WO2003051902A1 - Neurotransmission-associated proteins - Google Patents

Abstract

Various embodiments of the invention provide human neurotransmission-associated proteins (NTRAN) and polynucleotides which identify and encode NTRAN. 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 NTRAN.

Description

NEUROTRANSMISSION-ASSOCIATED PROTEINS

TECHNICAL FIELD

The invention relates to novel nucleic acids, neurotransmission-associated proteins encoded by these nucleic acids, and to the use of these nucleic acids and proteins in the diagnosis, treatment, and prevention of autoirrimune/inflammatory, cardiovascular, neurological, developmental, cell proliferative, transport, psychiatric, metabolic, and endocrine disorders. The invention also relates to the assessment of the effects of exogenous compounds on the expression of nucleic acids and neurotransmission-associated proteins.

BACKGROUND OF THE INVENTION

The human nervous system, which regulates all bodily functions, is composed of the central nervous system (CNS), consisting of the brain and spinal cord, and the peripheral nervous system (PNS), consisting of afferent neural pathways for conducting nerve impulses from sensory organs to the CNS, and efferent neural pathways for conducting motor impulses from the CNS to effector organs. The PNS can be further divided into the somatic nervous system, which regulates voluntary motor activity such as for skeletal muscle, and the autonomic nervous system, which regulates involuntary motor activity for internal organs such as the heart, lungs, and viscera. CNS-associated proteins function in neuronal signaling, cell adhesion, nerve regeneration, axon guidance, neurogenesis, and other processes.

The cerebral cortex or higher brain is the largest structure, consisting of a right and a left hemisphere interconnected by the corpus callosum. The cerebral cortex is involved in sensory, motor, and integrative functions related to perception, voluntary musculoskeletal movements, and the broad range of activities associated with consciousness, language, emotions, and memory. The cerebrum functions in association with the lower centers of the nervous system. The lower areas of the brain such as the medulla, pons, mesencephalon, cerebellum, basal ganglia, substantia nigra, hypothalamus, and thalamus control unconscious activities including arterial pressure and respiration, equilibrium, and feeding reflexes, such as salivation.

The central nervous system (CNS) is composed of more than 100 billion neurons at the spinal cord level, the lower brain level, and the higher brain or cortical level. Neurons transmit electric or chemical signals between cells. The spinal cord, a thin, tubular extension of the central nervous system within the bony spinal canal, contains ascending sensory and descending motor pathways, and is covered by membranes continuous with those of the brainstem and cerebral hemispheres. The spinal cord contains almost the entire motor output and sensory input systems of the trunk and limbs, and neuronal circuits in the cord also control rhythmic movements, such as walking, and a variety of reflexes. The lower areas of the brain such as the medulla, pons, mesencephalon, cerebellum, basal ganglia, substantia nigra, hypothalamus, and thalamus control unconscious activities including arterial pressure and respiration, equilibrium, and feeding reflexes, such as salivation. Emotions, such as anger, excitement, sexual response, and reaction to pain or pleasure, originate in the lower brain. The cerebral cortex or higher brain is the largest structure, consisting of a right and a left hemisphere interconnected by the corpus callosum. The cerebral cortex is involved in sensory, motor, and integrative functions related to perception, voluntary musculoskeletal movements, and the broad range of activities associated with consciousness, language, emotions, and memory. The cerebrum functions in association with the lower centers of the nervous system. Nervous system organization and development

A nerve cell (neuron) contains four regions, the cell body, axon, dendrites, and axon terminal. The cell body contains the nucleus and other organelles. The dendrites are processes which extend outward from the cell body and receive signals from sense organs or from the axons of other neurons. These signals are converted to electrical impulses and transmitted to the cell body. The axon, whose size can range from one rmllimeter to more than one meter, is a single process that conducts the nerve impulse away from the cell body. Cytoskeletal fibers, including microtubules and neurofilaments, run the length of the axon and function in transporting proteins, membrane vesicles, and other macromolecules from the cell body along the axon to the axon terminal. Some axons are surrounded by a myelin sheath made up of membranes from either an oligodendrocyte cell (CNS) or a Schwann cell (PNS). Myelinated axons conduct electrical impulses faster than unmyelinated ones of the same diameter. The axon terminal is at the tip of the axon away from the cell body. (See Lodish, H. et al. (1986) Molecular Cell Biology Scientific American Books New York NY, pp. 715-719.) CNS-associated proteins have roles in neuronal signaling, cell adhesion, nerve regeneration, axon guidance, neurogenesis, and other functions. Certain CNS-associated proteins form an integral part of a membrane or are attached to a membrane. For example, neural membrane protein 35 (NMP35) is closely associated with neuronal membranes and is known to be highly expressed in the rat adult nervous system (Schweitzer, B. et al. (1998) Mol. Cell. Neurosci. 11:260-273). Synaptophysin (SY) is a major integral membrane protein of small synaptic vesicles. The chromosomal location of SY inhuman and mouse is on the X chromosome in subbands Xpll.22- pl 1.23. This region has been implicated in several inherited diseases including Wiskott- Aldrich syndrome, three forms of X-linked hypercalciuric neplirolithiaisis, and the eye disorders retinitis pigmentosa 2, congenital stationary night blindness, and Aland Island eye disease (Fisher, S.E. et al. (1997) Genomics 45:340-347). Peripherin, or retinal degeneration slow protein (rds), is an integral membrane glycoprotein that is present in the rims of photoreceptor outer segment disks. In mammals, rds is thought to stabilize the disk rim through heterophilic interactions with related nonglycosylated proteins. Rds is a mouse neurological mutation that is characterized by abnormal development of rod and cone photoreceptors followed by their slow degeneration (Kedzierski, W.J. et al. (1999) Neurochem. 72:430-438).

Each of over a trillion neurons in adult humans connects with over a thousand target cells (Tessier-Lavigne, M. et al. (1996) Science 274:1123-1133). These neuronal connections form during embryonic development. Each differentiating neuron sends out an axon tipped at the leading edge by a growth cone. Aided by molecular guidance cues, the growth cone migrates through the embryonic environment to its synaptic target. Progressive axon outgrowth occurs during neural development but not in the mature mammalian CNS. Following CNS injury, expression of grow -inMbiting molecules is enhanced while availability of their growth-promoting counterparts diminishes. Proteins governing developmental axon guidance contribute to the failure of injured central neurons to regenerate. These proteins include Semaphorin3A and the Semaphorin3A receptor proteins neuropilin-1 and plexin-Al (Pasterkamp, RJ. and J. Verhaagen (2001) Brain Res. Brain Res. Rev. 35:36-54).

Semaphorins function during embryogenesis by providing local signals to specify territories inaccessible to growing axons (Puschel, A.W. et al. (1995) Neuron 14:941-948). They consist of at , least 30 different members and are found in vertebrates, invertebrates, and even certain viruses. All semaphorins contain the sema domain which is approximately 500 amino acids in length. Neuropilin, a semaphorin receptor, has been shown to promote neurite outgrowth in vitro. The extracellular region of neuropilins consists of three different domains: CUB, discoidin, and MAM domains. The CUB and the MAM motifs of neuropilin have been suggested to have roles in protein-protein interactions and are thought to be involved in the binding of semaphorins through the sema and the C-terminal domains (reviewed in Raper, J. A. (2000) Curr. Opin. Neurobiol. 10:88-94).

The guidance of axons during development involves both positive and negative effects (i.e., chemoattraction and chemorepulsion). The Slit family of proteins have been implicated in promoting axon branching, elongation, and repulsion. Members of the Slit family have been identified in a variety of organisms, including insects, amphibians, birds, rodents and humans (Guthrie, S. (1999) Current Biology 9:R432-R435). Slit proteins are ligands for the repulsive guidance receptor, Roundabout (Robo); however, Slit proteins also cause elongation in some assays. A post-translationally processed form of Slit appears to be the active form of the protein (Guthrie, S. supra; and Brose, K. et al. (1999) Cell 96:795-806).

Axon growth is also guided in part by contact-mediated mechanisms involving cell surface and extracellular matrix (ECM) molecules. Many ECM molecules, including fibronectin, vitronectin, members of the laminin, tenascin, collagen, and thrombospondin families, and a variety of proteoglycans, can act either as promoters or inhibitors of neurite outgrowth and extension (Tessier- Lavigne et al., supra). Receptors for ECM molecules include integrins, immunoglobulin superfamily members, and proteoglycans. ECM molecules and their receptors have also been implicated in the adhesion, maintenance, and differentiation of neurons (Reichardt, L.F. et al. (1991) Ann. Rev. Neurosci. 14:531-571). The proteoglycan testican is localized to the post-synaptic area of pyramidal cells of the hippocampus and may play roles in receptor activity, neuromodulation, synaptic plasticity, and neurotransmission (Bonnet, F. et al. (1996) J. Biol. Chem. 271:4373-4380).

Other proteins involved in morphogenetic processes in neural tissues include members of the striatin family such as zinedin and striatin, and members of the sialoadhesins such as myelin-associated glycoprotein (MAG), Schwann cell myelin protein (SMP) and sialoadhesin. Zinedin and striatin are calmodulin-binding, WD repeat proteins primarily expressed in the brain (Castets, F. et al. (2000) J. Biol. Chem. 275:19970-19977). These two proteins share four homologous stretches of amino acids that have been determined to be involved in protein-protein interactions such as caveolin-binding and cahnodulin-binding. Both zinedin and striatin have been shown to bind calmodulin in a Ca²⁺-dependent manner. That striatin is possibly involved in the formation of dendrites has been demonstrated in cultured embryonic motoneurons in which striatin synthesis was specifically blocked. These cells had a reduced number of poorly branched dendrites, whereas the growth of axons appeared normal (Bartoli, M. et al. (1999) J. Neurobiol. 40:234-243). The role of striatin in the control of locomotion has been demonstrated in rats (Bartoli, M. et al. supra). The sialoadhesins are a major subgroup of the I-type lectins which are themselves a subfamily of the lectins. I-type lectins consist mainly of transmembranous glycoproteins belonging to the immunoglobulin superfamily. The sialoadhesins, myelin-associated glycoprotein (MAG), Schwann cell myelin protein (SMP) and sialoadhesin, are expressed in the nervous system and are generally characterized as sialic acid-binding adhesion molecules. MAG is expressed by oligodendrocytes in the CNS in L (large) and S (small) forms that are developmentally regulated such that the L form is expressed predominantly during early stages of myelination and the S form is primarily expressed in adulthood (Tropak, M.B. et al. (1988) Mol. Brain Res. 4:143-155; Pedraza, L. et al. (1991) J. Neurosci. Res. 29:141-148). MAG is also expressed in the Schwann cells in the peripheral nervous system (PNS) and has been implicated in cell recognition mechanisms underlying either the promotion or inhibition of cell adhesion and neurite outgrowth (Martini, R (1994) J. Neurocytol. 21:1-28; Bartsch, U. (1996) J. Neurocytol. 25:303-313). SMP is expressed by oligodendrocytes and in Schwann cells, has similar sialic acid-binding properties to MAG and shares 43.5% sequence identity with MAG (Dulac, C. et al. (1992) Neuron 8:323-334; Collins, B. E. et al. (1997) J. Biol. Chem. 272:16889-16895). Sialoadhesin is a membrane glycoprotein originally described as a cell surface receptor expressed by murine macrophages (Crocker, P.R. and Gordon, S. (1986) J. Exp. Med. 164:1862-1875; Crocker, P.R. and Gordon, S. (1989) J. Exp. Med. 169:1333- 1346). Expression of sialoadhesin has since been demonstrated in rat macrophages which have been induced by glucocorticoids and can be further enhanced by the addition of interferon β or γ, interleukin 4 or lipopolysaccharides (Nan den Berg T.K. et al. (1996) J. Immunol. 157:3130-3138). Expression patterns and binding specificities of sialoadhesin suggest a possible role in regulation of myeloid cell development (Crocker, P.R. et al. (1990) Blood 76:1131-1138; Crocker P.R. et al. (1995) J. Clin. Invest. 95:635-643).

