatcctgc actacagccc cttcaaggcc gtgtgggact 840 ggctcatcct gctgctggtc atctacacgg ctgtcttcac gccctactca gccgccttcc 900 tgctcagcga ccaggacgaa tcacggcgtg gggcctgcag ctatacctgc agtcccctca 960 ctgtggtgga tctcatcgtg gacatcatgt tcgtcgtgga catcgtcatc aacttccgca 1020 ccacctatgt caacaccaat gatgaggtgg tcagccaccc ccgccgcatc gccgtccact 1080 acttcaaggg ctggttcctc attgacatgg tggccgccat ccctttcgac ctcctgatct 1140 tccgcactgg ctccgatgag accacaaccc tgattgggct attgaagaca gcgcggctgc 1200 tgcggctggt gcgcgtagca cggaagctgg actgctactc tgagtatggg gcggctgtgc 1260 tcttcttgct catgtgcacc ttcgcgctca tagcgcactg gctggcctgc atctggtacg 1320 ccatcggcaa tgtggagcgg ccctacctag aacacaagat cggctggctg gacagcctgg 1380 gtgtgcagct tggcaagcgc tacaacggca gcgacccagc ctcgggcccc tcggtgcagg 1440 acaagtatgt cacagccctc tacttcacct tcagcagcct caccagcgtg ggcttcggca 1500 atgtctcgcc caacaccaac tccgagaagg tcttctccat ctgcgtcatg ctcatcggct 1560 ccctgatgta cgccagcatc ttcgggaacg tgtccgcgat catccagcgc ctgtactcgg 1620 gcaccgcgcg ctaccacacg cagatgctgc gtgtcaagga gttcatccgc ttccaccaga 1680 tccccaaccc actgcgccag cgcctggagg agtatttcca gcacgcctgg tcctacacca 1740 atggcattga catgaacgcg gtgctgaagg gcttccccga gtgcctgcag gctgacatct 1800 gcctgcacct gcaccgcgca ctgctgcagc actgcccagc tttcagcggc gccggcaagg 1860 gctgcctgcg cgcgctagcc gtcaagttca agaccaccca cgcgccgcct ggggacacgc 1920 tggtgcacct cggcgacgtg ctctccaccc tctacttcat ctcccgaggc tccatcgaga 1980 tcctgcgcga cgacgtggtc gtggccatcc taggaaagaa tgacatcttt ggggaacccg 2040 tcagcctcca tgcccagcca ggcaagtcca gtgcagacgt gcgggctctg acctactgcg 2100 acctgcacaa gatccagcgg gcagatctgc tggaggtgct ggacatgtac ccggcctttg 2160 cggagagctt ctggagtaag ctggaggtca ccttcaacct gcgggacgct cctggcagcc 2220 aagaccacca aggtttcttt ctcagtgaca accagtcaga tgcagcccct cccctgagca 2280 tctcagatgc atctggcctc tggcctgagc tactgcagga aatgccccca aggcacagcc 2340 cccaaagccc tcaggaagac ccagattgct ggcctctgaa gctgggctcc aggctagagc 2400 agctccaggc ccagatgaac aggctggagt cccgcgtgtc ctcagacctc agccgcatct 2460 tgcagctcct ccagaagccc atgccccagg gccacgccag ctacattctg gaagcccctg 2520 cctccaatga cctggccttg gttcctatag cctcggagac gacgagtcca gggcccaggc 2580 tgccccaggg ctttctgcct cctgcacaga ccccaagcta tggagacttg gatgactgta 2640 gtccaaagca caggaactcc tcccccagga tgcctcacct ggctgtggca atggacaaaa 2700 ctctggcacc atcctcagaa caggaacagc ctgaggggct ctggccaccc ctagcctcac 2760 ctctacatcc cctggaagta caaggactca tctgtggtcc ctgcttctcc tccctccctg 2820 aacaccttgg ctctgttccc aagcagctgg acttccagag acatggctca gatcctggat 2880 ttgcagggag ttggggccac tgaactccaa gataaagaca ccatgagggg actgaaggtg 2940 ggcaagggga tttcctttag nctgggcatg gtggcgggcg cctgtaatcc cagctactca 3000 ggaggctgaa gcaagagaat cacttgaacc ctaaaggcag aggttgcagt gagccgagat 3060 agtgccactg cactacagcc cgggcgacag agtgagactc catctcaaaa ataaaataca 3120 attaacaaaa aaaga 3135 <210> 33 <211> 843 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7500819CB1 <400> 33 ctcctcggga ctcggcgggt cctcctggga gtctcggagg ggaccggctg tgcagacgcc 60 atggagttgg tgctggtctt cctctgcagc ctgctggccc ccatggtcct ggccagtgca 120 gctgaaaagg agaaggaaat ggaccctttt cattatgatt accagaccct gaggattggg 180 ggactggtgt tcgctgtggt cctcttctcg gttgggatcc tccttatcct aagtcgcagg 240 tgcaagtgca gcttctactc tgcccctggg gaatgtgtcc cctgcatatc ttctcagcaa 300 taactccatg ggctctggga ccctacccct tccaaccttc cctgcttctg agacttcaat 360 ctacagccca gctcatccag atgcagacta cagtccctgc aattgggtct ctggcaggca 420 atagttgaag gactcctgtt ccgttggggc cagcacaccg ggatggatgg agggagagca 480 gaggcctttg cttctctgcc tacgtcccct tagatgggca gcagaggcaa ctcccgcatc 540 ctttgctctg cctgtcagtg gtcagagcgg tgagcgaggt gggttggaga ctcagcaggc 600 tccgtgcagc ccttgggaac agtgagaggt tgaaggtcat aacgagagtg ggaactcaac 660 ccagatcccg cccctcctgt cctctgtgtt cccgcggaaa ccaaccaaac cgtgcgctgt 720 gacccattgc tgttctctgt atcgtgatct atcctcaaca acaacagaaa aaaggaataa 780 aatatccttt gtttcctaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 840 aaa 843 <210> 34 <211> 3159 <212> DNA
<213> Homo sapiens <220>
<22~> misc_feature <223> Incyte ID No: 7503413CB1 <400> 34 gcgcgatggc ctcggcgctg agctatgtct ccaagttcaa gtccttcgtg atcttgttcg 60 tcaccccgct cctgctgctg ccactcgtca ttctgatgcc cgccaagttt gtcaggtgtg 120 cctacgtcat catcctcatg gccatttact ggtgcacaga agtcatccct ctggctgtca 180 cctctctcat gcctgtcttg cttttcccac tcttccagat tctggactcc aggcaggtgt 240 gtgtccagta catgaaggac accaacatgc tgttcctggg cggcctcatc gtggccgtgg 300 ctgtggagcg ctggaacctg cacaagagga tcgccctgcg cacgctcctc tgggtggggg 360 ccaagcctgc acggctgatg ctgggcttca tgggcgtcac agccctcctg tccatgtgga 420 tcagtaacac ggcaaccacg gccatgatgg tgcccatcgt ggaggccata ttgcagcaga 480 tggaagccac aagcgcagcc accgaggccg gcctggagct ggtggacaag ggcaaggcca 540 aggagctgcc agggagtcaa gtgatttttg aaggccccac tctggggcag caggaagacc 600 aagagcggaa gaggttgtgt aaggccatga ccctgtgcat ctgctacgcg gccagcatcg 660 ggggcaccgc caccctgacc gggacgggac ccaacgtggt gctcctgggc cagatgaacg 720 agttgtttcc tgacagcaag gacctcgtga actttgcttc ctggtttgca tttgcctttc 780 ccaacatgct ggtgatgctg ctgttcgcct ggctgtggct ccagtttgtt tacatgagat 840 tcaattttaa aaagtcctgg ggctgcgggc tagagagcaa gaaaaacgag aaggctgccc 900 tcaaggtgct gcaggaggag taccggaagc tggggccctt gtccttcgcg gagatcaacg 960 tgctgatctg cttcttcctg ctggtcatcc tgtggttctc ccgagacccc ggcttcatgc 1020 ccggctggct gactgttgcc tgggtggagg aaaggaaaac tccattttat ccccctcccc 1080 tgctggattg gaaggtaacc caggagaaag tgccctgggg catcgtgctg ctactagggg 1140 gcggatttgc tctggctaaa ggatccgagg cctcggggct gtccgtgtgg atggggaagc 1200 agatggagcc cttgcacgca gtgcccccgg cagccatcac cttgatcttg tccttgctcg 1260 ttgccgtgtt cactgagtgc acaagcaacg tggccaccac caccttgttc ctgcccatct 1320 ttgcctccat gtctcgctcc atcggcctca atccgctgta catcatgctg ccctgtaccc 1380 tgagtgcctc ctttgccttc atgttgcctg tggccacccc tccaaatgcc atcgtgttca 1440 cctatgggca cctcaaggtt gctgacatgg tgaaaacagg agtcataatg aacataattg 1500 gagtcttctg tgtgtttttg gctgtcaaca cctggggacg ggccatattt gacttggatc 1560 atttccctga ctgggctaat gtgacacata ttgagactta ggaagagcca caagaccaca 1620 cacacagccc ttaccctcct caggactacc gaaccttctg gcacaccttg tacagagttt 1680 tggggttcac accccaaaat gacccaacga tgtccacaca ccaccaaaac ccagccaatg 1740 ggccacctct tcctccaagc ccagatgcag agatggtcat gggcagctgg agggtaggct 1800 cagaaatgaa gggaacccct cagtgggctg ctggacccat ctttcccaag ccttgccatt 1860 atctctgtga gggaggccag gtagccgagg gatcaggatg caggctgctg tacccgctct 1920 gcctcaagca tcccccacac agggctctgg ttttcactcg cttcgtccta gatagtttaa 1980 atgggaatca gatcccctgg ttgagagcta agacaaccac ctaccagtgc ccatgtccct 2040 tccagctcac cttgagcagc ctcagatcat ctctgtcact ctggaaggga caccccagcc 2100 agggacggaa tgcctggtct tgagcaacct cccactgctg gagtgcgagt gggaatcaga 2160 gcctcctgaa gcctctggga actcctcctg tggccaccac caaaggatga ggaatctgag 2220 ttgccaactt caggacgaca cctggcttgc cacccacagt gcaccacagg ccaacctacg 2280 cccttcatca cttggttctg ttttaatcga ctggccccct gtcccacctc tccagtgagc 2340 ctccttcaac tccttggtcc cctgttgtct gggtcaacat ttgccgagac gccttggctg 2400 gcaccctctg gggtccccct tttctcccag gcaggtcatc ttttctggga gatgcttccc 2460 ctgccatccc caaatagcta ggatcacact ccaagtatgg gcagtgatgg cgctctgggg 2520 gccacagtgg gctatctagg ccctccctca cctgaggccc agagtggaca cagctgttaa 2580 tttccactgg ctatgccact tcagagtctt tcatgccagc gtttgagctc ctctgggtaa 2640 aatcttccct ttgttgactg gccttcacag ccatggctgg tgacaacaga ggatcgttga 2700 gattgagcag cgcttggtga tctctcagca aacaacccct gcccgtgggc caatctactt 2760 gaagttactc ggacaaagac cccaaagtgg ggcaacaact ccagagaggc tgtgggaatc 2820 ttcagaagcc cccctgtaag agacagacat gagagacaag catcttcttt cccccgcaag 2880 tccattttat ttccttcttg tgctgctctg gaagagaggc agtagcaaag agatgagctc 2940 ctggatggca ttttccaggg caggagaaag tatgagagcc tcaggaaacc ccatcaagga 3000 ccgagtatgt gtctggttcc ttgggtggga cgattcctga ccacactgtc cagctcttgc 3060 tctcattaaa tgctctgtct cccgcggaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3120 aaaaaaaaaa aaattctcgg acgcaaggaa ttcagctgg 3159 <210> 35 <211> 1883 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7500007CB1 <400> 35 ccacgcgtcc gtaaggtggg atggatagca gggtctcagg cacaaccagt aatggagaga 60 caaaaccagt gtatccagtc atggaaaaga aggaggaaga tggcaccctg gagcgggggc 120 actggaacaa caagatggag tttgtgctgt cagtggctgg ggagatcatt ggcttaggca 180 acgtctggag gtttccctat ctctgctaca aaaatggggg agaacactgt atggagttcc 240 agaagaccaa cggctccctg aatggtacct ctgagaatgc cacctctcct gtcatcgagt 300 tctgggagcg gcgggtcttg aagatctctg atgggatcca gcacctgggg gccctgcgct 360 gggagctggc tctgtgcctc ctgctggcct gggtcatctg ctacttctgc atctggaagg 420 gggtgaagtc cacaggcaag gtggtgtact tcacggccac atttccttac ctcatgctgg 480 tggtcctgtt aattcgaggg gtgacgttgc ctggggcagc ccaaggaatt cagttttacc 540 tgtacccaaa cctcacgcgt ctgtgggatc cccaggtgtg gatggatgca ggcacccaga 600 tattcttctc cttcgccatc tgtcttgggt