Νeurotrophins regulate development, maintenance, and function of vertebrate nervous systems. Νeurotrophins activate two different classes of receptors, the Trk family of receptor tyrosine kinases and p75ΝTR, a member of the TNF receptor superfamily. Through these receptors, neurotrophins activate many signaling pathways, including those mediated by ras and members of the cdc-42/ras/rho G protein families, and by the MAP kinase, PI-3 kinase, and Jun kinase cascades. During development, limiting amounts of neurotrophins function as survival factors to ensure a match between the number of surviving neurons and the requirement for appropriate target innervation. They also regulate cell fate decisions, axon growth, dendrite pruning, the patterning of innervation, and the expression of proteins crucial for normal neuronal function, such as neurotransmitters and ion channels. These proteins also regulate many aspects of neural function. In the mature nervous system, they control synaptic function and synaptic plasticity, while continuing to modulate neuronal survival (Huang, E.J. and L.F. Reichardt (2001) Ann. Rev. Neurosci. 24:677-736). Neuritin is a protein induced by neural activity and by neurotrophins which promote neuritogenesis.

The neurexophilins are neuropeptide-like proteins which are proteolytically processed after synthesis. They are ligands for the neuron-specific cell surface proteins, the α-neurexins. Neurexophilins and neurexins may participate in a neuron signaling pathway (Missler, M. and T.C. Sudhof (1998) J. Neurosci. 18:3630-3638; Missler, M. et al. (1998) J. Biol. Chem. 273:34716-34723). Ninjurin is a neuron cell surface protein which plays a role in cell adhesion and in nerve regeneration following injury. Ninjurin is up-regulated after nerve injury in dorsal root ganglion neurons and in Schwann cells (Araki, T. and J. Milbrandt (1996) Neuron 17:353-361). Ninjurirώ is expressed in mature sensory and enteric neurons and promotes neurite outgrowth. Ninjurirώ is upregulated in Schwann cells surrounding the distal segment of injured nerve with a time course similar to that of ninjurinl, neural CAM, and LI (Araki, T. and J. Milbrandt (2000) J. Neurosci. 20:187-195).

Neurexin IN is essential for axonal insulation in the PΝS in embryos and larvae. Axonal insulation is of key importance for the proper propagation of action potentials. Caspr, a vertebrate homolog of Neurexin IV — also named paranodin — is found in septate-like junctional structures localized to the paranodal region of the nodes of Ranvier, between axons and Schwann cells. Caspr/paranodin is implicated in blood-brain barrier formation, and linkage of neuronal membrane components with the axonal cytoskeletal network (Bellen, HJ. et al. (1998) Trends Neurosci. 21:444-449). Mammalian Numb is a phosphotyrosine-binding (PTB) domam-containing protein which may be involved in cortical neurogenesis and cell fate decisions in the mammalian nervous system. Numb's binding partner, the LNX protein, contains four PDZ domains and a ring finger domain and , may participate in a signaling pathway involving Numb. PDZ domains have been found in proteins which act as adaptors in the assembly of multifunctional protein complexes involved in signaling events at surfaces of cell membranes (Ponting, C.P. (1997) Bioessays 19:469-479). LNX contains a tyrosine phosphorylation site which may be important for the binding of other PTB-containing proteins such as SHC, an adaptor protein which associates with tyrosine-phosphorylated growth factor receptors and downstream effectors (Dho, S.E. et al. (1998) J. Biol. Chem. 273:9179-9187).

Nogo has been identified as a component of the central nervous system myelin that prevents axonal regeneration in adult vertebrates. Cleavage of the Nogo-66 receptor and other , glycophosphatidylmositol-linked proteins from axonal surfaces renders neurons insensitive to Nogo-66, facilitating potential recovery from CNS damage (Fournier, A.E. et al. (2001) Nature 409:341-346).

Homeobox transcription factors direct nerve-cell associated tissue patterning and differentiation. The presence and function of these proteins appears to be ubiquitous in nematodes, arthropods, and vertebrates. One example of these proteins is DRG11, a homeobox transcription factor expressed in mammalian sensory neurons, and which appears to be involved in neural crest development (Saito, T. et al. (1995) Mol. Cell Neurosci. 6:280-292). Cutaneous sensory neurons that detect noxious stimuli project to the dorsal horn of the spinal cord, while those innervating muscle stretch receptors project to the ventral horn. DRG11 is required for the formation of spatio-temporally appropriate projections from nociceptive sensory neurons to their central targets in the dorsal horn of the spinal cord (Chen, Z.F. et al. (2001) Neuron 31:59-73). Synapses

Contact between one neuron and another occurs at a specialized site called the synapse. Many nervous system functions are regulated by diverse synaptic proteins such as synaptophysin, the synapsins, growth associated protein 43 (GAP-43), SV-2, and p65, which are distributed in subcellular compartments of the synapse. Synaptic terminals also contain many other proteins involved in calcium transport, neurotransmission, signaling, growth, and plasticity. At this site, the axon terminal from one neuron (the presynaptic cell) sends a signal to. another neuron (the postsynaptic cell). Synapses may be connected either electrically or chemically. An electrical synapse consists of gap junctions connecting the two neurons, allowing electrical impulses to pass directly from the presynaptic to the postsynaptic cell. In a chemical synapse, the axon terminal of the presynaptic cell contains membrane vesicles containing a particular neurotransmitter molecule. A change in electrical potential at the nerve terminal results in the influx of calcium ions through voltage-gated channels which triggers the release of the neurotransmitter from the synaptic vesicle by exocytosis. The neurotransmitter rapidly diffuses across the synaptic cleft separating the presynaptic nerve cell from the postsynaptic cell. The neurotransmitter then binds receptors and opens transmitter-gated ion channels located in the plasma membrane of the postsynaptic cell, provoking a change in the cell's electrical potential. This change in membrane potential of the postsynaptic cell may serve either to excite or inhibit further transmission of the nerve impulse.

Presynaptic calcium channel activity is modulated by cysteine-string proteins (CSPs). CSPs are secretory vesicle proteins that function in neurotransmission as well as in exocytosis in other cell- types. CSPs belong to the DnaJ/hsp40 (heat shock protein) chaperone family. The effect of CSPs on calcium levels is likely to be downstream of calcium release and is likely to involve exocytosis, possibly in connection with G-proteins (Braun, J.E. et al. (1995) Neuropharmacology 34:1361-9136; Magga, J.M. et al. (2000) Neuron 28:195-204; Dawson-Scully, K. et al. (2000) J. Neurosci. 20:6039-6047; and Chamberlain, L.H. et al. (2001) J. Cell Sci. 114:445-455). Neuregulins (NRGs) mediate between the electrical neural activity and molecular components by regulating the expression of ion channel receptors or transmitter release in synapses. NRGs may also be signaling factors involved in tuning locomotion or other higher functions by coordinating excitatory and inhibitory neurons (Ozaki, M. (2001) Neuroscientist 7:146-154).

N- and P/Q-type Ca²⁺ channels are localized in high density in presynaptic nerve terminals and are crucial elements in neuronal excitation-secretion coupling. In addition to mediating Ca²⁺ entry to initiate transmitter release, they are thought to interact directly with proteins of the synaptic vesicle docking/fusion machinery. N-type and P/Q-type Ca²⁺ channels are colocalized with syntaxin in high-density clusters in nerve terminals. The synaptic protein interaction (synprint) sites in the intracellular loop II-DI (Lπ-HI) of both alpha IB and alpha IA subunits of N-type and P/Q-type Ca²⁺ channels bind to syntaxin, SNAP-25, and synaptotagmin. Presynaptic Ca²⁺ channels not only provide the Ca²⁺ signal required by the exocytotic machinery, but also contain structural elements that are integral to vesicle docking, priming, and fusion processes (Catterall, W.A. (1999) Ann. NY Acad. Sci. 868:144-159). Synaptotagmins are a large family of proteins involved in both regulated and constitutive vesicular trafficking. They include a neuronal type (synaptotagmin I-V, X, and XI) and a ubiquitous type (synaptotagmin VI-LX). Ca²⁺-dependent synaptotagmin activation is involved in neurite outgrowth (Mikoshiba, K. et al. (1999) Chem. Phys. Lipids 98:59-67).

Proteins associated with the membranes of synaptic vesicles include vamp (synaptobrevin), rab3A, synaptophysin, synaptotagmin (ρ65) and SV2. These membrane proteins function in regulated exocytosis by regulating neurotransmitter uptake, vesicle targeting, and fusion with the presynaptic plasma membrane (Elferink, L.A. and R.H. Scheller (1993) J. Cell Sci. Suppl.17:75-79).

Physophilin, also known as the Ac39 subunit of the V- ATPase, is an oligomeric protein that binds the synaptic vesicle protein synaptophysin, constituting a complex that may form the exocytotic fusion pore. Ac39 is present in a synaptosomal complex which, in addition to synaptophysin, includes the bulk of synaptobrevin π, and subunits c and Acl 15 of the V0 sector of the V- ATPase. In situ hybridization in rat brain reveals a largely neuronal distribution of Ac39/ρhysophilin mRNA which correlates spatio-temporaUy with those of subunit c and synaptophysin. Immunohistochemical analysis shows that Ac39/physophilin is mostly concentrated in the neuropil with a pattern identical to subunit A and very similar to synaptophysin. Double-labeling immunofluorescence shows a complete colocalization of Ac39/ρhysophilin with subunit A and a partial colocalization with synaptophysin in the neuropil (Carrion- Vazquez M. et al. (1998) Eur. J. Neurosci.10: 1153-1166).