gcctgacagc cctgggcagc tacaacaagt 660 accacaacaa ctgctacagg gactgcatcg ccctctgctt cctcaacagc ggcaccagct 720 ttgtggccgg ctttgccatc ttctccatcc tgggcttcat gtctcaggag cagggggtgc 780 ccatttctga ggtggccgag tcaggccctg gcctggcttt catcgcttac ccgcgggctg 840 tggtgatgct gcccttctct cctctctggg cctgctgttt cttcttcatg gtcgttctcc 900 tgggactgga tagccagttt gtgtgtgtag aaagcctggt gacagcgctg gtggacatgt 960 accctcacgt gttccgcaag aagaaccgga gggaagtcct catccttgga gtatctgtcg 1020 tctccttcct tgtggggctg atcatgctca cagagggcgg aatgtacgtg ttccagctct 1080 ttgactacta tgcggccagt ggcatgtgcc tcctgttcgt ggccatcttc gagtccctct 1140 gtgtggcttg ggtttacgga gccaagcgct tctacgacaa catcgaagac atgattgggt 1200 acaggccatg gcctcttatc aaatactgtt ggctcttcct cacaccagct gtgtgcacag 1260 ccacctttct cttctccctg ataaagtaca ctccgctgac ctacaacaag aagtacacgt 1320 acccgtggtg gggcgatgcc ctgggctggc tcctggctct gtcctccatg gtctgcattc 1380 ctgcctggag cctctacaga ctcggaaccc tcaagggccc cttcagagag agaatccgtc 1440 agctcatgtg cccagccgag gacctgcccc agcggaaccc agcaggaccc tcggctcccg 1500 ccacccccag gacctcactg ctcagactca cagagctaga gtctcactgc tagggggcag 1560 gcccttggat ggtgcctgtg tgcctggcct tggggatggc tgtggaggga acgtggcaga 1620 agcagcccca tgtgcttccc tgcccccgac ctggagtgga taagacaaga ggggtatttt 1680 ggagtccacc tgctgagctg gaggcctccc actgcaactt ttcagctcag gggttgttga 1740 acagatgtga aaggccagtg ccaagagtgt ccctctgaga cccttgggaa gctgggtggg 1800 ggctggtagg tggggcgaga cttgctggct tcgggccctc tcatccttca ttccattaaa 1860 tccacattct tcccgctgaa aaa 1883 <210> 36 <211> 2746 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7500025CB1 <400> 36 cgcctgtggc tccgggcagg ggccgcggcc gaaagatgtc ggtccgcagg ggccacgtcg 60 ctccccaaaa cacttacctg gacaccatca tccgcaagtt cgagggccaa agtcggaagt 120 tcctgattgc caatgctcag atggagaact gcgccatcat ttactgcaac gacggcttct 180 gcgaactctt cggctactcc cgagtggagg tgatgcagca accctgcacc tgcgacttcc 240 tcacaggccc caacacacca agcagcgccg tgtcccgcct agcgcaggcc ctgctggggg 300 ctgaggagtg caaggtggac atcctctact accgcaagga tgcctccagc ttccgctgcc 360 tggtagatgt ggtgcccgtg aagaacgagg acggggctgt catcatgtcc attctcaact 420 tcgaggacct ggcccagctc ctggccaagt gcagcagccg cagcttgtcc cagcgcctgt 480 tgtcccagag cttcctgggc tccgagggct ctcatggcag gccaggcgga ccaggcccag 540 gcacaggcag gggcaagtac aggaccatca gccagatccc acagttcacg ctcaacttcg 600 tggagttcaa cttggagaag caccgctcca gctccaccac ggagattgag atcatcgcgc 660 cccataaggt ggtggagcgg acacagaacg tcactgagaa ggtcacccag gtcctgtccc 720 tgggcgcaga tgtgctgccg gagtacaagc tgcaggcgcc gcgcatccac cgctggacca 780 tcctgcacta cagccccttc aaggccgtgt gggactggct catcccgctg ctggtcatct 840 acacggctgt cttcacgccc tactcagccg ccttcctgct cagcgatcag gacgaatcac 900 ggcgtggggc ctgcagctat acctgcagtc ccctcactgt ggtggatctc atcgtggaca 960 tcatgttcgt cgtggacatc gtcatcaact tccgcaccac ctatgtcaac accaatgatg 1020 aggtggtcag ccacccccgc cgcatcgccg tccactactt caagggctgg ttcctcattg 1080 acatggttJgc cgccatccct ttcgacctcc tgatcttccg cactggctcc gatgagacca 1140 caaccctgat tgggctattg aagacagcgc ggctgctgcg gctggtgcgc gtagcacgga 1200 agctggaccg ctactctgag tatggggcgg ctgtgctctt cttgctcatg tgcaccttcg 1260 cgctcatagc gcactggctg gcctgcatct gcagcctcac cagcgtgggc ttcggcaatg 1320 tctcgcccaa caccaactcc gagaaggtct tctccatctg cgtcatgctc atcggctccc 1380 tgatgtacgc cagcatcttc gggaacgtgt ccgcgatcat ccagcgcctg tactcgggca 1440 ccgcgcgcta ccacacgcag atgctgcgtg tcaaggagtt catccgcttc caccagatcc 1500 ccaacccact gcgccagcgc ctggaggagt atttccagca cgcctggtcc tacaccaatg 1560 gcattgacat gaacgcggtg ctgaagggct tccccgagtg cctgcaggct gacatctgcc 1620 tgcacctgca ccgcgcactg ctgcagcact gcccagcttt cagcggcgcc ggcaagggct 1680 gcctgcgcgc gctagccgtc aagttcaaga ccacccacgc gccgcctggg gacacgctgg 1740 tgcacctcgg cgacgtgctc tccaccctct acttcatctc ccgaggctcc atcgagatcc 1800 tgcgcgacga cgtggtcgtg gccatcctag gaaagaatga catctttggg gaacccgtca 1860 gcctccatgc ccagccaggc aagtccagtg cagacgtgcg ggctctgacc tactgcgacc 1920 tgcacaagat ccagcgggca gatctgctgg aggtgctgga catgtacccg gcctttgcgg 1980 agagcttctg gagtaagctg gaggtcacct tcaacctgcg ggacgctcct ggcagccaag 2040 accaccaagg tttctttctc agtgacaacc agtcagatgc agcccctccc ctgagcatct 2100 cagatgcatc tggcctctgg cctgagctac tgcaggaaat gcccccaagg cacagccccc 2160 aaagccctca ggaagaccca gattgctggc ctctgaagct gggctccagg ctagagcagc 2220 tccaggccca gatgaacagg ctggagtccc gcgtgtcctc agacctcagc cgcatcttgc 2280 agctcctcca gaagcccatg ccccagggcc acgccagcta cattctggaa gcccctgcct 2340 ccaatgacct ggccttggtt cctatagcct cggagacgac gagtccaggg cccaggctgc 2400 cccagggctt tctgcctcct gcacagaccc caagctatgg agacttggat gactgtagtc 2460 caaagcacag gaactcctcc cccaggatgc ctcacctggc tgtggcaatg gacaaaactc 2520 tggcaccatc ctcagaacag gaacagcctg aggggctctg gccaccccta gcctcacctc 2580 tacatcccct ggaagtacaa ggactcatct gtggtccctg cttctcctcc ctccctgaac 2640 accttggctc tgttcccaag cagctggact tccagagaca tggctcagat cctggatttg 2700 cagggagttg gggccactga actccaagat aaagacacca tgaggg 2746 <210> 37 <211> 2868 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7502736CB1 <400> 37 caaaacactt acctggacac catcatccgc aagttcgagg gccaaagtcg gaagttcctg 60 attgccaatg ctcagatgga gaactgcgcc atcatttact gcaacgacgg cttctgcgaa'120 ctcttcggct actcccgagt ggagcctcca gcttccgctg cctggtagat gtggtgcccg 180 tgaagaacga ggacggggct gtcatcatgt tcattctcaa cttcgaggac ctggcccagc 240 tcctggccaa gtgcagcagc cgcagcttgt cccagcgcct gttgtcccag agcttcctgg 300 gctccgaggg ctctcatggc aggccaggcg gaccagggcc aggcacaggc aggggcaagt 360 acaggaccat cagccagatc ccacagttca cgctcaactt cgtggagttc aacttggaga 420 agcaccgctc cagctccacc acggagattg agatcatcgc gccccataag gtggtggagc 480 ggacacagaa cgtcactgag aaggtcaccc aggtcctgtc cctgggcgcg gatgtgctgc 540 cggagtacaa gctgcaggcg ccgcgcatcc accgctggac catcctgcac tacagccccc 600 ttcaaggccg tgtgggactg gctcatcctg ctgctggtca tctacacggc tgtcttcacg 660 ccctactcag ccgccttcct gctcagcgat caggacgaat cacggcgtgg ggcctgcagc 720 tatacctgca gtcccctcac tgtggtggat ctcatcgtgg acatcatgtt cgtcgtggac 780 atcgtcatca acttccgcac cacctatgtc aacaccaatg atgaggtggt cagccacccc 840 cgccgcatcg ccgtccacta cttcaagggc tggttcctca ttgacatggt ggccgccatc 900 cctttcgacc tcctgatctt ccgcactggc tccgatgaga ccacaaccct gattgggcta 960 ttgaagacag cgcggctgct gcggctggtg cgcgtagcac ggaagctgga ccgctactct 1020 gagtatgggg cggctgtgct cttcttgctc atgtgcacct tcgcgctcat agcgcactgg 1080 ctggcctgca tctggtacgc catcggcaat gtggagcggc cctacctaga acacaagatc 1140 ggctggctgg acagcctggg tgtgcagctt ggcaagcgct acaacggcag cgacccagcc 1200 tcgggcccct cggtgcagga caagtatgtc acagccctct acttcacctt cagcagcctc 1260 accagcgtgg gcttcggcaa tgtctcgccc aacaccaact ccgagaaggt cttctccatc 1320 tgcgtcatgc tcatcggctc cctgatgtac gccagcatct tcgggaacgt gtccgcgatc 1380 atccagcgcc tgtactcggg caccgcgcgc taccacacgc agatgctgcg tgtcaaggag 1440 ttcatccgct tccaccagat ecccaaccca ctgcgccagc gcctggagga gtatttccag 1500 cacgcctggt cctacaccaa tggcattgac atgaacgcgg tgctgaaggg cttccccgag 1560 tgcctgcagg ctgacatctg cctgcacctg caccgcgcac tgctgcagca ctgcccagct 1620 ttcagcggcg ccggcaaggg ctgcctgcgc gcgctagccg tcaagttcaa gaccacccac 1680 gcgccgcctg gggacacgct ggtgcacctc ggcgacgtgc tctccaccct ctacttcatc 1740 tcccgaggct ccatcgagat cctgcgcgac gacgtggtcg tggccatcct aggaaagaat 1800 gacatctttg gggaacccgt cagcctccat gcccagccag gcaagtccag tgcagacgtg 1860 cgggctctga cctactgcga cctgcacaag atccagcggg cagatctgct ggaggtgctg 1920 gacatgtacc cggcctttgc ggagagcttc tggagtaagc tggaggtcac cttcaacctg 1980 cgggacgctc ctggcagcca agaccaccaa ggtttctttc tcagtgacaa ccagtcagat 2040 gcagcccctc ccctgagcat ctcagatgca tctggcctct ggcctgagct actgcaggaa 2100 atgcccccaa ggcacagccc ccaaagccct caggaagacc cagattgctg gcctctgaag 2160 ctgggctcca ggctagagca gctccaggcc cagatgaaca ggctggagtc ccgcgtgtcc 2220 tcagacctca gccgcatctt gcagctcctc cagaagccca tgccccaggg ccacgccagc 2280 tacattctgg aagcccctgc ctccaatgac ctggccttgg ttcctatagc ctcggagacg 2340 acgagtccag ggcccaggct gccccagggc tttctgcctc ctgcacagac cccaagctat 2400 ggagacttgg atgactgtag tccaaagcac aggaactcct cccccaggat gcctcacctg 2460 gctgtggcaa tggacaaaac tctggcacca tcctcagaac aggaacagcc tgaggggctc 2520 tggccacccc tagcctcacc tctacatccc ctggaagtac aaggactcat ctgtggtccc 2580 tgcttctcct ccctccctga acaccttggc tctgttccca agcagctgga cttccagaga 2640 catggctcag atcctggatt tgcagggagt tggggccact gaactccaag ataaagacac 2700 catgagggga ctgaaggtgg gcaaggggat ttcctttagc tgggcatggt ggcgggcgcc 2760 tgtaatccca gctactcagg aggctgaagc aagagaatca cttgaaccct aaaggcagag 2820 gttgcagtga gccgagatag tgccactgca ctacagcccg ggcgacag 2868 <210> 38 <211> 1906 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503570CB1 <400> 38 ccttaagggg cgggccgggg cggggctccg ctgccccttc ccagaggccg cgcctgctgc 60 tgagcagatg cagtagccga aactgcgcgg aggcacagag gccggggaga gcgttctggg 120 tccgagggtc