The plasma membrane dopamine transporter (DAT) is essential for the reuptake of released dopamine from the synapse. Uptake of dopamine is temperature- and time-dependent, and is inhibited by a variety of compounds, such as cocaine. DAT-knockout mice have been shown to exhibit extreme hyperactivity and resistance to both cocaine and amphetamine, consistent with the primary action of cocaine on DAT (Giκ>s, B. et al. (1996) Nature 379:606-612). The perturbation of the tightly regulated DAT also predisposes neurons to damage by a variety of insults. Most notable is the selective degeneration of DAT-expressing dopamine nerve terminals in the striatum thought to underlie Parkinson's disease. DAT expression can predict the selective vulnerability of neuronal populations, which suggests that therapeutic strategies aimed at altering DAT function could have significant benefits in a variety of disorders (Gary, W.M. et al. (1999) Trends Pharmacol. Sci. 20:424-429).

43 KD postsynaptic protein or acetylcholine receptor-associated 43 KD protein (RAPSYN) is thought to play a role in anchoring or stabilizing the nicotinic acetylcholine receptor at synaptic sites. RAPSYN is involved in membrane association and may link the nicotinic acetylcholine receptor to the underlying postsynaptic cytoskeleton (Buckel, A. et al. (1996) Genomics 35:613-616). Neuritin is a protein whose gene is known to be induced by neural activity and by neurotrophins which promote neuritogenesis. Neuraxin is a structural protein of the rat central nervous system that is believed to be immunologically related to microtubule-associated protein 5 (MAP5). Neuraxin is a novel type of neuron-specific protein which is characterized by an unusual amino acid composition, 12 central heptadecarepeats and putative protein and membrane interaction sites. The gene encoding neuraxin is unique in the haploid rat genome and is conserved in higher vertebrates. Neuraxin is implicated in neuronal membrane-microtubule interactions and is expressed throughout the rodent CNS (Rienitz, A. et al. (1989) EMBO J. 8:2879-2888).

Synaptic nuclear envelope-1 (Syne-1) protein is selectively associated with synaptic nuclei and may be involved in the formation or maintenance of nuclear aggregates at the neuromuscular junction. Syne-1 contains multiple spectrin repeats similar to those found in dystrophin and utrophin, as well as a domain homologous to the carboxyl-terminal of Klarsicht, a protein associated with nuclei and required for a subset of nuclear migrations in Drosophila (Apel, E. D. et al. (2000) J. Biol. Chem. 275:31986-31995.).

Clq-Related Factor (CRF) is found to be expressed at highest levels in the brain, particularly in the brainstem. In situ hybridization to mouse brain sections demonstrated that CRF transcripts are most abundant in areas of the nervous system involved in motor function, such as the Purkinje cells of the cerebellum, the accessory olivary nucleus, the pons and the red nucleus. CRF is predicted to encode a polypeptide with a hydrophobic signal sequence, a collagenous region, and a globular domain at the carboxy terminus that shares homology to the Clq signature domain, a subunit of the Cl enzyme complex that activates the serum complement system (Berube, N. G. et al. (1999) Brain Res. Mol. Brain Res. 63:233-240).

Neurotransmitters and neurotransmitter transport proteins

Contact from one neuron to another occurs at a specialized site called the synapse. At this site, the axon terminal from one neuron (the presynaptic cell) sends a signal to another neuron (the postsynaptic cell). Synapses maybe connected either electrically or chemically. An electrical synapse consists of gap junctions connecting the two neurons, allowing electrical impulses to pass directly from the presynaptic to the postsynaptic cell. In a chemical synapse, the axon terminal of the presynaptic cell contains membrane vesicles containing a particular neurotransmitter molecule. A change in electrical potential at the nerve terminal results in the influx of calcium ions through voltage- gated channels which triggers the release of the neurotransmitter from the synaptic vesicle by exocytosis. The neurotransmitter rapidly diffuses across the synaptic cleft separating the presynaptic nerve cell from the postsynaptic cell. The neurotransmitter then binds receptors and opens transmitter-gated ion channels located in the plasma membrane of the postsynaptic cell, provoking a change in the cell's electrical potential. This change in membrane potential of the postsynaptic cell may serve either to excite or inhibit further transmission of the nerve impulse. Presynaptic calcium channel activity is modulated by cysteine-string proteins (CSPs). CSPs are secretory vesicle proteins that function in neurotransmission as well as in exocytosis in other cell-types. CSPs belong to the DnaJ/hsp40 (heat shock protein) chaperone family. The effect of CSPs on calcium levels is likely to be downstream of calcium release and is likely to involve exocytosis, possibly in connection with G- proteins (Braun, J.E. et al. (1995) Neuropharmacology 34:1361-9136; Magga, J.M. et al. (2000) Neuron 28:195-204; Dawson-Scully, K. et al. (2000) J. Neurosci. 20:6039-6047; and Chamberlain, L.H. et al. (2001) J. Cell Sci. 114:445-455).

Neurotransmitters comprise a diverse group of some 30 small molecules which include acetylcholine, monoamines such as serotonin, dopamine, and Mstamine, and amino acids such as gamma-aminobutyric acid (GABA), glutamate, and aspartate, and neuropeptides such as endorphins and enkephalins (McCance, K.L. and S.E. Huether (1994) PATHOPHYSIOLOGY. The Biologic Basis for Disease in Adults and Children, 2nd edition, Mosby, St. Louis, MO, pp. 403-404). Many of these molecules have more than one function and the effects may be excitatory, e.g. to depolarize the postsynaptic cell plasma membrane and stimulate nerve impulse transmission, or inhibitory, e.g. to hyperpolarize the plasma membrane and inhibit nerve impulse transmission.

Neurotransmitters and their receptors are targets of pharmacological agents aimed at controlling neurological function. For example, GABA is the major inhibitory neurotransmitter in the CNS, and GABA receptors are the principal target of sedatives such as benzodiazepines and barbiturates which act by enhancing GABA-mediated effects (Katzung, B.G. (1995) Basic and Clinical Pharmacology, 6th edition, Appleton & Lange, Norwalk, CT, pp. 338-339).

Two major classes of neurotransmitter transporters are essential to the function of the nervous system. The first class is uptake carriers in the plasma membrane of neurons and glial cells, which pump neurotransmitters from the extracellular space into the cell. This process relies on the Na⁺ gradient across the plasma membrane, particularly the co-transport of Na⁺. Two families of proteins have been identified. One family includes the transporters for GABA, monoamines such as noradrenaline, dopamine, and serotonin, and amino acids such as glycine and proline. Common structural components include twelve putative transmembrane a-helical domains, cytoplasmic N- and C- termini, and a large glycosylated extracellular loop separating transmembrane domains three and four. This family of homologous proteins derives their energy from the co-transport of Na⁺ and Cl" ions with the neurotransmitter into the cell (Na⁺/Cl" neurotransmitter transporters). The second family includes transporters for excitatory amino acids such as glutamate. Common structural components include 6-10 putative transmembrane domains, cytoplasmic N- and C- termini, and glycosylations in the extracellular loops. The excitatory amino acid transporters are not dependent on Cl", and may require intracellular K⁺ ions (Na⁺/K⁺- neurotransmitter transporters) (Liu, Y. et al. (1999) Trends Cell Biol. 9:356-363).

The second class of neurotransmitter transporters is present in the vesicle membrane, and concentrates neurotransmitters from the cytoplasm into the vesicle, before exocytosis of the vesicular contents during synaptic transmission. Vesicular transport uses the electrochemical gradient across the vesicular membrane generated by a H⁺- ATPase. Two families of proteins are involved in the transport of neurotransmitters into vesicles. One family uses primarily proton exchange to drive transport into secretory vesicles and includes the transporters for monoamines and acetylcholine. For example, the monoamine transporters exchange two luminal protons for each molecule of cytoplasmic transmitter. The second family includes the GABA transporter, which relies on the positive charge inside synaptic vesicles. The two classes of vesicular transporters show no sequence similarity to each other and have structures distinct from those of the plasma membrane carriers (Schloss, P. et al. (1994) Curr. Opin. Cell Biol. 6:595-599; Liu et al., supra). GABA is the predominant inhibitory neurotransmitter and is widely distributed in the mammalian nervous system. GABA is cleared from the synaptic cleft by specific, Mgh-affinity, Na⁺- and Cl- dependent transporters, which are thought to be localized to both pre- and postsynaptic neurons, as well as to surrounding glial cells. At least four GABA transporters (GAT1-GAT4) have been cloned (Liu, Q.-R. et al. (1993) J. Biol. Chem. 268:2106-2112). Studies of [³H]-GABA uptake into cultured cells and plasma-membrane vesicles isolated from various tissues revealed considerable differences in GABA transporter heterogeneity. GABA transporters exhibit differences in substrate affinity and specificity, distinct blocker pharmacologies, and different tissue localization. For example, the K^ values of GABA uptake of the expressed GATl to GAT4 are 6, 79, 18, and 0.8 mM, respectively. In addition to transporting GABA, GAT2 also transports betaine; GAT3 and GAT4 also transport β-alanine and taurine. Pharmacological studies revealed that GABA transport by GATl and GAT4 is more sensitive to 2,4-diaminobutyric acid and guavicine than that by GAT2 and GAT3. In situ hybridization showed that GATl and GAT4 expression is brain specific. GAT2 and GAT3 mRNAs were detected in tissues such as liver and kidney (Schloss et al., supra; Borden, L.A. (1996) Neurochem. Int. 29:335-356; Nelson, N. (1998) J. Neurochem. 71:1785-1803).

Human studies indicated that GABA transporter function is reduced in epileptic hippocampi. Decreased GABAergic neurotransmission has also been implicated in the pathophysiology of schizophrenia (Simpson, M.D. et al. (1992) Psychiatry Res. 42:273-282). Diazepam binding inhibitor (DBI), also known as endozepine and acyl-Coenzyme (CoA)- binding protein, is an endogenous GABA receptor ligand which is thought to down-regulate the effects of GABA. DBI binds medium- and long-chain acyl-CoA esters with very high affinity and may function as an intracellular carrier of acyl-CoA esters (* 125950 Diazepam Binding Inhibitor; DBI, Online Mendelian Inheritance in Man (OMIM); PROSITE PDOC00686 Acyl-CoA-binding protein signature).

Glycine serves as one of the major inhibitory neurotransmitters in the mammalian nervous system by activating chloride-channel receptors, which are members of a ligand-gated ion-channel superfamily (Betz, H. (1990) Neuron 5:383-392). Glycine also facilitates excitatory transmission through an allosteric activation of the N-methyl-D-aspartate (NMD A) receptor (Johnson, J.W. and P. Ascher (1987) Nature 325:529-531). Forms of glycine transporter include GLYT 1 and GLYT 2. Variants of GLYT1 (GLYT1 a/b) are generated by alternative splicing (Liu, Q.-R. et al. (1993) J. Biol. Chem. 268:22802-22808). GLYTla is transcribed in both neural and non-neural tissues, whereas GLYTlb was detected only in neural tissues (Borowsky, B. et al. (1993) Neuron 10:851-863). High levels of GLYTla/b mRNA were found in hippocampus and cortex, implying its involvement in the regulation of excitatory synaptic transmission. It is not clear whether GLYTla is expressed in neurons, in glia or in both. In contrast, GLYTlb is found almost exclusively in fiber tracts, suggesting its localization in glial cells (Schloss et al., supra). GLYT2 is expressed mainly in brainstem and spinal cord (Schloss et al., supra).

The second identified glycine transporter (GLYT2) differs from GLYTla/b by its extended intracellular amino tenninus. The predominant locahzation of its mRNA in brainstem and spinal cord and its insensitivity to N-memyl-aminoacetic acid suggests that GLYT2 terminates signal transduction at the strychnine-sensitive inhibitory glycine receptor. It has been proposed that, upon depolarization of cells harboring GLYTlb, the transporter runs backwards and releases glycine to act as a neuromodulatory amino acid at the NMDA receptor (Attwell, D. and M. Bouvier (1992) Curr. Biol. 2:541-543). Such a Ca²⁺-independent, non-vesicular release of neurotransmitters by reverse transport was demonstrated for glutamate and serotonin. This evidence suggests that the transmitter transporters may be important for both the initiation and teπriination of neurotransmitter action (Schloss et al., supra). The plasma membrane dopamine transporter (DAT) is essential for the reuptake of released dopamine from the synapse. Uptake of dopamine is temperature- and time-dependent, and is inhibited by a variety of compounds, such as cocaine. DAT- knockout mice have been shown to exhibit extreme hyperactivity and resistance to both cocaine and amphetamine, consistent with the primary action of cocaine on DAT (Giros, B. et al. (1996) Nature 379:606-612). The perturbation of the tightly regulated DAT also predisposes neurons to damage by a variety of insults. Most notable is the selective degeneration of DAT-expressing dopamine nerve terminals in the striatum thought to underlie Parkinson's disease. DAT expression can predict the selective vulnerability of neuronal populations, which suggests that therapeutic strategies aimed at altering DAT function could have significant benefits in a variety of disorders (Gary, W.M. et al. (1999) Trends Pharmacol. Sci. 20:424-429).

Creatine transporters are strongly related to transporters for GABA. The primary sequence identity between creatine transporter species homologs is very high (98-99%). Pharmacological characterization demonstrated high affinity creatine uptake (27-43 mM), which was blocked by creatine analogs with high affinity. Creatine transporters are widely expressed in a variety of mammalian tissues, including brain, adrenal gland, intestine, colon, prostate, thymus, ovary, spleen, pancreas, placenta, umbilical cord, thyroid, tongue, pharynx, vertebral discs, jaw, and nasal epithelium. Genetic mapping in the mouse localizes the creatine transporter to a region on the X chromosome in linkage conservation with the human region Xq28, the location of the genes for several neuromuscular diseases (Nash, S.R. et al. (1994) Receptors Channels 2:165-174).

The substrates of a number of cDNA clones encoding proteins of the Na⁺ /Cl"-dependent transporter families are still not identified. These are orphan transporters. Identification of the substrates for orphan transporters has been difficult because in situ hybridization and immunohistochemistry indicate that the transporters are synthesized by phenotypically different neuronal populations, for example glutaminergic, GABAergic, Mstaminergic, or serotoninergic neurons. One of the transporters, NTT4, exhibits the highest homology to the creatine transporter. It differs structurally from other members of this family in having an unusually long loop between transmembranes seven and eight (Liu, Q.-R. et al. (1993) FEBS Lett. 315:114-118; Schloss et al., supra). Glutamate is a major excitatory neurotransmitter in the mammalian central nervous system.

Electrogenic (Na⁺ /K⁺)-coupled glutamate transporters, located in the plasma membranes of nerve teπninals and glial cells, mediate removal of glutamate released at excitatory synapses and maintain extracellular concentrations below neurototoxic levels. Glutamate transporters achieve this process by co-transport with three sodium ions and one proton, followed by translocation of a potassium ion in the opposite direction (Zerangue, N. and M.P. Kavanaugh (1996) Nature 383:634-637).

Glutamate transporters belong to a large family of transport proteins. The membrane topology of the glutamate transporters reveals six membrane-spanning helices in the N-terminal part of the proteins (Slotboom, D.J. et al. (1999) Microbiol. Mol. Biol. Rev. 63:293-307). The C-terminal half of the glutamate transporters is well conserved and constitutes a major part of the translocation pathway and contains the binding sites for the substrate and co-transported ions (Zhang, Y. and B.I. Kanner (1999) Proc. Natl. Acad. Sci. USA 96:1710-1715).

The membrane topology of the glutamate transporters reveals six membrane-spanning helices in the N-terminal part of the proteins (Slotboom, D.J. et al. (1999) Microbiol. Mol. Biol. Rev.

63:293-307). The C-terminal half of the glutamate transporters is well conserved and constitutes a major part of the translocation pathway and contains the binding sites for the substrate and co- transported ions (Zhang, Y. and B.I. Kanner (1999) Proc. Natl. Acad. Sci. USA 96:1710-1715).

Impaired re-uptake of synaptic glutamate, and a reduced expression of glutamate transporters have been found in the motor cortex of patients with amyofrophic lateral sclerosis (ALS). Inhibition of the synthesis of each glutamate transporter subtype using chronic antisense oligonucleotide administration, in vitro and in vivo, selectively and specifically reduced the protein expression and function of glutamate transporters. The loss of glial glutamate transporters produced elevated extracellular glutamate levels, neurodegeneration characteristic of excitotoxicity, and a progressive paralysis. The loss of the neuronal glutamate transporter did not elevate extracellular glutamate in the striatumbut produced mild neurotoxicity and resulted in epilepsy (Rothstein, J.D. et al. (1996) Neuron 16:675-686).

The vesicular monoamine transporters (VMAT) package cytoplasmic monoamine neurotransmitters into secretory vesicles for regulated exocytotic release. VMAT acts as an electrogenic exchanger of protons and monoamines, using a proton electrochemical gradient. VMAT transporters include VMAT1 and VMAT2. The VMAT proteins possess twelve transmembrane segments, with both extremities lying on the cytoplasmic side. VMAT proteins are associated with distinct vesicle populations in neurons and neuroendocrine cells (Henry, J.-P. et al. (1994) J. Exp. Biol. 196:251-262). Vesicular transport is inhibited by the antihypertensive drug reserpine and the related but more centrally acting drug tetrabenazine. The mechanism of transport and the biochemistry of VMAT have been analyzed with these drugs, using mainly the chromaffin granules from bovine adrenal glands as a source of transporters (Peter, D. et al. (1994) J. Biol. Chem. 269:7231-7237). Human studies indicated that reserpine can cause a syndrome resembling depression, indicating the importance of vesicular transport activity for the control of mood and behavior. The psychostimulant amphetamine also disrupts the storage of amines in secretory vesicles, further indicating that alterations in vesicular monoamine transport can affect behavior (Sulzer, D. and S. Rayport (1990) Neuron 5:797-808).

Human diseases caused by defects in neurotransmitter transporters include schizophrenia, Tourette's syndrome, Parkinson's disease, brain ischemia, amyofrophic lateral scerlosis, depression, and epilepsy. For example, decreased GABAergic neurotransmission has been implicated in the pathophysiology of CNS disorders such as epilepsy and schizophrenia. Impaired re-uptake of synaptic glutamate, and a reduced expression of the glutamate fransporter have been found in the motor cortex of patients with amyofrophic lateral sclerosis (ALS). The loss of glial glutamate transporters produces elevated extracellular glutamate levels, neurodegeneration characteristic of excitotoxicity, and a progressive paralysis. The loss of neuronal glutamate transporters produces mild neurotoxicity and result in epilepsy (Rothstein, J.D. et al. (1996) Neuron 16:675-686). Transporters for dopamine, norepinephrine, and serotonin have particular significance as targets for clinically relevant psychoactive agents including cocaine, antidepressants, and amphetamines. Cocaine and antidepressants are transporter antagonists that act with varying degrees of specificity to enhance synaptic concentrations of amines by limiting clearance. Amphetamines enhance transporter mediated efflux in concert with a depletion of vesicular amine stores (Barker, , E.L. and R.D. Blakely (1995) Psychopharmacology 28:321-333; Sulzer, D. and S. Rayport (1990) Neuron 5:797-808; Wall, S.C. et al. (1995) Mol. Pharmacol. 47:544-550).

Each of over a trillion neurons in adult humans connects with over a thousand target cells (Tessier-Lavigne, M. et al. (1996) Science 274:1123-1133). These neuronal connections form during embryonic development. Each differentiating neuron sends out an axon tipped at the leading edge by a growth cone. Aided by molecular guidance cues, the growth cone migrates through the embryonic environment to its synaptic target. Semaphorins are growth cone guidance signals that may function during embryogenesis by providing local signals to specify territories inaccessible to growing axons (Puschel, A.W. et al. (1995) Neuron 14:941-948).

The guidance of axons during development involves both positive and negative effects (Le., chemoattraction and chemorepulsion). The Slit family of proteins have been implicated in promoting axon branching, elongation, and repulsion. Members of the Slit family have been identified in a variety of organisms, including insects, amphibians, birds, rodents and humans (Guthrie, S. (1999) Current Biology 9:R432-R435). Slit proteins appear to be ligands for the repulsive guidance receptor, Roundabout (Robo); however, Slit proteins also cause elongation in some assays. A post- translationally processed form of Slit appears to be the active form of the protein (Guthrie, S. supra and Brose, K. et al. (1999) Cell 96:795-806).

Axon growth is also guided in part by contact-mediated mechanisms involving cell surface and extracellular matrix (ECM) molecules. Many ECM molecules, including fibronectin, vifronectin, members of the laminin, tenascin, collagen, and thrombospondin families, and a variety of proteoglycans, can act either as promoters or inhibitors of neurite outgrowth and extension (Tessier- Lavigne et al., supra). Receptors for ECM molecules include integrins, immunoglobulin superfamily members, and proteoglycans. ECM molecules and their receptors have also been implicated in the adhesion, maintenance, and differentiation of neurons (Reichardt, L.F. et al. (1991) Ann. Rev.