caggtagggg ttgagccacc atctgaccgc aagctgcgtc gtgtcgccgg 180 ttctgcaggc accatgagcc aggacaccga ggtggatatg aaggaggtgg agctgaatga 240 gttagagccc gagaagcagc cgatgaacgc ggcgtctggg gcggccatgt ccctggcggg 300 agccgagaag aatggtctgg tgaagatcaa ggtggcggaa gacgaggcgg aggcggcagc 360 cgcggctaag ttcacgggcc tgtccaagga ggagctgctg aaggtggcag gcagccccgg 420 ctgggtacgc acccgctggg cactgctgct gctcttctgg ctcggctggc tcggcatgct 480 tgctggtgcc gtggtcataa tcgtgcgagc gccgcgttgt cgcgagctac cggcgcagaa 540 gtggtggcac acgggcgccc tctaccgcat cggcgacctt caggccttcc agggccacgg 600 cgcgggcaac ctggcgggtc tgaaggggcg tctcgattac ctgagctctc tgaaggtgaa 660 gggccttgtg ctgggtccaa ttcacaagaa ccagaaggat gatgtcgctc agactgactt 720 gctgcagatc gaccccaatt ttggctccaa ggaagatttt gacagtctct tgcaatcggc 780 taaaaaaaag agcatccgtg tcattctgga ccttactccc aactaccggg gtgagaactc 840 gtggttctcc actcaggttg acactgtggc caccaaggtg aaggatgctc tggagttttg 900 gctgcaagct ggcgtggatg ggttccaggt tcgggacata gagaatctga aggatgcatc 960 ctcattcttg gctgagtggc aaaatatcac caagggcttc agtgaagaca ggctcttgat 1020 tgcggggact aactcctccg accttcagca gatcctgagc ctactcgaat ccaacaaaga 1080 cttgctgttg actagctcat acctgtctga ttctggttct actggggagc atacaaaatc 1140 cctagtcaca cagtatttga atgccactgg caatcgctgg tgcagctgga gtttgtctca 1200 ggcaaggctc ctgacttcct tcttgccggc tcaacttctc cgactctacc agctgatgct 1260 cttcaccctg ccagggaccc ctgttttcag ctacggggat gagattggcc tggatgcagc 1320 tgcccttcct ggacagggcc agagtgaaga ccctggctcc ctcctttcct tgttccggcg 1380 gctgagtgac cagcggagta aggagcgctc cctactgcat ggggacttcc acgcgttctc 1440 cgctgggcct ggactcttct cctatatccg ccactgggac cagaatgagc gttttctggt 1500 agtgcttaac tttggggatg tgggcctctc ggctggactg caggcctccg acctgcctgc 1560 cagcgccagc ctgccagcca aggctgacct cctgctcagc acccagccag gccgtgagga 1620 gggctcccct cttgagctgg aacgcctgaa actggagcct cacgaagggc tgctgctccg 1680 cttcccctac gcggcctgac ttcagcctga catggaccca ctacccttct cctttccttc 1740 ccaggccctt tggcttctga tttttctctt ttttaaaaac aaacaaacaa actgttgcag 1800 attatgagtg aacccccaaa tagggtgttt tctgccttca aataaaagtc acccctgcat 1860 ggtgaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa attcgg 1906 <210> 39 <211> 2506 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7504008CB1 <400> 39 cggctctagc tcgagcagca ggagcagccc gcaccggaca acttgcgagc catggggctg 60 gcggatgcgt cgggaccgag ggacacacag gcactgctgt ctgcaacaca agcaatggac 120 ctgcggaggc gagactacca catggaacgg ccgctgctga accaggagca tttggaggag 180 ctggggcgct ggggctcagc acctaggacc caccagtggc ggacctggtt gcagtgctcc 240 cgtgctcggg cctatgccct tctgctccaa cacctcccgg ttttggtctg gttaccccgg 300 tatcctgtgc gtgactggct cctgggtgac ctgttatccg gcctgagtgt ggccatcatg 360 cagcttccgc agggcttggc ctacgccctc ctggctggat tgccccccgt gtttggcctc 420 tatagctcct tctaccctgt cttcatctac ttcctgtttg gcacttcccg gcacatctcc 480 gtggggacct ttgctgtcat gtctgtgatg gtgggcagtg tgacagaatc cctggccccg 540 caggccttga acgactccat gatcaatgag acagccagag atgctgcccg ggtacaggtg 600 gcctccacac tcagtgtcct ggttggcctc ttccaggtgg ggctgggcct gatccacttc 660 ggcttcgtgg tcacctacct gtcagaacct cttgtccgag gctataccac agctgcagct 720 gtgcaggtct tcgtctcaca gctcaagtat gtgtttggcc tccatctgag cagccactct 780 gggccactgt ccctcatcta tacagtgctg gaggtctgct ggaagctgcc ccagagcaag 840 ctcatcgggg ccacaggcat ctcctatggc atgggtctaa agcacagatt tgaggtagat 900 gtcgtgggca acatccctgc agggctggtg cccccagtgg cccccaacac ccagctgttc 960 tcaaagctcg tgggcagcgc cttcaccatc gctgtggttg ggtttgccat tgccatctca 1020 ctggggaaga tcttcgccct gaggcacggc taccgggtgg acagcaacca ggagctggtg 1080 gccctgggcc tcagtaacct tatcggaggc atcttccagt gcttccccgt gagttgctct 1140 atgtctcgga gcctggtaca ggagagcacc gggggcaact cgcaggttgc tggagccatc 1200 tcttcccttt tcatcctcct catcattgtc aaacttgggg aactcttcca tgacctgccc 1260 aaggcggtcc tggcagccat catcattgtg aacctgaagg gcatgctgag gcagctcagc 1320 gacatgcgct ccctctggaa ggccaatcgg gcggatctgc ttatctggct ggtgaccttc 1380 acggccacca tcttgctgaa cctggacctt ggcttggtgg ttgcggtcat cttctccctg 1440 ctgctcgtgg tggtccggac acagatgccc cactactctg tcctggggca ggtgccagac 1500 acggatattt acagagatgt ggcagagtac tcagaggcca aggaagtccg gggggtgaag 1560 gtcttccgct cctcggccac cgtgtacttt gccaatgctg agttctacag tgatgcgctg 1620 aagcagaggt gtggtgtgga tgtcgacttc ctcatctccc agaagaagaa actgctcaag 1680 aagcaggagc agctgaagct gaagcaactg cagaaagagg agaagcttcg gaaacaggct 1740 gcctccccca agggcgcctc agtttccatt aatgtcaaca ccagccttga agacatgagg 1800 agcaacaacg ttgaggactg caagatgatg caggtgagct caggagataa gatggaagat 1860 gcaacagcca atggtcaaga agactccaag gccccagatg ggtccacact gaaggccctg 1920 ggcctgcctc agccagactt ccacagcctc atcctggacc tgggtgccct ctcctttgtg 1980 gacactgtgt gcctcaagag cctgaagaat attttccatg acttccggga gattgaggtg 2040 gaggtgtaca tggcggcctg ccacagccct gtggtcagcc agcttgaggc tgggcacttc 2100 ttcgatgcat ccatcaccaa gaagcatctc tttgcctctg tccatgatgc tgtcaccttt 2160 gccctccaac acccgaggcc tgtccccgac agccctgttt cggtcaccag actctgaaca 2220 tgctacatcc tgcccaagac tgcacctctg gaggtgcagg gcacccttga gaagcccctc 2280 acccctaggc cgcctccagg tgctacccag gagtcccctc catgtacaca cacacaactc 2340 agggaaggag gtcctgggac tccaagttca gcgctccagg tctgggacag ggcctgcatg 2400 cagtcaggct ggcagtggcg cggtacaggg agggaactgg tgcatatttt agcctcagga 2460 ataaagattt gtctgctcaa aaaaaaaaaa aaaaaaaacc gcggtc 2506 <210> 40 <211> 3836 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503559CB1 <400> 40 ctggaccttt aatccactgt aggtatggac agggaagaaa ggaagaccat caatcagggt 60 caagaagatg aaatggagat ttatggttac aatttgagtc gctggaagct tgccatagtt 120 tctttaggag tgatttgctc tggtgggttt ctcctcctcc tcctctattg gatgcctgag 180 tggcgggtga aagcgacctg tgtcagagct gcaattaaag actgtgaagt agtgctgctg 240 aggactactg atgaattcaa aatgtggttt tgtgcaaaaa ttcgcgttct ttctttggaa 300 acttacccag tttcaagtcc aaaatctatg tctaataagc tttcaaatgg ccatgcagtt 360 tgtttaattg agaatcccac tgaagaaaat aggcacagga tcagtaaata ttcacagact 420 gaatcacaac agattcgtta tttcacccac catagtgtaa aatatttctg gaatgatacc 480 attcacaatt ttgatttctt aaagggactg gatgaaggtg tttcttgtac gtcaatttat 540 gaaaagcata gtgcaggact gacaaagggg atgcatgcct acagaaaact gctttatgga 600 gtaaatgaaa ttgctgtaaa agtgccttct gtttttaagc ttctaattaa agaggttctc 660 aacccatttt acattttcca gctgttcagt gttatactgt ggagcactga tgaatactat 720 tactatgctc tagctattgt ggttatgtcc atagtatcaa tcgtaagctc actatattcc 780 attagaaagc aatatgttat gttgcatgac atggtggcaa ctcatagtac cgtaagagtt 840 tcagtttgta gagtaaatga agaaatagaa gaaatctttt ctaccgacct tgtgccagga 900 gatgtcatgg tcattccatt aaatgggaca ataatgcctt gtgatgctgt gcttattaat 960 ggtacctgca ttgtaaacga aagcatgtta acaggagaaa gtgttccagt gacaaagact 1020 aatttgccaa atccttcagt ggatgtgaaa ggaataggag atgaattata taatccagaa 1080 acacataaac gacatacttt gttttgtggg acaactgtta ttcagactcg tttctacact 1140 ggagaactcg tcaaagccat agttgttaga acaggattta gtacttccaa aggacagctt 1200 gttcgttcca tattgtatcc caaaccaact gattttaaac tctacagaga tgcctacttg 1260 tttctactat gtcttgtggc agttgctggc attgggttta tctacactat tattaatagc 1320 attttaaatg aggtacaagt tggggtcata attatcgagt ctcttgatat tatcacaatt 1380 actgtgcccc ctgcacttcc tgctgcaatg actgctggta ttgtgtatgc tcagagaaga 1440 ctgaaaaaaa tcggtatttt ctgtatcagt cctcaaagaa taaatatttg tggacagctc 1500 aatcttgttt gctttgacaa gactggaact ctaactgaag atggtttaga tctttggggg 1560 attcaacgag tggaaaatgc acgatttctt tcaccagaag aaaatgtgtg caatgagatg 1620 ttggtaaaat cccagtttgt tgcttgtatg gctacttgtc attcacttac aaaaattgaa 1680 ggagtgctct ctggtgatcc acttgatctg aaaatgtttg aggctattgg atggattctg 1740 gaagaagcaa ctgaagaaga aacagcactt cataatcgaa ttatgcccac agtggttcgt 1800 cctcccaaac aactgcttcc tgaatctacc cctgcaggaa accaagaaat ggagctgttt 1860 gaacttccag ctacttatga gataggaatt gttcgccagt tcccattttc ttctgctttg 1920 caacgtatga gtgtggttgc cagggtgctg ggggatagga aaatggacgc ctacatgaaa 1980 ggagcgcccg aggccattgc cggtctctgt aaacctgaaa cagttcctgt cgattttcaa 2040 aacgttttgg aagacttcac taaacagggc ttccgtgtga ttgctcttgc acacagaaaa 2100 ttggagtcaa aactgacatg gcataaagta cagaatatta gcagagatgc aattgagaac 2160 aacatggatt ttatgggatt aattataatg cagaacaaat taaagcaaga aacccctgca 2220 gtacttgaag atttgcataa agccaacatt cgcaccgtca tggtcacagg tgacagtatg 2280 ttgactgctg tctctgtggc cagagattgt ggaatgattc tacctcagga taaagtgatt 2340 attgctgaag cattacctcc aaaggatggg aaagttgcca aaataaattg gcattatgca 2400 gactccctca cgcagtgcag tcatccatca gcaattgacc cagaggctat tccggttaaa 2460 ttggtccatg atagcttaga ggatcttcaa atgactcgtt atcattttgc aatgaatgga 2520 aaatcattct cagtgatact ggagcatttt caagaccttg ttcctaagtt gatgttgcat 2580 ggcaccgtgt ttgcccgtat ggcacctgat cagaagacac agttgataga agcattgcaa 2640 aatgttgatt attttgttgg gatgtgtggt gatggcgcaa atgattgtgg tgctttgaag 2700 agggcacacg gaggcatttc cttatcggag ctcgaagctt cagtggcatc tccctttacc 2760 tctaagactc ctagtatttc ctgtgtgcca aaccttatca