Neurosci. 14:531-571). The proteoglycan testican is localized to the post-synaptic area of pyramidal cells of the hippocampus and may play roles in receptor activity, neuromodulation, synaptic plasticity, and neurotransmission (Bonnet, F. et al. (1996) J. Biol. Chem. 271:4373-4380).

Other nervous system-associated proteins have roles in neuron signaling, cell adhesion, nerve regeneration, axon guidance, and neurogenesis. The neurexophilins are neuropeptide-like proteins which are proteolytically processed after synthesis. They are ligands for the neuron-specific cell surface proteins, the α-neurexins. NeurexopMlins and neurexins may participate in a neuron signaling pathway (Missler, M. and T.C. Sudhof (1998) J. Neurosci. 18:3630-3638; Missler, M. et al. (1998) J. Biol. Chem. 273 :34716-34723). Ninjurin is a neuron cell surface protein which plays a role in cell adhesion and in nerve regeneration following injury. Ninjurin is up-regulated after nerve injury in dorsal root ganglion neurons and in Schwann cells (*602062 Ninjurin; NINJ1 OMTM; Araki, T. and Milbrandt, J. (1996) Neuron 17:353-361). Mammalian Numb is a phosphotyrosine-binding (PTB) domam-containing protein which may be involved in cortical neurogenesis and cell fate decisions in the mammalian nervous system. Numb's binding partner, the LNX protein, contains four PDZ domains and a ring finger domain and may participate in a signaling pathway involving Numb. PDZ domains have been found in proteins which act as adaptors in the assembly of multifunctional protein complexes involved in signaling events at surfaces of cell membranes (Ponting, C.P. (1997) Bioessays 19:469-479). LNX contains a tyrosine phosphorylation site which may be important for the binding of other PTB-containing proteins such as SHC, an adaptor protein which associates with tyrosine- phosphorylated growth factor receptors and downstream effectors (Dho, S.E. et al. (1998) J. Biol. Chem. 273:9179-9187).

Another family of molecules that appear to be important for neurotransmission are the choHne-transporter-like CTLl proteins. The prototypic CTLl was identified in yeast as a suppressor of a choline transport mutation; however, mammalian homologues have been identified. The proteins comprise approximately ten putative transmembrane domains in addition to transporter-like motifs but do not appear to be canonical choline transporters. Choline transport is important to neurotransmission because choline is a precursor of acetylcholine, required in abundance by cholinergic neurons (ORegan, S. et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97:1835-1840).

Transcriptional regulatory proteins are also essential for the development of the nervous system and elements of neurotransmission. A specific class of transcription factors, homeobox transcription factors, directs nerve cell-associated tissue patterning and differentiation. The presence and function of these proteins appears to be ubiquitous in nematodes, arthropods, and vertebrates. One example of these proteins is DRGl 1, a homeobox transcription factor expressed in mammalian sensory neurons, and which appears to be involved in neural crest development (Saito, T. et al. (1995) Mol. Cell Neurosci. 6:280-292). cDNA clones were selected by comparing libraries of normal mouse cerebellar cDNA and cerebellar cDNA from Purkinje cell degeneration (pcd) mice. An mRNA present in Purkinje neurons encodes PCD5, a protein of 99 amino acids. PCD5's expression is restricted to the cerebellum and the eye. The gene encoding PCD5 was localized to mouse chromosome 8 (Nordquist, D.T. et al. (1988) J. Neurosci. 8(12):4780-4789).

Neuronal signals are transmitted across the neuromuscular junction (NMJ). Motor axons release the molecule agrin to induce the formation of the postsynaptic apparatus in muscle fibers. Proteins such as dystroglycan, MuSK, and rapsyn participate in the transduction of agrin signals. Agrin also functions in the upregulation of gene transcription in myonuclei and the control of presynaptic differentiation (Ruegg, M.A. and J.L. Bixby (1998) Trends Neurosci. 21:22-27). Neurological protein domains

CNS-associated proteins can be phosphoproteins. For example, ARPP-21 (cyclic AMP-regulated phosphoprotein) is a cytosolic neuronal phosphoprotem that is highly enriched in the striatum and in other dopaminoceptive regions of the brain. The steady-state level of ARPP-21 mRNA is developmentally regulated. But, in the neonatal and mature animal, ARPP-21 mRNA is not altered following 6-hydroxydopamine lesions of the substantia nigra or by pharmacologic treatments that upregulate the DI- or D2-dopamine receptors (Ehrlich, M.E. et al. (1991) Neurochem. 57:1985- 1991).

CNS-associated signaling proteins may contain PDZ domains. PDZ domains have been found in proteins which act as adaptors in the assembly of multifunctional protein complexes involved in signaling events at surfaces of cell membranes. PDZ domains are generally found in membrane- associated proteins including neuronal nitric oxide synthase (NOS) and several dystrophin-associated proteins (Ponting, C.P. et al. (1997) Bioessays 19:469-479). PSD-95/SAP90 is a membrane- associated guanylate kinase found in neuronal cells at the postsynaptic density (PSD) (Takeuchi, M. et al. (1997) J. Biol. Chem. 272:11943-11951). PSD-95/SAP90 contains three PDZ domains, one SH3 domain, and one guanylate kinase domain. The PDZ domains mediate interactions with NMDA receptors, Shaker-type potassium channels, and brain nitric oxide synthase. SAPAPs (SAP90/PSD- 95-Associated Proteins) promote localization of PSD-95/SAP90 at the plasma membrane.

CNS-associated proteins may also contain epidermal growth factor (EGF) domains. The Notch proteins are transmembrane proteins which contain extracellular regions of repeated EGF domains. Notch proteins, such as the Drosophila melanogaster neurogenic protein Notch, are generally involved in the inhibition of developmental processes. Other members of the Notch family are the lin-12 and glp-1 genes of Caenorhabditis elegans. Genetic studies indicate that the lin-12 and glρ-1 proteins act as receptors in specific developmental cell interactions which may be involved in certain embyronic defects (Tax, F. E. et al. (1994) Nature 368:150-154). Pecanex, a maternal-effect neurogenic locus of D. melanogaster, is believed to encode a large transmembrane protein. In the absence of maternal expression of the pecanex gene, an embryo develops severe hyperneuralization similar to that characteristic of Notch mutant embryos (LaBonne, S. G. et al. (1989) Dev. Biol. 136:1-116).

Other CNS-associated signaling proteins contain WW domains. The WW domain is a protein motif with two highly conserved tryptophans. It is present in a number of signaling and regulatory proteins, including Huntingtin interacting protein. Several fibroblast growth factor (FGF) homologous factors (i.e., FHF polypeptides) have also been implicated in nervous system development based on mRNA expression patterns in mouse and human tissues. Members of the FHF family of polypeptides are structurally distinct from prototypic FGFs, consistent with the unusual role of these FGF-related proteins (SmaUwood, P.M. et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:9850-9857 and Hartung, H. et al. (1997) Mech. Dev. 64:31-39).

CNS-associated proteins may also contain leucine rich repeats (LRR) which are short motifs found in numerous proteins from a wide range of species. LRR motifs are of variable length, most commonly 20-29 amino acids, and multiple repeats are typically present in tandem. LRR motifs are important for protein/protein interactions, and LRR proteins are involved in cell/cell interactions, morphogenesis, and development (Kobe, B. and Deisenhofer, J. (1995) Curr. Opin. Struct. Biol. 5:409- 416). The human ISLR (immunoglobulin superfamily containing leucine-rich repeat) protein contains a C2-type immunoglobulin domain as well as LRR motifs. The ISLR gene is linked to the critical region for Bardet-Biedl syndrome, a developmental disorder of which the most common feature is retinal dystrophy (Nagasawa, A. et al. (1999) Genomics 61:37-43). Brain leucine rich repeat protein, a member of the LRR family of proteins, possibly functions in nervous system development and maintenance (Taniguchi, H. et al. (1996) Brain Res. Mol. Brain Res. 36:45-52). Disorders associated with neurological processes

Alzheimer's disease (AD) is a degenerative disorder of the CNS which causes progressive memory loss and cognitive decline during mid to late adult life. AD is characterized by a wide range of neuropathologic features including amyloid deposits and infra-neuronal neurofibrillary tangles. Although the pathogenic pathway leading to neurodegeneration and AD is not well understood, at least three genetic loci that confer genetic susceptibility to the disease have been identified (Schellenberg, GD. (1995) Proc. Natl. Acad. Sci. 92:8552-8559; Sherr ngton, R. et al. (1995) Nature 375:754-760). Familial British dementia (EBD), is an autosomal dominant disease featuring amyloid plaques surrounded by astrocytes and microglia, neurofibrillary tangles, neuronal loss, and progressive dementia. The BRI gene on chromosome 13 encodes a 4 kD peptide, A-Bri. This membrane- anchored protein is a primary constituent of amyloid deposits, and its presence in lesions from the CNS of EBD patients maybe a contributive factor of this disease (El-Agnaf, O.M.A. et al. (2001) Biochemistry 40:3449-3457).

Astrocytomas, and the more malignant glioblastomas, are the most common primary tumors of the brain, accounting for over 65% of primary brain tumors. These tumors arise in glial cells of the astrocyte lineage. Following infection by pathogens, astrocytes function as antigen-presenting cells and modulate the activity of lymphocytes and macrophages. Astrocytomas constitutively express many cytokines and interleukins that are normally produced only after infection by a pathogen (de Micco, C. (1989) J. Neuroimmunol. 25:93-108). In the course of identifying genes related to astrocyte differentiation, one cDNA was isolated from an astrocytoma cDNA library that encodes a protein structurally related to the plant pathogenesis-related (PR) proteins (Murphy, EN. et al. (1995) Gene 159:131-135). The glioma pathogenesis-related protein (GHPR) is highly expressed in glioblastoma, but not in fetal or adult brain, or in other nervous system tumors. PR proteins are a family of small (10-20 kDa), protease resistant proteins induced in plants by viral infections, such as tobacco mosaic virus. The synthesis of PR proteins is believed to be part of a primitive immunological response in plants (van Loon, L.C. (1985) Plant Mol. Biol. 4:111-116). GliPR shares up to 50% homology with the PR-1 protein family over a region that comprises almost two thirds of the protein, including a conserved triad of amino acids, His-Glu-His, appropriately spaced to form a metal-binding domain (Murphy et al., supra). Signaling initiated by the Trk family receptors plays a dynamic role in neurogenic tumors. The proto-oncogene Trks encode the mgh-affinity receptor tyrosine kinases for nerve growth factor (NGF) neurotoophins. A rearranged Trk oncogene is often observed in non-neuronal neoplasms such as colon and papillary thyroid cancers. The proto-oncogene Trks regulates growth, differentiation and apoptosis of tumors of neuronal origin, such as neuroblastoma and medulloblastoma (Nakagawara, A. (2001) Cancer Lett.l69:107-114).