gggaaggccg tgctgcttta 2820 ataacttcct tctgtgtgtt taaattcatg gcattgtaca gcattatcca gtacttcagt 2880 gttactctgc tgtattctat cttaagtaac ctaggagact tccagtttct cttcattgat 2940 ctggcaatca ttttggtagt ggtatttaca atgagtttaa atcctgcctg gaaagaactt 3000 gtggcacaaa gaccaccttc gggtcttata tctggggccc ttctcttctc cgttttgtct 3060 cagattatca tctgcattgg atttcaatct ttgggttttt tttgggtcaa acagcaacct 3120 tggtatgaag tgtggcatcc aaaatcagat gcttgtaata caacaggaag cgggttttgg 3180 aattcttcac acgtagacaa tgaaaccgaa cttgatgaac ataatataca aaattatgaa 3240 aataccacag tgttttttat ttccagtttt cagtacctca tagtggcaat tgccttttca 3300 aaaggaaaac ccttcaggca accttgctac aaaaattatt tttttgtttt ttctgtgatt 3360 tttttatata tttttatatt attcatcatg ttgtatccag ttgcctctgt tgaccaggtt 3420 cttcagatag tgtgtgtacc atatcagtgg cgtgtaacta tgctcatcat tgttcttgtc 3480 aatgcctttg tgtctatcac agtggaggag tcagtggatc ggtggggaaa atgctgctta 3540 ccctgggccc tgggctgtag aaagaagaca ccaaaggcaa agtacatgta tctggcgcag 3600 gagctcttgg ttgatccaga atggccacca aaacctcaga caaccacaga agctaaagct 3660 ttagttaagg agaatggatc atgtcaaatc atcaccataa catagcagtg aatcagtctc 3720 agtggtattg ctgatagcag tattcaggaa tatgtgattt taggagtttc tgatcctgtg 3780 tgtcagaatg gcactagttc agtttatgtc ccttctgata tagtagctta tttgac 3836 <210> 41 <211> 2240 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 6243872CB1 <400> 41 agcgggtttt gcctcctgcc ctaggagaaa cgttcgctct ggaggcttgg gcggcaagag 60 ccccttgtgg ccaccgagtc ctccgacgcc ctcggccagg ctggcctttg ggttggccca 120 ggcaggacgg gcagccgaga gcactcgggc cgcgtcgcca ggagccgccc agggtgagcc 180 atgttcgtag gcgtcgcccg gcactctggg agccaggatg aagtctcaag gggagtagag 240 ccgctggagg ccgcgcgggc ccagcctgct aaggacagga gggccaaggg aaccccgaag 300 tcctcgaagc ccgggaaaaa acaccggtat ctgagactac ttccagaggc cttgataagg 360 ttcggcggtt tccgaaaaag gaaaaaagcc aagtcctcag tttccaagaa gccgggagaa 420 gtggatgaca gtttggagca gccctgtggt ttgggctgct tagtcagcac ctgctgtgag 480 tgttgcaata acattcgctg cttcatgatt ttctactgca tcctgctcat atgtcaaggt 540.
gtggtgtttg gtcttataga tgtcagcatt ggtgattttc agaaggaata tcaactgaaa 600 accattgaga agttggcatt ggaaaagagt tacgatattt catctggcct gactgtgcag 660 ggaatagcag gaatgcctct ttatatcctt ggaataacct ttattgatga gaatgttgct 720 acacactcag ctggtatcta tttaggtatt gcagaatgta catcaatgat tggatatgct 780 ctgggttatg tgctaggagc accactagtt aaagtccctg agaatactac ttctgcaaca 840 aacactacag tcaataatgg tagtccagaa tggctatgga cttggtggat taattttctt 900 tttgccgctg tcgttgcatg gtgtacatta ataccattgt catgctttcc aaacaatatg 960 ccaggttcaa cacggataaa agctaggaaa cgtaaacagc ttcatttttt tgacagcaga 1020 cttaaagatc tgaaacttgg aattaatatc aaggatttat gtgctgctct ttggattctg 1080 atgaagaatc cagtgctcat atgcctagct ctgtcaaaag ctacagaata tttagttatt 1140 attggagctt ctgaattttt gcctatatat ttagaaaatc agtttatatt aacacccact 1200 gtggcaacta cacttgcagg acttgtttta attccaggag gtgcacttgg ccagcttctg 1260 ggaggtgtca ttgtttccac attagaaatg tcttgtaaag cccttatgag atttataatg 1320 gttacatctg tgatatcact tatactgctt gtgtttatta tttttgtacg ctgtaatcca 1380 gtgcaatttg ctgggatcaa tgaagattat gatggaacag ggaagttggg aaacctcacg 1440 gctccttgca atgaaaaatg tagatgctca tcttcaattt attcttctat atgtggaaga 1500 gatgatattg aatatttttc tccctgcttt gcaggtattg tttcctgtct tcaatactca 1560 cagatgtact acaattgttc ttgcattaaa gaaggattaa taactgcaga tgcagaaggt 1620 gattttattg atgccagacc cgggaaatgt gatgcaaagt gctataagtt acctttgttc 1680 attgctttta tcttttctac acttatattt tctggttttt ctggtgtacc aatcgtcttg 1740 gccatgacgc gggttgtacc tgacaaactg cgttctctgg ccttgggtgt aagctatgtg 1800 attttgagaa tatttgggac tattcctgga ccatcaatct ttaaaatgtc aggagaaact 1860 tcttgtattt tacgggatgt taataaatgt ggacacacag gacgttgttg gatatataac 1920 aagacaaaaa tggctttctt attggtagga atatgttttc tttgcaaact atgcactatc 1980 atcttcacta ctattgcatt tttcatatac aaacgtcgtc taaatgagaa cactgacttc 2040 ccagatgtaa ctgtgaagaa tccaaaagtt aagaaaaaag aagaaactga cttgtaactg 2100 gatcatcatt gtgattgcag atcatttgag gatcagagtg tgaaaaggag tttctctttt 2160 acagattctc caagatttgt ttctgtgccc aactttcaga agaggaaaat cacacattat 2220 gtttacataa gtagcaaaaa 2240 <210> 42 <211> 2807 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 90011608CB1 <400> 42 gggagtcaga agtcaagtga actcagcccg cctctgtgta ctttgcactt ttccatttcc 60 cttggtacca ggcactttca tacttaatcc atagtggagc tgtcacagtg agcaactctg 120 acaatgacag cttctacccc agaggcgacc ccaaacatgg agctaaaggc tccagctgca 180 ggaggtctta atgctggccc tgtcccccca gctgccttgt ccacgcagag acttcggaat 240 gaagactacc acgactacag ctccacggac gtgagccctg aggagagccc gtcggaaggc 300 ctcaacaacc tctcctcccc gggctcctac cagcgctttg gtcaaagcaa tagcacaaca 360 tggttccaga ccttgatcca cctgttaaaa ggcaacattg gcacaggact cctgggactc 420 cctctggcgg tgaaaaatgc aggcatcgtg atgggtccca tcagcctgct gatcataggc 480 atcgtggccg tgcactgcat gggtatcctg gtgaaatgtg ctcaccactt ctgccgcagg 540 ctgaataaat cctttgtgga ttatggtgat actgtgatgt atggactaga atccagcccc 600 tgctcctggc tccggaacca cgcacactgg ggaagacgtg ttgtggactt cttcctgatt 660 gtcacccagc tgggattctg ctgtgtctat tttgtgtttc tggctgacaa ctttaaacag 720 gtgatagaag cggccaatgg gaccaccaat aactgccaca acaatgagac ggtgattctg 780 acgcctacca tggactcgcg actctacatg ctctccttcc tgcccttcct ggtgctgctg 840 gttttcatca ggaacctccg agccctgtcc atcttctccc tgttggccaa catcaccatg 900 ctggtcagct tggtcatgat ctaccagttc attgttcaga ggatcccaga ccccagccac 960 ctccccttgg tggccccttg gaagacctac cctctcttct ttggcacagc gattttttca 1020 tttgaaggca ttggaatggt tctgcccctg gaaaacaaaa tgaaggatcc tcggaagttc 1080 ccactcatcc tgtacctggg catggtcatc gtcaccatcc tctacatcag cctggggtgt 1140 ctggggtacc tgcaatttgg agctaatatc caaggcagca taaccctcaa cctgcccaac 1200 tgctggttgt accagtcagt taagctgctg tactccatcg ggatcttttt cacctacgca 1260 ctccagttct acgtcccggc tgagatcatc atccccttct ttgtgtcccg agcgcccgag 1320 cactgtgagt tagtggtgga cctgtttgtg cgcacagtgc tggtctgcct gacatgcatc 1380 ttggccatcc tcatcccccg cctggacctg gtcatctccc tggtgggctc cgtgagcagc 1440 agcgccctgg ccctcatcat cccaccgctc ctggaggtca ccaccttcta ctcagagggc 1500 atgagccccc tcaccatctt taaggacgcc ctgatcagca tcctgggctt cgtgggcttt 1560 gtggtgggga cctatgaggc tctctatgag ctgatccagc caagcaatgc tcccatcttc 1620 atcaattcca cctgtgcctt catataggga tctgggttcg tctctgcagc tgcctacccc 1680 tgccccatgt gtcccccgtt acctgtcctc agagcctcag gtatggtcca ggctctgagg 1740 aaagtcaggg ttgctgtgtg ggaacccctc tgcctggcac ctggataccc tgggccaggt 1800 aacctgaggg caggggagag gtggggtggc agacacgcag aagtgctact agtgacaggg 1860 ctgccatcgc tcacctgtac ctatttacac ccagaacttt ccagctcccc ctcatcatgc 1920 ctcctccttc ctacctgcct cccctctgct ggtgcacctc gcccaactca ttcttactgc 1980 acagttcact ttatttaaca attttcatgt cccccacctc atgttttcac cttttctggg 2040 ccaggcatag attaagtaac tgggaacgcc ccctctttat aaagctgggc ttctttctca 2100 tctctctccc aaatgttgta tctcagtatt cttcctattc gagtctccag ggggtggctg 2160 gacctacctg gtcatttgaa acaggccccc aagctggagt tttttaatct ggactctctg 2220 gcttgctgtg acccctaagg caatgcttct cttccctgga ttccttagtg tgggtcacag 2280 tactgtgttc ttagttgctt tagctcttaa aacatacgaa gtgttgccta aactgaaaat 2340 atttatcttt tatttaaaat cagatttttg tttttagact gtcttagatc tggggctatt 2400 acgaatcact tcttcttcag taaactttga ctcaacttct cctgctgaaa agaagctcgc 2460 tccagatgtc tgcatgggtc ctcggcactc ttggctgagg actcaaaggt tttaatcagg 2520 atcgtctaaa aatgtacctc ggtgaggagg cacagatttt gcctcctgtt gaccagcctg 2580 gtttcatacc gaaaagacat tgaaggactg cagaaatgta tgggtgcacc gggccgaggg 2640 aagggtggct gagtgagagg catataaaat ggggctgtgt gcatgcaggc ccatgtttca 2700 gcctcagccc acgccaggtg aaaggatcag caatgctctg tcgccatcgt gctgggacga 2760 caccagctct attgccaccg atgagtagct gaggtcagtg tgcacag 2807 <210> 43 <211> 3201 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 90024583CB1 <400> 43 atggcctcgg cgctgagcta tgtctccaag ttcaagtcct tcgtgatctt gttcgtcacc 60 ccgctcctgc tgctgccact cgtcattctg atgcccgcca agtttgtcag gtgtgcctac 120 gtcatcatcc tcatggccat ttactggtgc acagaagtca tccctctggc tgtcacctct 180 ctcatgcctg tcttgctttt cccactcttc cagattctgg actccaggca ggtatgtgtc 240 cagtacatga aggacaccaa catgctgttc ctgggcggcc tcatcgtggc cgtggctgtg 300 gagcgctgga acctgcacaa gaggatcgcc ctgcgcacgc tcctctgggt gggggccaag 360 cctgcacggc tgatgctggg cttcatgggc gtcacagccc ccctgtccat gtggatcagt 420 aacacggcaa ccacggccat gatggtgccc atcgtggagg ccatattgca gcagatggaa 480 gccacaagcg cagccaccga ggccggcctg gagctggtgg acaagggcaa ggccaaggag 540 ctgccaggga gtcaagtgat ttttgaaggc cccactctgg ggcagcagga agaccaagag 600 cggaagaggt tgtgtaaggc catgaccctg tgcatctgct acgcggccag catcgggggc 660 accgccaccc tgaccgggac gggacccaac gtggtgctcc tgggccagat gaacgagttg 720 tttcctgaca gcaaggacct cgtgaacttt gcttcctggt ttgcatttgc ctttcccaac 780 atgctggtga tgctgctgtt cgcctggctg tggctccagt ttgtttacat gagattcaat 840 tttaaaaagt cctggggctg cgggctagag agcaagaaaa acgagaaggc tgccctcaag 900 gtgctgcagg aggagtaccg gaagctgggg cccttgtcct tcgcggagat caacgtgctg 960 atctgcttct tcctgctggt catcctgtgg~ttctcccgag accccggctt catgcccggc 1020 tggctgactg ttgcctgggt ggagggtgag acaaagtatg tctccgatgc cactgtggcc 1080 atctttgtgg ccaccctgct attcattgtg ccttcacaga agcccaagtt taacttccgc 1140 agccagactg aggaagaaag gaaaactcca ttttatcecc ctcccctgct ggattggaag 1200 gtaacccagg agaaagtgcc ctggggcatc gtgctgctac tagggggcgg atttgctctg 1260 gctaaaggat ccgaggcctc ggggctgtcc gtgtggatgg ggaagcagat ggagcccttg 1320 cacgcagtgc ccccggcagc catcaccttg atcttgtcct tgctcgttgc cgtgttcact 1380 gagtgcacaa gcaacgtggc caccaccacc ttgttcctgc ccatctttgc ctccatgtct 1440 cgctccaacg gcctcaatcc gctgtacatc atgctgccct gtaccctgag tgcctccttt 1500 gccttcatgt tgcctgtggc cacccctcca aatgccatcg tgttcaccta tgggcacctc 1560 aaggttgctg acatggtgaa aacaggagtc ataatgaaca taattggagt cttctgtgtg 1620 tttttggctg tcaacacctg gggacgggcc atatttgact tggatcattt ccctgactgg 1680 gctaatgtga cacatattga gacttaggaa gagccacaag accacacaca cagcccttac 1740 cctcctcagg actaccgaac cttctggcac accttgtaca gagttttggg gttcacaccc 1800 caaaatgacc caacgatgtc cacacaccac caaaacccag ccaatgggcc acctcttcct 1860 ccaagcccag atgcagagat ggtcatgggc agctggaggg taggctcaga aatgaaggga 1920 acccctcagt gggctgctgg acccatcttt cccaagcctt gccattatct ctgtgaggga 1980 ggccaggtag ccgagggatc aggatgcagg ctgctgtacc cgctctgcct caagcatccc 2040 ccacacaggg ctctggtttt cactcgcttc gtcctagata gtttaaatgg gaatcggatc 2100 ccctggttga gagctaagac aaccacctac cagtgcccat gtcccttcca gctcaccttg 2160 agcagcctca gatcatctct gccactctgg aagggacacc ccagccaggg acggaatgcc 2220 tggtcttgag caacctccca ctgctggagt gcgagtggga atcagagcct cctgaagcct 2280 ctgggaactc ctcctgtggc caccaccaaa ggatgaggaa tctgagttgc caacttcagg 2340 acgacacctg gcttgccacc cacagtgcac cacaggccaa cctacgccct tcatcacttg 2400 gttctgtttt aatcgactgg ccccctgtcc~cacctctcca gtgagcctcc ttcaactcct 2460 tggtcccctg ttgtctgggt caacatttgc cgagacgcct tggctggcac cctctggggt 2520 cccccttttc tcccaggcag gtcatctttt ctgggagatg cttcccctgc catccccaaa 2580 tagctaggat cacactccaa gtatgggcag tgatggcgct ctgggggcca cagtgggcta 2640 tctaggccct ccctcacctg aggcccagag tggacacagc tgttaatttc cactggctat 2700 gccacttcag agtctttcat gccagcgttt gagctcctct gggtaaaatc ttccctttgt 2760 tgactggcct tcacagccat ggctggtgac aacagaggat cgttgagatt gagcagcgct 2820 tggtgatctc tcagcaaaca acccctgccc gtgggccaat ctacttgaag ttactcggac 2880 aaagacccca aagtggggca acaactccag agaggctgtg ggaatcttca gaacccccct 2940 gtaagagaca gacatgagag acaagcatct tctttccccc gcaagtccat tttatttcct 3000 tcttgtgctg ctctggaaga gaggcagtag caaagagatg agctcctgga tggcattttc 3060 cagggcagga gaaagtatga gagcctcagg aaaccccatc aaggaccgag tatgtgtctg 3120 gttccttggg tgggacgatt cctgaccaca ctgtccagct cttgctctca ttaaatgctc 3180 tgtctcccgc ggaaaaaaaa a 3201 <210> 44 <211> 3688 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 90113658CB1 <400> 44 gctgtgtcca agaaaggagt cagcaaaggg tgca~tgggatt atcattagtt cttataggtt 60 tgggataggc ggtggagtta ggagcaattt tttataggca gggggtggat ctcacaaagt 120 acattctcaa gggcggggag aatattacaa agtaccttct taagggcggg ggagtatgac 180 aaagtatatt attgcaaggg cggggagggg gtattgtcat aaggtcaatt gatcagttag 240 ggtgggtagg aacagatcac aatggtggaa tgttattttt tgtggttctt catttgcttc 300 aggtcatctg gatgtatatg tgcaggtcac aggagatgtg atggcttagc ttgggctcag 360 aggcctgaca gtttgtatcc tcgtctccgt aatggtgcaa tgatccttcc ttcgcgtagg 420 gtaccatgtg tctcagggct ggaggaccac ctggccttgc tggcctctta caccagagcc 480 tgggccatag ctcataattt tgctgatatt ggcatctttt aacctcagaa agtatactct 540 gggtctaata atctgcctcc ttcaggaata ttcattcctc ttgttgcccg ctagcttcca 600 gtcataaaaa aaaatccgtt taataacttt agatttggtt acatccatct gtgtgttttt 660 cttcacctcc atttagaatt agtaggaaaa gcattagaac aaaagggttg tttaaagaac 720 attagtttat ctcatttgaa aagtgaaact tctttcatgt aagctctggt tacttcattg 780 ttctttccac aagcccttct aacaatgatt atctgcagtt tgcccaccca ctgtcaagag 840 ctcagcatcc atacttgaaa ctcaagcttc atatgccaac tttgggatga ttctcatcaa 900 acagaagagg ctgtttccat gctggatccc ggcgctgttc atcggcttca gccagttctc 960 ggactcgttc ctcctggacc agcccaactt ctggtgccgc ggggccggca aaggcaccga 1020 gctggcaggg gtcaccacca caggccgggg cggggacatg ggcaactgga ccagcctccc 1080 caccaccccc ttcgccactg ccccctggga ggctgcgggc aaccggagca acagcagcgg 1140 cgcggacgga ggcgacacac cacccctgcc atcccctccg gacaaggggg acaacgcctc 1200 caactgtgac tgccgcgcat gggactacgg catccgcgcc ggcctcgtcc agaacgtggt 1260 cagcaagtgg gatcttgtgt gtgataatgc ctggaaggtc catatcgcta agttctcctt 1320 actggttgga ttaatctttg gctacctaat aactggatgc attgctgact gggtcggccg 1380 gcggcctgtg ctgctgtttt ccatcatctt cattctgatc tttggactga ctgtggcact 1440 gtcagtgaat gtgacaatgt tcagcacact caggttcttt gaaggatttt gcctggctgg 1500 aatcattctc accttgtatg ctttacgaat agagctgtgc ccccctggaa aacggttcat 1560 gattacgatg gtggcgagct tcgtggccat ggcgggccag ttcctcatgc ctgggctagc 1620 cgccctgtgc cgggattggc aggtgctgca ggccctcatc atctgcccct tcctgctcat 1680 gctgctctac tggtcgatat tccccgagtc cctccggtgg ctaatggcca cccagcagtt 1740 tgagtctgca aagaggctga tcctccactt cacacagaag aatcgcatga accctgaggg 1800 cgacatcaag ggtgtgatac cagagctgga gaaagagctt tcccggaggc ccaagaaggt 1860 ctgcatcgtg aaggtggtgg ggacacggaa cctgtggaag aacattgtgg tcctgtgtgt 1920 gaactcgctg acggggtacg ggatccacca ctgctttgcc aggagcatga tgggccacga 1980 ggtgaaggtg ccgctcctgg agaacttcta tgctgactac tataccacgg ccagcatcgc 2040 gctggtgtcc tgcctggcca tgtgcgtggt ggtccgattc ctcgggcgca ggggagggct 2100 gctgctcttc atgatcctca ccgccctggc ctcgctcctg cagctcggcc tcctcaacct 2160 gattggaaag tacagccagc acccagactc agggatgagt gacagcgtca aggacaaatt 2220 ttccatcgcg ttttccatcg tgggcatgtt tgcctcccat gcggtgggga gcctcagcgt 2280 gttcttctgt gcggagatca ccccgacggt gataaggtgt ggcgggctgg ggctggtgct 2340 ggccagcgcg ggcttcggca tgctgacggc acccatcatc gagctgcaca accagaaagg 2400 ctacttcctg caccacatca tctttgcctg ctgcacgctc atctgcatca tctgcatcct 2460 cctgctgccc gagagcaggg accagaacct gcctgagaac atttctaacg gggagcacta 2520 cacgcgccag ccgctgctgc cgcacaagaa gggggagcag ccactgctgc tcaccaacgc 2580 cgagctcaag gactactcgg gcctccacga tgccgcagcc gcgggtgaca cactgcccga 2640 ggg.tgccacg gccaacggca tgaaggccat gtagcccggc ctgcggaacc cggggctcca 2700 gggtctgggg cagcttgggc acaggtttac agaccaggga ccgaacacgc agccaggggt 2760 gggaaagctg cctcagccaa gctgagcctc tcaactggtg tggggaaatc ctgtctttcc 2820 aaaagtccaa ggagcgcggg tcggaggaga caaactcttt ggaaataacc ctttcaagac 2880 tttcttttct gccgttaaat gtgtgtattt attttggtca tttttacgag aagcacttta 2940 ttccctctcc ctctcactga tcacaaatgg aatcacctcc ctgggcagcg agacgcagtt 3000 gctctgggaa gatgccacag tgaccagggc catggccggt ccctctgggg agatgggaca 3060 cggctcctgg cagcacccag gagcccccca ggctgcctgc ctgcgtgcag aggcaggaga 3120 ggaccgtgga gcgtttccgg gactgcattt tagacggagt gaaatgtaca tgaatttggc 3180 ttttgctaga gtctgtgtat ggttttttaa ggtccttttt ccctttctgt ttgtaaggta 3240 agagcttctg ttcgtgtgca gggaaagcag ctcacagacg ccgttaaaac cagcttcatc 3300 tttccttcag gatcatcctt ttgtacttga tgctggaagc tcttggagaa aaagcttaaa 3360 catttcacca gaaatcttaa ttgagcagca gtcatatcgc cacagctttg tgagtacaca 3420 gctaacagat gcgtcgagac ctgagatgtg ctcgttttta ttcctcctct cccagattgg 3480 ctccagcaga aaagtccctc gtgcacaggc agctcttgtg ccggcactac ttgaaaacat 3540 ggctactttc taagctacaa accattagaa aataactcaa aaagatggca gaggcaaatc 3600 cacagaaggg gggctgccct ccacacacac acgcccgtca cccacacctt gagcacacac 3660 catgaagatc acctactgag gaggatcc 3688 <210> 45 <211> 2402 <212> DNA
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 3942766CB1 <400> 45 gcccgtaccg ccaggcgatc gcgctgatgg cggcgctggc agcagcggcc aagaaggtgt 60 ggagcgcgcg gcggctgctg gtgctgctgt tcacgccgct cgcgctgctg ccggtggtct 120 tcgccctccc gcccaaggaa ggccgctgct tgtttgtcat cctgctcatg gcggtgtact 180 ggtgcacgga ggccctgccg ctctcagtga cggcgctgct gcccatcgtc ctcttcccct 240 tcatgggcat cttgccctcc aacaaggtct gcccccagta cttcctcgac accaacttcc 300 tcttcctcag tgggctgatc atggccagcg ccattgagga gtggaacctg caccggcgaa 360 tcgccctcaa gatcctgatg cttgttggag tccagccggc caggctcatc ctggggatga 420 tggtgaccac ctcgttcttg tccatgtggc tgagcaacac cgcctccact gccatgatgc 480 ttcccattgc caatgccatc ctgaaaagtc tctttggcca gaaggaggtt cgaaaggacc 540 ccagccagga gagtgaagag aacacagctg ctgtgcggag aaacggccta cacactgtgc 600 ccacggagat gcagtttctc gccagcacag aagccaaaga ccaccctggg gagacagagg 660 ttccactgga tctgccggct gactccagga aggaggatga atatcgtcgg aacatctgga 720 agggcttcct catctccatc ccctactcag ccagtattgg gggcacagcc acactcacgg 780 gcacagcccc taacctcatc ctgcttggcc agctcaagag tttctttccg cagtgtgacg 840 tggtgaattt cggctcctgg ttcattttcg ccttccctct tatgctgttg ttcctgttgg 900 caggctggct ctggatctcc ttcctgtacg ggggactgag cttcaggggc tggaggaaga 960 ataaatctga gataagaacc aatgcagaag atagggctcg agctgtaatt cgggaagaat 1020 accagaacct ggggcccatc aagtttgccg aacaggctgt tttcatcctt ttctgcatgt 1080 ttgccatcct