Neuronal thread proteins (NTP) are a group of immunologically related molecules found in the brain and neuroectodermal tumor cell lines. NTP expression is increased in neuronal cells during proliferation, differentiation, brain development, in Alzheimer's disease (AD) brains, and in pathological states associated with regenerative nerve sprouting (de la Monte, S.M. et al. (1996) J. Neuropathol. Exp. Neurol. 55:1038-1050). Monoclonal antibodies generated to a recombinant NTP, AD7C-NTP, isolated from an end-stage AD brain library, showed high levels of NTP immunoreactivity in perikarya, neuropil fibers, and white matter fibers of AD brain tissue. In vitro studies also demonstrated NTP upregulation, phosphorylation, and translocation from the perikarya to cell processes and growth cones during growth factor-induced neuritic sprouting and neuronal differentiation. Additionally, increased NTP immunoreactivity was found in Down syndrome brains beginning in the second decade, prior to establishment of widespread AD neurodegeneration, and at an age when a low-level or an absence of NTP expression was observed in control brains. These findings indicated that abnormal expression and accumulation of NTP in brain may be an early marker of AD neurodegeneration in Down syndrome (de la Monte, S.M. et al. (1996) J. Neurol. Sci. 135:118- 125). Furthermore, the increased expression and accumulation of NTP in AD brain tissue was paralleled by corresponding elevations of NTP in cerebrospinal fluid (CSF), and elevated levels of NTP were detectable in the CSF early in the course of the disease.

Ubiquilin is a presenilin-interacting protein. Ubiquilin contains numerous ubiquitin-like domains thought to be involved in targeting proteins for degradation. However, ubiquilin promotes increased presenilin protein accumulation. Ubiquilin, as a modulator of presenilin levels may have implications involving various cellular functions. Presenilins are linked to a variety of biological processes, including calcium regulation, Notch signaling, apoptosis, regulation of the cell cycle, including the unfolded-protein response and β-amyloid precursor protein-associated gamma secretase activity. Expression of ubiquilin is highest in human brain neurons and is associated with neurofibrillary tangles and Lewy bodies of Alzheimer's disease and Parkinson's disease brains, respectively (Mah,A.L., et al. (2000) J. Cell Biol. 151:847-862).

Fe65-like protein (Fe65L2), a new member of the Fe65 protein family, is one of the ligands that interacts with the cytoplasmic domain of Alzheimer beta-amyloid precursor protein (APP). Transgenic mice expressing APP simulate some of the prominent behavioral and pathological features of Alzheimer's disease, including age-related impairment in learning and memory, neuronal loss, gliosis, neuritic changes, amyloid deposition, and abnormal tau phosphorylation (Duilio, A. et al. (1998) Biochem. J. 330:513-519).

Amyofrophic lateral sclerosis (ALS) is characterized by motor neuron death, altered peroxidase activity of mutant SOD1, changes in intracellular copper homeostasis, protein aggregation, and changes in the function of glutamate transporters leading to excitotoxicity. Neurofilaments and peripherin appear to play some part in motor neuron degeneration. ALS is occasionally associated with mutations of the neurofilament heavy chain gene (Al-Chalabi, A. and P.N. Leigh (2000) Curr. Opin. Neurol. 13:397-405). Cytoskeletal abnormalities such as abnormal inclusions containing neurofilaments (NFs) and/or peripherin, reduced mRNA levels for the NF light (NF-L) protein and mutations in the NF heavy (NF-H) gene have been observed in ALS. Intermediate filament inclusions containing peripherin may play a contributory role in ALS (Julien, J.P. and J.M. Beaulieu (2000) J. Neurol. Sci.180:7-14).

Miller-Dieker syndrome (MDS) or isolated lissencephaly syndrome (ILS) are characterized by a smooth cerebral surface, a thickened cortex with four abnormal layers, and misplaced neurons. Both conditions may result from deletion or mutation in the LIS1 gene. The lissencephaly gene product Lisl is a component of evolutionarily conserved intracellular multiprotein complexes essential for neuronal migration, and which may be components of the machinery for cell proliferation and intracellular transport (Leventer, RJ. et al. (2001) Trends Neurosci. 24:489-492). NudC, a nuclear movement protein, interacts with Lisl (Morris, S.M. et al. (1998) Curr. Biol. 8:603-606).

CNS-associated proteins can also be phosphoproteins. For example, ARPP-21 (cyclic AMP-regulated phosphoprotein) is a cytosolic neuronal phosphoprotein that is highly enriched in the striatum and in other dopaminoceptive regions of the brain. The steady-state level of ARPP-21 mRNA is developmentally regulated. But, in the neonatal and mature animal, ARPP-21 mRNA is not altered following 6-hydroxydopamine lesions of the substantia nigra or by pharmacologic treatments that upregulate the DI- or D2-dopamine receptors. (Ehrlich, M. E. et al. (1991) Neurochem. 57:1985- 1991.) Retinitis pigmentosa comprises a group of slowly progressive, inherited disorders of the retina that cause loss of night vision and peripheral visual field in adolescence. A recessive nonsense mutation in the Drosophila opsin gene causes photoreceptor degeneration. In some families, genes encoding rhodopsin and peripherin/RDS map very close to the disease loci. Rhodopsin and peripherin/RDS mutations have been found in approximately 30% of all autosomal dominant cases (Shastry, B.S. (1994) Am. J. Med. Genet. 52:467-474).

Astrocytomas, and the more malignant glioblastomas are primary tumors of the brain. The glioma pathogenesis-related protein (GliPR) is highly expressed in ghoblastoma, but not in fetal or adult brain, or in other nervous system tumors (Murphy, EN. et al. (1995) Gene 159:131-135). Signaling initiated by the Trk family receptors plays a dynamic role in neurogenic tumors. The proto-oncogene Trks encode the Hgh-affinity receptor tyrosine kinases for nerve growth factor (ΝGF) neurotrophins. A rearranged Trk oncogene is often observed in non-neuronal neoplasms such as colon and papillary thyroid cancers. The proto-oncogene Trks regulates growth, differentiation and apoptosis of tumors of neuronal origin, such as neuroblastoma and medulloblastoma (Nakagawara, A. (2001) Cancer Lett.l69:107-114).

Synaptic proteins are involved in Alzheimer's disease (AD) and other disorders including ischemia, a variety of disorders where synapse-associated proteins are abnormally accumulated in the nerve terminals or synaptic proteins are altered after denervation, and neoplastic disorders (Masliah, E. and R. Terry (1993) Brain Pathol. 3:77-85). Synaptophysin (SY), a major integral membrane protein of small synaptic vesicles, is on the X chromosome in subbands Xpll.22-pll.23, a region implicated in several inherited diseases including Wiskott-Aldrich syndrome, three forms of X-linked hypercalciuric nephrolithiaisis, and the eye disorders retinitis pigmentosa 2, congenital stationary night blindness, and Aland Island eye disease (Fisher, S.E. et al. (1997) Genomics 45:340-347). Mutations in the BRI2 isoform of the BRI gene family are associated with dementia in humans (Vidal, R et al. (2001) Gene 266:95-102).

Changes in the molecular and cellular components of neuronal signaling systems correlate with the effects on mood and cognition observed after long-term treatment with antidepressant drugs. Two serine/threonine kinases, Ca²⁺/calmodulin-dependent protein kinase U and cyclic AMP-dependent protein kinase, are activated in the brain following antidepressant treatment. Associated changes in the phosphorylation of selected protein substrates in subcellular compartments including presynaptic terminals and microtubules may contribute to the modulation of synaptic transmission observed with antidepressants (Popoli, M. et al. (2001) Pharmacol. Ther. 89:149-170). Reserpine can cause a syndrome resembling depression, indicating the importance of vesicular transport activity for the control of mood and behavior. The psychostimulant amphetamine also disrupts the storage of amines in secretory vesicles, further indicating that alterations in vesicular monoamine transport can affect behavior (Sulzer, D. and S. Rayport (1990) Neuron 5:797-808).

Decreased GABAergic neurotransmission has been implicated in the pathophysiology of CNS disorders such as epilepsy and schizophrenia. Impaired re-uptake of synaptic glutamate and a reduced expression of the glutamate transporter have been found in the motor cortex of patients with amyotrophic lateral sclerosis (ALS). The loss of glial glutamate transporters produces elevated extracellular glutamate levels, neurodegeneration characteristic of excitotoxicity, and a progressive paralysis. The loss of neuronal glutamate transporters produces mild neurotoxicity and results in epilepsy (Rothstein, J.D. et al. (1996) Neuron 16:675-686). GABA transporter function is reduced in epileptic hippocampi. Transporters for dopamine, norepinephrine, and serotonin have particular significance as targets for clinically relevant psychoactive agents including cocaine, antidepressants, and amphetamines. Cocaine and antidepressants are transporter antagonists that act with varying degrees of specificity to enhance synaptic concentrations of amines by limiting clearance.

Amphetamines enhance transporter mediated efflux in concert with a depletion of vesicular amine stores (Barker, E.L. and R.D. Blakely (1995) Psychopharmacology 28:321-333; Sulzer, D. and S. Rayport (1990) Neuron 5:797-808; Wall, S.C. et al. (1995) Mol. Pharmacol. 47:544-550).

The μ-opioid receptor (MOR) mediates the actions of analgesic agents including morphine, codeine, methadone, and fentanyl as well as heroin. MOR is functionally coupled to a G-protein- activated potassium channel (Mestek A. et al. (1995) J. Neurosci. 15:2396-2406). A variety of MOR subtypes exist. Alternative splicing has been observed with MOR-1 as with a number of G protein-coupled receptors including somatostatin 2, dopamine D2, prostaglandinEP3, and serotonin receptor subtypes 5-hydroxyfryptamine4 and 5-hydroxytryptamine7 (Pan, Y.X. et al. (1999) Mol. Pharm. 56:396-403).

The central nervous system regulates the innate immune system by elaborating anti-inflammatory hormone cascades in response to bacterial products and immune mediators. The central nervous system also responds via acetylcholine-mediated efferent signals carried through the vagus nerve. Nicotinic cholinergic receptors expressed on macrophages detect these signals and respond with a dampened cytokine response (Tracey KJ. et al. (2001) FASEB J.15:1575-1576). Machado-Joseph disease (MJD) is an autosomal dominant, neurodegenerative disorder characterized by cerebellar ataxia, pyramidal and extra-pyramidal signs, peripheral nerve palsy, external ophthalmoplegia, facial and lingual fasciculation and bulging. The MJD josephin protein (amino acid residues 29-62) is a predominantly cytoplasmic protein associated with human neurons, but also detected in the nuclei of neurons and glial cells (Mruk, D.D. and Cheng, C. Y. (1999) J. Biol. Chem. 274:27056-27068).