cctcttcacc cgggacccga agttcatccc tggctgggcc agcctcttca 1140 atcctgggtt tctttctgat gctgtcaccg gcgtggctat tgtcaccatc ttgttcttct 1200 tcccgtccca aaggccctct ctcaagtggt ggtttgactt caaagctccc aacacagaga 1260 cagagccctt gctgacctgg aagaaggccc aggagacagt gccctggaac atcatccttc 1320 tcctgggagg gggcttcgcc atggccaaag gctgtgagga atcggggctg tctgtatgga 1380 ttggtgggca gctgcacccc ctggagaatg tgccccccgc cctggctgtg ctgctcatca 1440 ctgtggtcat cgccttcttc actgagtttg ccagcaacac ggcgaccatc atcatcttcc 1500 tgccggtcct ggcagagctg gccatccgcc tgagagtgca ccccctgtat ctgatgattc 1560 cgggcacagt cggctgctcc tttgccttca tgctcccggt ctcaacgccc cccaactcca 1620 tcgccttcgc ctctggacac ttgctggtca aagacatggt gcggacaggc ctcctgatga 1680 acctgatggg tgtcctgctg ctcagtttgg ctatgaatac ctgggcacag accatcttcc 1740 agctgggcac cttcccggac tgggctgata tgtactcggt caatgtcaca gcattgccac 1800 ccaccttggc caatgacaca tttcggaccc tctgagtccc cctggaggac tcccttgagg 1860 ctgaaccctc tcaaagggct gtcaccatca cctgctgcta ggacactaca aaacaatcaa 1920 ataattcttt tctgtaatcc aatatgcagc aagcaagggt gacctccagt ggcccactca 1980 agtccatgag ccattatcta ggatactttc tctctctttc atgcagttca aagcccaggt 2040 atctctcaga tctgctgcct gagaaataag ctcctttatc agttagctgt tttatcatta 2100 ggatacaaga cagcccagtg tcatcaacag tgagcaaatc tggcatggtg ttgtctcgta 2160 cagtgggata ggaggccatt cattcccatg ggcacagcta acattatccc ccagtgatta 2220 ctttcgatta cactgaagag acacctgtct taaagatcac tttcctgggc tggcatgtcc 2280 aaccagttgt tccagcctgt tcacaccagg gggggattgc ttagttggtg cccatctgcc 2340 cagtgtttac cctgcgatac caggagcaga gatcctttaa tcctgggacc ggaaggcagt 2400 ac 2402 <210> 46 <211> 2410 <212> DNA
<213> Homo Sapiens <220>
<221> misc-feature <223> Incyte ID No: 7501987CB1 <400> 46 catccgctca caatgccaca tcaatgatac gagcacgtag cctcactgct tgcacagtgc 60 atggcagagt cggctgcgag caggcgaggt ggcctgaggg aggtcactag gctggctgag 120 ggctttttgc tgtggttctg agccggcctg cttccaggca ccgtgtccat gcgggtaagc 180 ggtctccctg ggtgcccact cttgcgcccg gagatcctga gtttggtcct gtctggccat 240 gaagctcagc ctgctgggag gccacaggga gatgcaggct gggcggcggg tggatggttc 300 cagccggttg ggtccggggc ctggagctca gcctgtgggg tggggaccca gtggtgccct 360 ggagctgccg cttctgctct cagcaggatg atgggcagga cagggagagg ctgacctact 420 tccagaacct gcctgagtct ctgacttccc tcctggtgct gctgaccacg gccaacaacc 480 ccgatgtgat gattcctgcg tattccaaga accgggccta tgccatcttc ttcatagtct 540 tcactgtgat aggaagcctg tttctgatga acctgctgac agccatcatc tacagtcagt 600 tccggggcta cctgatgaaa tctctccaga cctcgctgtt tcggaggcgg ctgggaaccc 660 gggctgcctt tgaagtccta tcctccatgg tgggggaggg aggagccttc cctcaggcag 720 ttggggtgaa gccccagaac ttgctgcagg tgcttcagaa ggtccagctg gacagctccc 780 acaaacaggc catgatggag aaggtgcgtt cctatggcag tgttctgctc tcagctgagg 840 agtttcagaa gctcttcaac gagcttgaca gaagtgtggt taaagagcac ccgccgaggc 900 ccgagtacca gtctccgttt ctgcagagcg cccagttcct cttcggccac tactactttg 960 actacctggg gaacctcatc gccctggcaa acctggtgtc catttgcgtg ttcctggtgc 1020 tggatgcaga tgtgctgcct gctgagcgtg atgacttcat cctggggatt ctcaactgcg 1080 tcttcattgt gtactacctg ttggagttgc tgctcaaggt ctttgccctg ggcctgcgag 1140 ggtacctgtc ctaccccagc aacgtgtttg acgggctcct caccgttgtc ctgctggagg 1200 ccggagatgg tgggcctgct gtcgctgtgg gacatgaccc gcatgctgaa catgctcatc 1260 gtgttccgct tcctgcgtat catccccagc atgaagccga tggccgtggt ggccagtacc 1320 gtcctgggcc tggtgcagaa catgcgtgcg tttggcggga tcctggtggt ggtctactac 1380 gtatttgcca tcattgggat caacttgttt agaggcgtca ttgtggctct tcctggaaac 1440 agcagcctgg cccctgccaa tggctcggcg ccctgtggga gcttcgagca gctggagtac 1500 tgggccaaca acttcgatga ctttgcggct gccctggtca ctctgtggaa cttgatggtg 1560 gtgaacaact ggcaggtgtt tctggatgca tatcggcgct actcaggccc gtggtccaag 1620 atctattttg tgttgtggtg gctggtgtcg tctgtcatct gggtcaacct gtttctggcc 1680 ctgattctgg agaacttcct tcacaagtgg gacccccgca gccacctgca gccccttgct 1740 gggaccccag aggccaccta ccagatgact gtggagctcc tgttcaggga tattctggag 1800 gagcccgggg aggatgagct cacagagagg ctgagccagc acccgcacct gtggctgtgc 1860 aggtgacgtc cgggctgccg tcccagcagg ggcggcagga gagagaggct ggcctacaca 1920 ggtgcccatc atggaagagg cggccatgct gtggccagcc aggcaggaag agacctttcc 1980 tctgacggac cactaagctg gggacaggaa ccaagtcctt tgcgtgtggc ccaacaacca 2040 tctacagaac agctgctggt gcttcaggga ggcgccgtgc cctccgcttt cttttatagc 2100 tgcttcagtg agaattccct cgtcgactcc acagggacct ttcagacaaa aatgcaagaa 2160 gcagcggcct cccctgtccc ctgcagcttc cgtggtgcct ttgctgccgg cagcccttgg 2220 ggaccacagg cctgaccagg gcctgcacag gttaaccgtc agacttccgg ggcattcagg 2280 tggggatgct ggtggtttga catggagaga accttgactg tgttttatta tttcatggct 2340 tgtatgagtg tgactgggtg tgtttcttta gggttctgat tgccagttat tttcatcaat 2400 aagtcttgca 2410 <210> 47 <211> 968 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 750.3223CB1 <400> 47 gaggagatgg gttccaattt tagggagcag gctgcctaat gaaggagcca ggcttgcaca 60 cagacaattc tagaactggt ggcccgagag ggatgtgaag gcccaaaatg accctcttac 120 cgggagacaa ttctgactac gactacagcg cgctgagctg cacctcggac gcctccttcc 180 acccggcctt cctcccgcag cgccaggcca tcaagggcgc gttctaccgc cgggcgcagc 240 ggctgcggcc gcaggatgag ccccgccagg gctgtcagcc cgaggaccgc cgccgtcgga 300 tcatcatcaa cgtaggcggc atcaagtact cgctgccctg gaccacgctg gacgagttcc 360 cgctgacgcg cctgggccag ctcaaggcct gcaccaactt cgacgacatc ctcaacgtgt 420 gcgatgacta cgacgtcacc tgcaacgagt tcttcttcga ccgcaacccg ggggccttcg 480 gcactatcct gaccttcctg cgcgcgggca agctgcggct gctgcgcgag atgtgcgcgc 540 tgtccttcca ggacagtgac atcttgttcg gaagtgcctc ctcggacacc agagacaata 600 actgagcgcg gaggacacgc ctgccctgcc tgccatctgt ggcccgaagc cattgccatc 660 cactgcagac gcctggagag ggacaggccg cttccgagtg cagtcctggc gcagcaccga 720 ctcccacgca cccggggaag gacaccctca ctcccacacc ccgggaagaa cactagaaca 780 tcagcagagg ggccctgccc ctccgcctgc agccgtgaaa ggaagctggg tcatcagccc 840 agccccgccc accccagccc ctatgtgtgt ttccctcaat aaggagatgc cttgttcttt 900 tcaccatgca aataacatgc ccagcaaaaa cttgctttat gggtctgcct ggagaaaaaa 960 aaaaaaaa 968 <210> 48 <211> 2267 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503566CB1 <400> 48 gtcttgctaa cagctgccaa tacctcactg agtgcctcac accaacatgg gctccaagtg 60 agtttccttc gtctgggcag actccctccc ctcttccata aaggctgcag gagacctgta 120 gctgtcacag gaccttccct aagagcccgc aggggaagac tgccccagtc cggccatcac 180 catgctccgg accattctgg atgctcccca gcggttgctg aaggagggga gagcgtcccg 240 gcagctggtg ctggtggtgg tattcgtcgc tttgctcctg gacaacatgc tgtttactgt 300 ggtggtgcca attgtgccca ccttcctata tgacatggag ttcaaagaag tcaactcttc 360 tctgcacctc ggccatgccg gaagttcccc acatgccctc gcctctcctg ccttttccac 420 catcttctcc ttcttcaaca acaacaccgt ggctgttgaa gaaagcgtac ctagtggaat 480 agcatggatg aatgacactg ccagcaccat cccacctcca gccactgaag ccatctcagc 540 tcataaaaac aactgcttgc aaggcacagg tttcttggag gaagagacta cccgggtcgg 600 ggttctgttt gcttcaaagg ctgtgatgca acttctggtc aacccattcg tgggccctct 660 caccaacagg attggatatc atatccccat gtttgctggc tttgttatca tgtttctctc 720 cacagttagt cttggaatgc tggccagtgt ctacactgat gaccatgaga gaggacgagc 780 catgggaact gctctggggg gcctggcctt ggggttgctg gtgggagctc cctttggaag 840 tgtaatgtac gagtttgttg ggaagtctgc acccttcctc atcctggcct tcctggcact 900 actggatgga gcactccagc tttgcatcct acagccttcc aaagtctctc ctgagagtgc 960 caaggggact cccctcttta tgcttctcaa agacccttac atcctggtgg ctgcagggtc 1020 catctgcttt gccaacatgg gggtggccat cctggagccc acactgccca tctggatgat 1080 gcagaccatg tgctccccca agtggcagct gggtctagct ttcttgcctg ccagtgtgtc 1140 ctacctcatt ggcaccaacc tctttggtgt gttggccaac aagatgggtc ggtggctgtg 1200 ttccctaatc gggatgctgg tagtaggtac cagcttgctc tgtgttcctc tggctcacaa 1260 tatttttggt ctcattggcc ccaatgcagg gcttggcctt gccataggca tggtggattc 1320 ttctatgatg cccatcatgg ggcacctggt ggatctacgc cacacctcgg tgtatgggag 1380 tgtctacgcc atcgctgatg tggctttttg catgggcttt gctataggtc catccaccgg 1440 tggtgccatt gtaaaggcca tcggttttcc ctggctcatg gtcatcactg gggtcatcaa 1500 catcgtctat gctccactct gctactacct gcggagcccc ccggcaaagg aagagaagct 1560 tgctattctg agtcaggact gccccatgga gacccggatg tatgcaaccc agaagcccac 1620 gaaggaattt cctctggggg aggacagtga tgaggagcct gaccatgagg agtagcagca 1680 gaaggtgctc cttgaattca tgatgcctca gtgaccacct ctttccctgg gaccagatca 1740 ccatggctga gcccacggct cagtgggctt cacatacctc tgcctgggaa tcttctttcc 1800 tcccctccca tggacactgt ccctgatact cttctcacct gtgtaacttg tagctcttcc 1860 tctatgcctt ggtgccgcag tggcccatct tttatgggaa gacagagtga tgcaccttcc 1920 cgctgctgtg aggttgatta aacttgagct gtgacgggtt ctgcaagggg tgactcattg 1980 catagaggtg gtagtgagta atgtgcccct gaaaccagtg gggtgactga caagcctctt 2040 taatctgttg cctgattttc tctggcatag tcccaacaga tcggaagagt gttaccctct 2100 tttcctcaac gtgttctttc ccgggttttc ccagccgagt tgagaaaatg ttctcagcat 2160 tgtcttgctg ccaaatgcca gcttgaagag ttttgttttg ttttttttcc atttattttt 2220 ttttttaata aagtgagtga tttttctgtg gctaaaaaaa aaaaaaa 2267 <210> 49 <211> 319 <212> DNA

<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7505122CB1 <400> 49 cggctcgagc tcgaccgaat cggctcgagc ggctcgagtg ctcagcctgg tgaaccacac 60 aggccagcgc tctgacatgc agaaggtgac cctgggcctg cttgtgttcc tggcaggctt 120 tcctgtcctg gacgccaatg acctagaaga taaaaacagt cctttctact atgactggca 180 cagcctccag gttggcgggc tcatctgcgc tggggttctg tgcatggcag ggcctcatct 240 cacctctcgc aagagggtct ctttgttcaa ttttttttaa tctaaaatga ttgtgcctct 300 gcccaaaaaa aaaaaaaaa 319 <210> 50 <211> 2510 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7511620CB1 <400> 50 ggagcagccc gcaccggaca acttgcgagc catggggctg gcggatgcgt cgggaccgag 60 ggacacacag gcactgctgt ctgcaacaca agcaatggac ctgcggaggc gagactacca 120 catggaacgg ccgctgctga accaggagca tttggaggag ctggggcgct ggggctcagc 180 acctaggacc caccagtggc ggacctggtt gcagtgctcc cgtgctcggg cctatgccct 240 tctgctccaa cacctcccgg ttttggtctg gttaccccgg tatcctgtgc gtgactggct 300 cctgggtgac ctgttatccg gcctgagtgt ggccatcatg cagcttccgc agggcttggc 360 ctacgccctc ctggctggat tgccccccgt gtttggcctc tatagctcct tctaccctgt 420 cttcatctac ttcctgtttg gcacttcccg gcacatctcc gtgggccttg aacgactcca 480 tgatcaatga gacagccaga gatgctgccc gggtacaggt ggcctccaca ctcagtgtcc 540 tggttggcct cttccaggtg gggctgggcc tgatccactt cggcttcgtg gtcacctacc 600 tgtcagaacc tcttgtccga ggctatacca cagctgcagc tgtgcaggtc ttcgtctcac 660 agctcaagta tgtgtttggc ctccatctga gcagccactc tgggccactg tccctcatct 720 atacagtgct ggaggtctgc tggaagctgc cccagagcaa ggttggcacc gtggtcactg 780 cagctgtggc tggggtggtg ctcgtggtgg tgaagctgtt gaatgacaag ctgcagcagc 840 agctgcccat gccgataccc ggggagctgc tcacgctcat cggggccaca ggcatctcct 900 atggcatggg tctaaagcac agatttgagg tagatgtcgt gggcaacatc cctgcagggc 960 tggtgccccc agtggccccc aacacccagc tgttctcaaa gctcgtgggc agcgccttca 1020 ccatcgctgt ggttgggttt gccattgcca tctcactggg gaagatcttc gccctgaggc 1080 acggctaccg ggtggacagc aaccaggagc tggtggccct gggcctcagt aaccttatcg 1140 gaggcatctt ccagtgcttc cccgtgagtt gctctatgtc tcggagcctg gtacaggaga 1200 gcaccggggg caactcgcag gttgctggag ccatctcttc ccttttcatc ctcctcatca 1260 ttgtcaaact tggggaactc ttccatgacc tgcccaaggc ggtcctggca gccatcatca 1320 ttgtgaacct gaagggcatg ctgaggcagc tcagcgacat gcgctccctc tggaaggcca 1380 atcgggcgga tctgcttatc tggctggtga ccttcacggc caccatcttg ctgaacctgg 1440 accttggctt ggtggttgcg gtcatcttct ccctgctgct cgtggtggtc cggacacaga 1500 tgccccacta ctctgtcctg gggcaggtgc cagacacgga tatttacaga gatgtggcag 1560 agtactcaga ggccaaggaa gtccgggggg tgaaggtctt ccgctcctcg gccaccgtgt 1620 actttgccaa tgctgagttc tacagtgatg cgctgaagca gaggtgtggt gtggatgtcg 1680 acttcctcat ctcccagaag aagaaactgc tcaagaagca ggagcagctg aagctgaagc 1740 aactgcagaa agaggagaag cttcggaaac aggctgcctc ccccaagggc gcctcagttt 1800 ccattaatgt caacaccagc cttgaagaca tgaggagcaa caacgttgag gactgcaaga 1860 tgatggtgag ctcaggagat aagatggaag atgcaacagc caatggtcaa gaagactcca 1920 aggccccaga tgggtccaca ctgaaggccc tgggcctgcc tcagccagac ttccacagcc 1980 tcatcctgga cctgggtgcc ctctcctttg tggacactgt gtgcctcaag agcctgaaga 2040 atattttcca tgacttccgg gagattgagg tggaggtgta catggcggcc tgccacagcc 2100 ctgtggtcag ccagcttgag gctgggcact tcttcgatgc atccatcacc aagaagcatc 2160 tctttgcctc tgtccatgat gctgtcacct ttgccctcca acacccgagg cctgtccccg 2220 acagccctgt ttcggtcacc agactctgaa catgctacat cctgcccaag actgcacctc 2280 tggaggtgca gggcaccctt gagaagcccc tcacccctag gccgcctcca ggtgctaccc 2340 aggagtcccc tccatgtaca cacacacaac tcagggaagg aggtcctggg actccaagtt 2400 cagcgctcca ggtctgggac agggcctgca tgcagtcagg ctggcagtgg cgcggtacag 2460 ggagggaact ggtgcatatt ttagcctcag gaataaagat ttgtctgctc 2510 <210> 51 <211> 2241 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7506995CB1 <400> 51 catccgctca caatgccaca tcaatgatac gagcacgtag cctcactgct tgcacagtgc 60 atggcagagt cggctgcgag caggcgaggt ggcctgaggg aggtcactag gctggctgag 120 ggctttttgc tgtggttctg agccggcctg cttccaggca ccgtgtccat gcgggtgagc 180 ggtctccctg ggtgcccact cttgcgcccg gagatcctga gtttggtcct gtctggccat 240 gaagctcagc ctgctgggag gccacaggga gatgcaggct gggcggcggg tggatggttc 300 cagccggttg ggtccggggc ctggagctca gcctgtgggg tggggaccca gtggtgccct 360 ggagctgccg cttctgctct cagcaggatg atgggcagga cagggagagg ctgacctact 420 tccagaacct gcctgagtct ctgacttccc tcctggtgct gctgaccacg gccaacaacc 480 ccgatgtgat gattcctgcg tattccaaga accgggccta tgccatcttc ttcatagtct 540 tcactgtgat aggaagcctg tttctgatga acctgctgac agccatcatc tacagtcagt 600 tccggggcta cctgatgaaa tctctccaga cctcgctgtt tcggaggcgg ctgggaaccc 660 gggctgcctt tgaagtccta tcctccatgg tgggggaggg aggagccttc cctcaggcca 720 cccgccgagg cccgagtacc agtctccgtt tctgcagagc gcccagttcc tcttcggcca 780 ctactacttt gactacctgg ggaacctcat cgccctggca aacctggtgt ccatttgcgt 840 gttcctggtg ctggatgcag atgtgctgcc tgctgagcgt gatgacttca tcctggggat 900 tctcaactgc gtcttcattg tgtactacct gttggagatg ctgctcaagg tctttgccct 960 gggcctgcga gggtacctgt cctaccccag caacgtgttt gacgggctcc tcaccgttgt 1020 cctgctggag gccggagatg gtgggcctgc tgtcgctgtg ggacatgacc cgcatgctga 1080 acatgctcat cgtgttccgc ttcctgcgta tcatccccag catgaagccg,atggccgtgg 1140 tggccagtac cgtcctgggc ctggtgcaga acatgcgtgc ttttggcggg atcctggtgg 1200 tggtctacta cgtatttgcc atcattggga tcaacttgtt tagaggcgtc attgtggctc 1260 ttcctggaaa cagcagcctg gcccctgcca atggctcggc gccctgtggg agcttcgagc 1320 agctggagta ctgggccaac aacttcgatg actttgcggc tgccctggtc actctgtgga 1380 acttgatggt ggtgaacaac tggcaggtgt ttctggatgc atatcggcgc tactcaggcc 1440 cgtggtccaa gatctatttt gtgttgtggt ggctggtgtc gtctgtcatc tgggtcaacc 1500 tgtttctggc cctgattctg gagaacttcc ttcacaagtg ggacccccgc agccacctgc 1560 agccccttgc tgggacccca gaggccacct accagatgac tgtggagctc ctgttcaggg 1620 atattctgga ggagcccggg gaggatgagc tcacagagag gctgagccag cacccgcacc 1680 tgtggctgtg caggtgacgt ccgggctgcc gtcccagcag gggcggcagg agagagaggc 1740 tggcctacac aggtgcccat catggaagag gcggccatgc tgtggccagc caggcaggaa 1800 gagacctttc ctctgacgga ccactaagct ggggacagga accaagtcct ttgcgtgtgg 1860 cccaacaacc atctacagaa cagctgctgg tgcttcaggg aggcgccgtg ccctccgctt 1920 tcttttatag ctgcttcagt gagaattccc tcgtcgactc cacagggacc tttcagacaa 1980 aaatgcaaga agcagcggcc tcccctgtcc cctgcagctt ccgtggtgcc tttgctgccg 2040 gcagcccttg gggaccacag gcctgaccag ggcctgcaca ggttaaccgt cagacttccg 2100 gggcattcag gtggggatgc tggtggtttg acatggagag aaccttgact gtgttttatt 2160 atttcatggc ttgtatgagt gtgactgggt gtgtttcttt agggttctga ttgccagtta 2220 ttttcatcaa taagtcttgc a 2241 <210> 52 <211> 2312 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7506996CB1 <400> 52 catccgctca caatgccaca tcaatgatac gagcacgtag cctcactgct tgcacagtgc 60 atggcagagt cggctgcaga gcaggcgagg tggcctgagg gaggtcacta ggctggctga 120 gggctttttg ctgtggttca tgagccggcc tgcttccagg caccgtgtcc atgcgggtga 180 gcggtctccc tgggtgccca ctcttgcgcc cggagatcct gagtttggtc ctgtctggcc 240 atgaagctca gcctgctggg aggccacagg gagatgcagg ctgggcggcg ggtggatggt 300 tccagccggt tgggtccggg gcctggagct cagcctgtgg ggtggggacc cagtggtgcc 360 ctggagctgc cgcttctgct ctcagcagga tgatgggcag gacagggaga ggctgaccta 420 cttccagaac ctgcctgagt ctctgacttc cctcctggtg ctgctgacca cggccaacaa 480 ccccgatgtg atgattcctg cgtattccaa gaaccgggcc tatgccatct tcttcatagt 540 cttcactgtg ataggaagcc tgtttctgat gaacctgctg acagccatca tctacagtca 600 gttccggggc tacctgatga aatctctcca gacctcgctg tttcggaggc ggctgggaac 660 ccgggctgcc tttgaagtcc tatcctccat ggtgggggag ggaggagcct tccctcaggc 720 agttggggtg aagccccaga acttgctgca ggtgcttcag aaggtccagc tggacagctc 780 ccacaaacag gccatgatgg agaaggtgcg ttcctatggc agtgttctgc tctcagctga 840 ggagtttcag aagctcttca acgagcttga cagaagtgtg gttaaagagc acccgccgag 900 gcccgagtac cagtctccgt ttctgcagag cgcccagttc ctcttcggcc actactactt 960 tgactacctg gggaacctca tcgccctggc aaacctggtg tccatttgcg tgttcctggt 1020 gctggatgca gatgtgctgc ctgctgagcg tgatgacttc atcctgggga ttctcaactg 1080 cgtcttcatt gtgtactacc tgttggagtt gctgctcaag gtctttgccc tgggcctgcg 1140 agggtacctg tcctacccca gcaacgtgtt tgacgggctc ctcaccgttg tcctgctgcc 1200 gatggccgtg gtggccagta ccgtcctggg cctggtgcag aacatgcgtg cttttggcgg 1260 gatcctggtg gtggtctact acgtatttgc catcattggg atcaacttgt ttagaggcgt 1320 cattgtggct cttcctggaa acagcagcct ggcccctgcc aatggctcgg cgccctgtgg 1380 gagcttcgag cagctggagt actgggccaa caacttcgat gactttgcgg ctgccctggt 1440 cactctgtgg aacttgatgg tggtgaacaa ctggcaggtg tttctggatg catatcggcg 1500 ctactcaggc ccgtggtcca agatctattt tgtgttgtgg tggctggtgt cgtctgtcat 1560 ctgggtcaac ctgtttctgg ccctgattct ggagaacttc cttcacaagt gggacccccg 1620 cagccacctg cagccccttg ctgggacccc agaggccacc taccagatga ctgtggagct 1680 cctgttcagg gatattctgg aggagcccgg ggaggatgag ctcacagaga ggctgagcca 1740 gcacccgcac ctgtggctgt gcaggtgacg tccgggctgc cgtcccagca ggggcggcag 1800 gagagagagg ctggcctaca caggtgccca tcatggaaga ggcggccatg ctgtggccag 1860 ccaggcagga agagaccttt cctctgacgg accactaagc tggggacagg aaccaagtcc 1920 tttgcgtgtg gcccaacaac catctacaga acagctgctg gtgcttcagg gaggcgccgt 1980 gccctccgct ttcttttata gctgcttcag tgagaattcc ctcgtcgact ccacagggac 2040 ctttcagaca aaaatgcaag aagcagcggc ctcccctgtc ccctgcagct tccgtggtgc 2100 ctttgctgcc ggcagccctt ggggaccaca ggcctgacca gggcctgcac aggttaaccg 2160 tcagacttcc ggggcattca ggtggggatg ctggtggttt gacatggaga gaaccttgac 2220 tgtgttttat tatttcatgg cttgtatgag tgtgactggg tgtgtttctt tagggttctg 2280 attgccagtt attttcatca ataagtcttg ca 2312