Juvenile neuronal ceroid lipofuscinosis (JNCL), also known as Batten disease, is an autosomal recessive lysosomal storage disease associated with mutations in CLN3. The predominant mutation in CLN3 is a 1.02 kb genomic deletion that accounts for nearly 85% of the disease alleles. Additional missense and nonsense mutations have been described. Some missense substitutions result in a protracted phenotype, with delays in the onset of classical clinical features, whereas others lead to classical JNCL. CLN3 is a hydrophobic protein containing 5 to 7 transmembrane domains. CLN3 is found to be highly associated with lysosome-associated membrane protein JJ in non-neuronal cells and with synaptophysin in neuronal cell lines (Haskell, R.E. et al. (2000) Hum. Mol. Genet. 9:735-744). Dysferlin is the protein product of the gene mutated in patients with an autosomal recessive limb-girdle muscular dystrophy type 2B (LGMD2B) and a distal muscular dystrophy, Miyoshi myopathy. Dysferlin is homologous to a Caenorhabditis elegans spermatogenesis factor, FER-1. Otoferlin, another human FER-1-like protein (ferlin), is responsible for autosomal recessive nonsyndromic deafness (DFNB9). All the ferlins are characterized by sequences corresponding to multiple C2 domains that share the highest level of homology with the C2A domain of rat synaptotagmin HI (Britton S. et al. (2000) Genomics 68:313-321). 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 find 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 cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder. Lung Cancer Lung cancer is the leading cause of cancer death for men and the second leading cause of cancer death for women in the U.S. The vast majority of lung cancer cases are attributed to smoking tobacco, and increased use of tobacco products in third world countries is projected to lead to an epidemic of lung cancer in these countries. Exposure of the bronchial epithelium to tobacco smoke appears to result in changes in tissue morphology, which are thought to be precursors of cancer. Lung cancers are divided into four histopathologically distinct groups. Three groups (squamous cell carcinoma, adenocarcinoma, and large cell carcinoma) are classified as non-small cell lung cancers (NSCLCs). The fourth group of cancers is referred to as small cell lung cancer (SCLC). Collectively, NSCLCs account for -70% of cases while SCLCs account for -18% of cases. The molecular and cellular biology underlying the development and progression of lung cancer are incompletely understood.

Deletions on chromosome 3 are common in this disease and are thought to indicate the presence of a tumor suppressor gene in this region. Activating mutations in K-ras are commonly found in lung cancer and are the basis of one of the mouse models for the disease. 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 rates for this disease is very low. Identification of early-stage markers for ovarian cancer would significantly increase the survival rate. The molecular events that lead to ovarian cancer are poorly understood. Some of the known aberrations include mutation of p53 and microsateUite instability. Since gene expression patterns are likely to vary when normal ovary is compared to ovarian tumors, examination of gene expression in these tissues to identify possible markers for ovarian cancer is particularly relevant to improving diagnosis, prognosis, and treatment of this disease. Colon Cancer

Colorectal cancer is the second leading cause of cancer deaths in the United States. Colon cancer is associated with aging, since 90% of the total cases occur in individuals over the age of 55. A widely accepted hypothesis is that several contributing genetic mutations must accumulate over time in an individual who develops the disease. To understand the nature of genetic alterations in colorectal cancer, a number of studies have focused on the inherited syndromes. The first known inherited syndrome, Familial Adenomatous Polyposis (FAP), is caused by mutations in the Adenomatous Polyposis Coli gene (APC), resulting in truncated or inactive forms of the protein. This tumor suppressor gene has been mapped to chromosome 5q. The second known inherited syndrome is hereditary nonpolyposis colorectal cancer (HNPCC), which is caused by mutations in mismatch repair genes. Although hereditary colon cancer syndromes occur in a small percentage of the population and most colorectal cancers are considered sporadic, knowledge from studies of the hereditary syndromes can be generally applied. For instance, somatic mutations in APC occur in at least 80% of ^discriminate colon tumors. APC mutations are thought to be the initiating event in the disease. Other mutations occur subsequently. Approximately 50% of colorectal cancers contain activating mutations in ras, while 85% contain inactivating mutations in p53. Changes in these genes lead to gene expression changes in colon cancer. Less is understood about downstream targets of these mutations and the role they may play in cancer development and progression. Tangier Disease Tangier disease (TD) is a rare genetic disorder characterized by near absence of circulating high density lipoprotein (HDL) and the accumulation of cholesterol esters in many tissues, including tonsils, lymph nodes, liver, spleen, thymus, and intestine. Low levels of HDL represent a clear predictor of premature coronary artery disease and homozygous TD correlates with a four- to six-fold increase in cardiovascular disease compared to controls. The major cardio-protective activity of HDL is ascribed to its role in reverse cholesterol transport, the flux of cholesterol from peripheral cells such as tissue macrophages, through plasma lipoproteins to the liver. The HDL protein, apolipoprotein A-I, plays a major role in this process, interacting with the cell surface to remove excess cholesterol and phospholipids. Recent studies have shown that this pathway is severely impaired in TD and the defect lies in a specific gene, the ABCl transporter. This gene is a member of the family of ATP-binding cassette transporters, which utilize ATP hydrolysis to transport a variety of substrates across membranes. RNA Expression

Atherosclerosis and the associated coronary artery disease and cerebral stroke represent the most common cause of death in industrialized nations. Although certain key risk factors have been identified, a full molecular characterization that elucidates the causes and provide care for this complex disease has not been achieved. Molecular characterization of growth and regression of atherosclerotic vascular lesions requires identification of the genes that contribute to features of the lesion including growth, stability, dissolution, rupture and, most lethally, induction of occlusive vessel thrombus. An early step in the development of atherosclerosis is formation of the "fatty streak".

Lipoproteins, such as the cholesterol-rich low-density lipoprotein (LDL), accumulate in the extracellular space of the vascular intima, and undergo modification. Oxidation of LDL occurs most avidly in the sub-endothelial space where circulating antioxidant defenses are less effective. The degree of LDL oxidation affects its interaction with target cells. "Minimally oxidized" LDL (MM- LDL) is able to bind to LDL receptor but not to the oxidized LDL (Ox-LDL) or "scavenger" receptors that have been identified, including scavenger receptor types A and B, CD36 , CD68/macrosialin and LOX-1 (Navab et al. (1994) Arterioscler Thromb Vase Biol 16:831-842; Kodama et al. (1990) Nature 343:531-535; Acton et al. (1994) J Biol Chem 269:21003-21009; Endemann et al. (1993) J Biol Chem 268:11811-11816; Ramprasad et al. (1996) Proc Natl Acad Sci 92:14833-14838; Kataoka et al. (1999) Circulation 99:3110-3117). MM-LDL can increase the adherence and penetration of monocytes, stimulate the release of monocyte chemotactic protein 1 (MCP-1) by endothelial cells, and induce scavenger receptor A (SRA) and CD36 expression in macrophages (Cushing et al. (1990) Proc Natl Acad Sci 87:5134-5138; Yoshida et al. (1998) Arterioscler Thromb Vase Biol 18:794-802; Steinberg (1997) J Biol Chem 272:20963-20966). SRA and the other scavenger receptors can bind Ox-LDL and enhance uptake of lipoprotein particles.

Mononuclear phagocytes enter the intima, differentiate into macrophages, and ingest modified lipids including Ox-LDL. In most cell types, cholesterol content is tightly controlled by feedback regulation of LDL receptors and biosynthetic enzymes (Brown and Goldstein (1986) Science 232:34- 47). In macrophages, however, the additional scavenger receptors lead to unregulated uptake of cholesterol (Brown and Goldstein (1983) Annu Rev Biochem 52:223-261) and accumulation of multiple intracellular hpid droplets producing a "foam cell" phenotype. Cholesterol-engorged and dead macrophages contribute most of the mass of early "fatty streak" plaques and typical "advanced" lesions of diseased arteries. Numerous studies have described a variety of foam cell responses that contribute to growth and rupture of atherosclerotic vessel wall plaques. These responses include production of multiple growth factors and cytokines, which promote proliferation and adherence of neighboring cells; chemokines, which further attract circulating monocytes into the growing plaque; proteins, which cause remodeling of the extracellular matrix; and tissue factor, which can trigger thrombosis (Ross (1993) Nature 362:801-809; Quin et al. (1987) Proc Natl Acad Sci 84:2995-2998). Thus, cholesterol-loaded macrophages which occur in abundance in most stages of the atherosclerotic plaque formation contribute to inception of the atheroscerotic process and to eventual plaque rupture and occlusive thrombus. During Ox-LDL uptake, macrophages produce cytokines and growth factors that elicit further cellular events that modulate atherogenesis such as smooth muscle cell proliferation and production of extracellular matrix. Additionally, these macrophages may activate genes involved in inflammation including inducible nitric oxide synthase. Thus, genes differentially expressed during foam cell formation may reasonably be expected to be markers of the atherosclerotic process. Association of NTRAN polynucleotides with Parkinson's Disease

Several genes have been identified as showing linkage to autosomal dominant forms of Parkinson's Disease (PD). PD is a common neurodegenerative disorder causing bradykinesia, resting tremor, muscular rigidity, and postural instability. Cytoplasmic eosinophilic inclusions called Lewy bodies, and neuronal loss especially in the substantia nigra pars compacta, are pathological hallmarks of PD (Valente, E.M. et al (2001) Am. J. Hum. Genet. 68:895-900). Lewy body Parkinson disease has been thought to be a specific autosomal dominant disorder (Wakabayashi, K. et al. (1998) Acta Neuropath. 96:207-210). Juvenile parkinsonism may be a specific autosomal recessive disorder

(Matsumine, H et al. (1997) Am. J. Hum. Genet. 60: 588-596, 1997). (Online Mendelian Inheritance in Man, OMM. Johns Hopkins University, Baltimore, MD. MTM Number: 168600: Sept. 9, 2002: . World Wide Web URL: http://www.ncbi.nlm.n .gov/omim/)

The discovery of new neurotransmission-associated proteins, and the polynucleotides encoding them, satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of autoimmune/inflammatory disorders, cardiovascular disorders, neurological diseases, developmental disorders, and cell prohferative diseases and disorders, including cancer, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of neurotransmission-associated proteins. There is a need in the art for new compositions, including nucleic acids and proteins, for the diagnosis, prevention, and treatment of autoimmune/inflammatory, cardiovascular, neurological, developmental, cell prohferative, fransport, psychiatric, metabolic, and endocrine disorders.