Claims (107)

What is claimed is:

1. 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:1-26, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90%
identical to an amino acid sequence selected from the group consisting of SEQ
ID
NO:1-8, 11, 14-18, 20, 22-23, and 25-26, c) a polypeptide comprising a naturally occurring amino acid sequence at least 93 %
identical to an amino acid sequence selected from the group consisting of SEQ
ID
NO:9 and SEQ ID NO:21, d) a polypeptide comprising a naturally occurring amino acid sequence at least 96%
identical to the amino acid sequence of SEQ ID NO:24, e) a polypeptide comprising a naturally occurring amino acid sequence at least 99%
identical to the amino acid sequence of SEQ ID NO:19, f) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-26, and g) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-26.

2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-26.

3. An isolated polynucleotide encoding a polypeptide of claim 1.

4. An isolated polynucleotide encoding a polypeptide of claim 2.

5. An isolated polynucleotide of claim 4 comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:27-52.

6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 3.

7. A cell transformed with a recombinant polynucleotide of claim 6.

8. A transgenic organism comprising a recombinant polynucleotide of claim 6.

9. A method of producing a polypeptide of claim 1, the method comprising:
a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed.

10. A method of claim 9, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-26.

11. An isolated antibody which specifically binds to a polypeptide of claim 1.

12. An isolated polynucleotide selected from the group consisting of:
a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:27-52, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ
ID NO:27-44, SEQ ID NO:46-47, SEQ ID NO:49, and SEQ ID NO:51-52, c) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 92% identical to the polynucleotide sequence of SEQ ID NO:48, d) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 99% identical to the polynucleotide sequence of SEQ ID NO:45, e) a polynucleotide consisting essentially of a naturally occurring polynucleotide sequence at least 90% identical to the polynucleotide sequence of SEQ ID
NO:50, d) a polynucleotide complementary to a polynucleotide of a), e) a polynucleotide complementary to a polynucleotide of b), f) a polynucleotide complementary to a polynucleotide of c), g) a polynucleotide complementary to a polynucleotide of d), h) a polynucleotide complementary to a polynucleotide of e), and i) an RNA equivalent of a)-h).

13. An isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide of claim 12.

14. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising:
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.

15. A method of claim 14, wherein the probe comprises at least 60 contiguous nucleotides.

16. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising:
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.

17. A composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable excipient.

18. A composition of claim 17, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-26.

19. A method for treating a disease or condition associated with decreased expression of functional TRICH, comprising administering to a patient in need of such treatment the composition of claim 17.

20. A method of screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising:
a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample.

21. A composition comprising an agonist compound identified by a method of claim 20 and a pharmaceutically acceptable excipient.

22. A method for treating a disease or condition associated with decreased expression of functional TRICH, comprising administering to a patient in need of such treatment a composition of claim 21.

23. A method of screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising:
a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample.

24. A composition comprising an antagonist compound identified by a method of claim 23 and a pharmaceutically acceptable excipient.

25. A method for treating a disease or condition associated with overexpression of functional TRICH, comprising administering to a patient in need of such treatment a composition of claim 24.

26. A method of screening for a compound that specifically binds to the polypeptide of claim 1, the method comprising:
a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1.

27. A method of screening for a compound that modulates the activity of the polypeptide of claim 1, the method comprising:
a) combining the polypeptide of claim 1 with at least one test compound under conditions permissive for the activity of the polypeptide of claim 1, b) assessing the activity of the polypeptide of claim 1 in the presence of the test compound, and c) comparing the activity of the polypeptide of claim 1 in the presence of the test compound with the activity of the polypeptide of claim 1 in the absence of the test compound, wherein a change in the activity of the polypeptide of claim 1 in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide of claim 1.

28. A method of screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence of claim 5, the method comprising:
a) exposing a sample comprising the target polynucleotide to a compound, under conditions suitable for the expression of the target polynucleotide, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.

29. A method of assessing toxicity of a test compound, the 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 of claim 12 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 12 or fragment thereof, c) quantifying the amount of hybridization complex, and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.

30. A diagnostic test for a condition or disease associated with the expression of TRICH in a biological sample, the method comprising:
a) combining the biological sample with an antibody of claim 11, under conditions suitable for the antibody to bind the polypeptide and form an antibody:polypeptide complex, and b) detecting the complex, wherein the presence of the complex correlates with the presence of the polypeptide in the biological sample.

31. The antibody of claim 11, wherein the antibody is:
a) a chimeric antibody, b) a single chain antibody, c) a Fab fragment, d) a F(ab')2 fragment, or e) a humanized antibody.

32. A composition comprising an antibody of claim 11 and an acceptable excipient.

33. A method of diagnosing a condition or disease associated with the expression of TRICH
in a subject, comprising administering to said subject an effective amount of the composition of claim 32.

34. A composition of claim 32, wherein the antibody is labeled.

35. A method of diagnosing a condition or disease associated with the expression of TRICH
in a subject, comprising administering to said subject an effective amount of the composition of claim 34.

36. A method of preparing a polyclonal antibody with the specificity of the antibody of claim 11, the method comprising:
a) immunizing an animal with a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1-26, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibodies from the animal, and c) screening the isolated antibodies with the polypeptide, thereby identifying a polyclonal antibody which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-26.

37. A polyclonal antibody produced by a method of claim 36.

38. A composition comprising the polyclonal antibody of claim 37 and a suitable carrier.

39. A method of making a monoclonal antibody with the specificity of the antibody of claim 11, the method comprising:
a) immunizing an animal with a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1-26, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibody producing cells from the animal, c) fusing the antibody producing cells with immortalized cells to form monoclonal antibody-producing hybridoma cells, d) culturing the hybridoma cells, and e) isolating from the culture monoclonal antibody which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-26.

40. A monoclonal antibody produced by a method of claim 39.

41. A composition comprising the monoclonal antibody of claim 40 and a suitable carrier.

42. The antibody of claim 11, wherein the antibody is produced by screening a Fab expression library.

43. The antibody of claim 11, wherein the antibody is produced by screening a recombinant immunoglobulin library.

44. A method of detecting a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-26 in a sample, the method comprising:
a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) detecting specific binding, wherein specific binding indicates the presence of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-26 in the sample.

45. A method of purifying a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-26 from a sample, the method comprising:
a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) separating the antibody from the sample and obtaining the purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID
NO:1-26.

46. A microarray wherein at least one element of the microarray is a polynucleotide of claim 13.

47. A method of generating an expression profile of a sample which contains polynucleotides, the method comprising:
a) labeling the polynucleotides of the sample, b) contacting the elements of the microarray of claim 46 with the labeled polynucleotides of the sample under conditions suitable for the formation of a hybridization complex, and c) quantifying the expression of the polynucleotides in the sample.

48. An array comprising different nucleotide molecules affixed in distinct physical locations on a solid substrate, wherein at least one of said nucleotide molecules comprises a first oligonucleotide or polynucleotide sequence specifically hybridizable with at least 30 contiguous nucleotides of a target polynucleotide, and wherein said target polynucleotide is a polynucleotide of claim 12.

49. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 30 contiguous nucleotides of said target polynucleotide.

50. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 60 contiguous nucleotides of said target polynucleotide.

51. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to said target polynucleotide.

52. An array of claim 48, which is a microarray.

53. An array of claim 48, further comprising said target polynucleotide hybridized to a nucleotide molecule comprising said first oligonucleotide or polynucleotide sequence.

54. An array of claim 48, wherein a linker joins at least one of said nucleotide molecules to said solid substrate.

55. An array of claim 48, wherein each distinct physical location on the substrate contains multiple nucleotide molecules, and the multiple nucleotide molecules at any single distinct physical location have the same sequence, and each distinct physical location on the substrate contains nucleotide molecules having a sequence which differs from the sequence of nucleotide molecules at another distinct physical location on the substrate.

56. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:1.

57. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:2.

58. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:3.

59. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:4.

60. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:5.

61. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:6.

62. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:7.

63. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:8.

64. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:9.

65. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:10.

66. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:11.

67. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:12.

68. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:13.

69. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:14.

70. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:15.

71. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:16.

72. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:17.

73. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:18.

74. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:19.

75. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:20.

76. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:21.

77. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:22.

78. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:23.

79. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:24.

80. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:25.

81. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:26.

82. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:27.

83. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:28.

84. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:29.

85. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:30.

86. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:31.

87. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:32.

88. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:33.

89. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:34.

90. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:35.

91. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:36.

92. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:37.

93. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:38.

94. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:39.

95. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:40.

96. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:41.

97. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:42.

98. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:43.

99. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:44.

100. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:45.

101. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:46.

102. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:47.

103. A polynucleotide of claim 12, comprising the, polynucleotide sequence of SEQ ID
NO:48.

104. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:49.

105. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:SO.

106. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:51.

107. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:52.