SUMMARY OF THE INVENTION Various embodiments of the invention provide purified polypeptides, neurotransmission-associated proteins, referred to collectively as 'NTRAN' and individually as 'NTRAN-1,' 'NTRAN-2,' 'NTRAN-3,' 'NTRAN-4,' 'NTRAN-5,' 'NTRAN-6,' 'NTRAN-7,' 'NTRAN-8,' 'NTRAN-9,' 'NTRAN-10,' 'NTRAN-11,' 'NTRAN-12,' 'NTRAN-13,' 'NTRAN-14,' 'NTRAN-15,' 'NTRAN-16,' 'NTRAN-17,' 'NTRAN-18,' 'NTRAN-19,' 'NTRAN-20,' 'NTRAN- 21,' and 'NTRAN-22' 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 neurotransmission-associated proteins 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 neurotransmission-associated proteins 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 JD NO:l- 22, 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 NO: 1-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22. Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 1-22.

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 ID NO:l-22, 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 NO: 1-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO:l-22. In another embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO: 1-22. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:23-44.

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 ID NO: 1-22, 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 NO: 1-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-22. 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 ID NO:l-22, 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 NO: 1-22, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22. The method comprises a) culturing a ceU under conditions suitable for expression of the polypeptide, wherein said ceU 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 specificaUy 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 RO NO: 1-22, b) a polypeptide comprising a naturaUy 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 NO:l-22, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consistmg of SEQ ID NO: 1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22. 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 JD NO:23-44, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consistmg of SEQ ID NO:23-44, 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.

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 JJD NO:23-44, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ DD NO:23-44, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specificaUy 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 stiU other embodiments, the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides. StiU 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 NO:23-44, b) a polynucleotide comprising a naturaUy 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 NO:23-44, 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) ampHfying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amphfied target polynucleotide or fragment thereof. In a related embodiment, the method can include detecting the amount of the amphfied 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 ID NO:l-22, b) a polypeptide comprising a naturaUy 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 NO: 1-22, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO: 1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO:l-22, and a pharmaceuticaUy acceptable excipient. In one embodiment, the composition can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22. Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional NTRAN, 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 ID NO: 1-22, b) a polypeptide comprising a naturaUy 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 NO:l-22, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO:l-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-22. 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 pharmaceuticaUy acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional NTRAN, comprising administering to a patient in need of such treatment the composition.

StiU 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 JD NO: 1-22, b) a polypeptide comprising a naturaUy 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 NO: 1-22, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO: 1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO:l-22. 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 pharmaceuticaUy acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional NTRAN, comprising administering to a patient in need of such treatment the composition.

Another embodiment provides a method of screening for a compound that specificaUy 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 JD NO:l-22, b) a polypeptide comprising a naturaUy 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 JD NO:l-22, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO: 1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-22. 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 specificaUy 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 ID NO: 1-22, b) a polypeptide comprising a naturaUy 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 NO:l-22, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO: 1-22. 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. StiU 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 ID NO:23-44, 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 ID NO:23-44, ii) a polynucleotide comprising a naturaUy 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 NO:23-44, 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 JD NO:23-44, ii) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ JD NO:23-44, 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 unfreated 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 fuU length polynucleotide and polypeptide embodiments of the invention. Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog, and the PROTEOME 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 with applicable descriptions, references, and threshold parameters.

Table 8 shows single nucleotide polymorphisms found in polynucleotide sequences of the invention, along with aUele 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 temoinology 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 ceU" includes a plurality of such host ceUs, and a reference to "an antibody" is a reference to one or more antibodies and equivalents thereof known to those skiUed in the art, and so forth. Unless defined otherwise, aU technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skiU 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. AU publications mentioned herein are cited for the purpose of describing and disclosing the ceU lines, protocols, reagents and vectors which are reported in the pubhcations 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

"NTRAN" refers to the amino acid sequences of substantiaUy purified NTRAN 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 NTRAN. Agonists may include proteins, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of NTRAN either by directly interacting with NTRAN or by acting on components of the biological pathway in which NTRAN participates.

An "aUelic variant" is an alternative form of the gene encoding NTRAN. AUelic 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 aUehc variants of its naturaUy occurring form. Common mutational changes which give rise to aUelic variants are generaUy 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 NTRAN include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as NTRAN or a polypeptide with at least one functional characteristic of NTRAN. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding NTRAN, and improper or unexpected hybridization to aUelic variants, with a locus other than the normal chromosomal locus for the polynucleotide encoding NTRAN. 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 functionaUy equivalent NTRAN. Deliberate amino acid substitutions may be made on the basis of one or more similarities in polarity, charge, solubility, hydrophobicity, hydrophihcity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of NTRAN 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 hydrophihcity values may include: asparagine and glutamine; and serine and threonine. Amino acids with uncharged side chains having similar hydrophihcity 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 naturaUy occurring or synthetic molecules. Where "amino acid sequence" is recited to refer to a sequence of a naturaUy 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 weU known in the art. The term "antagonist" refers to a molecule which inhibits or attenuates the biological activity of NTRAN. Antagonists may include proteins such as antibodies, anticalins, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of NTRAN either by directly interacting with NTRAN or by acting on components of the biological pathway in which NTRAN participates.

The term "antibody" refers to intact immunoglobulin molecules as weU as to fragments thereof, such as Fab, F(ab')₂, and Fv fragments, which are capable of binding an epitopic determinant. Antibodies that bind NTRAN polypeptides can be prepared using intact polypeptides or using fragments containing smaU 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 chemicaUy, and can be conjugated to a carrier protein if desired.

Commonly used carriers that are chemicaUy 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 specificaUy 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'-NH₂), which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood. Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.

Aptamers may be specificaUy 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 naturaUy occurring enzymes, which normaUy 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, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2 '-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'- deoxyguanosine. Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a ceU, the complementary antisense molecule base-pairs with a naturaUy occurring nucleic acid sequence produced by the ceU 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 "biologicaUy active" refers to a protein having structural, regulatory, or biochemical functions of a naturaUy occurring molecule. Likewise, "immunologicahy active" or "immunogenic" refers to the capability of the natural, recombinant, or synthetic NTRAN, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or ceUs 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 NTRAN or fragments of NTRAN maybe employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.). "Consensus sequence" refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncaUed 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 GELVTEW fragment assembly system (Accelrys,

Burlington MA) or Phrap (TJniversity of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.

"Conservative amino acid substitutions" are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especiaUy 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, Gin, His

Asp Asn, Glu

Cys Ala, Ser

Gin Asn, Glu, His Glu Asp, Gin, His

Gly Ala

His Asn, Arg, Gin, Glu

He Leu, Val

Lys Arg, Gin, Glu

Met Leu, Ue

Phe His, Met, Leu, Trp, Tyr

Ser Cys, Thr

Thr Ser, Val Trp Phe, Tyr

Tyr His, Phe, Trp Val He, Leu, Thr

Conservative amino acid substitutions generaUy maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha hehcal 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 chemicaUy modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.

A "detectable label" refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.

"Differential expression" refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons maybe 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 aUowing acceleration of the evolution of new protein functions.

A "fragment" is a unique portion of NTRAN or a polynucleotide encoding NTRAN 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, maybe 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 maybe preferentiaUy selected from certain regions of a molecule. For example, a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.

A fragment of SEQ JD NO:23-44 can comprise a region of unique polynucleotide sequence that specificaUy identifies SEQ ID NO:23-44, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ ID NO:23-44 can be employed 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 NO:23-44 from related polynucleotides. The precise length of a fragment of SEQ ID NO:23-44 and the region of SEQ ID NO:23-44 to which the fragment conesponds are routinely determinable by one of ordinary skiU in the art based on the intended purpose for the fragment. A fragment of SEQ ID NO: 1-22 is encoded by a fragment of SEQ ID NO:23-44. A fragment of SEQ ID NO: 1-22 can comprise a region of unique amino acid sequence that specificaUy identifies SEQ ID NO:l-22. For example, a fragment of SEQ ID NO:l-22 can be used as an immunogenic peptide for the development of antibodies that specificaUy recognize SEQ JD NO: 1-22. The precise length of a fragment of SEQ JD NO:l-22 and the region of SEQ JD NO:l-22 to which the fragment corresponds can be detennined based on the intended purpose for the fragment using one or more analytical methods described herein or otherwise known in the art.

A "fuU length" polynucleotide is one containing at least a translation initiation codon (e.g., methionine) fohowed 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, alternatively, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.

The terms "percent identity" and "% identity," as apphed to polynucleotide sequences, refer to the percentage of identical 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 ahgnment 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 ahgnment 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 foUows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The "weighted" residue weight table is selected as the default. 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 Ahgnment Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which is available from several sources, including the NCBI, Bethesda, MD, and on the Internet at http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various sequence analysis programs including "blastn," that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool caUed "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.n .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 2.0.12 (April-21-2000) set at default parameters. Such default parameters maybe, for example:

Matrix: BLOSUM62

Reward for match: 1

Penalty for mismatch: -2 Open Gap: 5 and Extension Gap: 2 penalties

Gap x drop-off: 50

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 JD number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that aU encode substantiaUy the same protein.

The phrases "percent identity" and "% identity," as applied to polypeptide sequences, refer to the percentage of identical residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence ahgnment are weU-known. Some ahgnment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generaUy preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. The phrases "percent similarity'' and "% similarity," as applied to polypeptide sequences, refer to the percentage of residue matches, including identical residue matches and conservative substitutions, between at least two polypeptide sequences aligned using a standardized algorithm. In contrast, conservative substitutions are not included in the calculation of percent identity between polypeptide sequences. 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 ahgnment program (described and referenced above). For pairwise ahgnments of polypeptide sequences using CLUSTAL V, the default parameters are set as foUows: Ktuple=l, gap penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as the default residue weight table.

Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences" tool Version 2.0.12 (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: 50 Expect: 10 Word Size: 3 Filter: on

Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ JD number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 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 aU of the elements required for chromosome replication, segregation and maintenance.

The term "humanized antibody" refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and stiU 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 step(s) is particularly important in determining the stringency of the hybridization process, with more stringent conditions aUowing 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 skiU 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 μg/ml sheared, denatured salmon sperm DNA.

GeneraUy, stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out. Such wash temperatures are typicaUy selected to be about 5°C to 20°C lower than the thermal melting point T^) for the specific sequence at a defined ionic strength and pH. The T_m 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 T_m and conditions for nucleic acid hybridization are weU known and can be found in Sambrook, J. and D.W. RusseU (2001; Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor Press, Cold Spring Harbor NY, ch. 9).

High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1% SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C maybe used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%. TypicaUy, 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 μg/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 wiU be readily apparent to those of ordinary skiU 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 maybe formed in solution (e.g., C₀t or R₀t 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 shdes, or any other appropriate substrate to which ceUs 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 ceUular and systemic defense systems.

An ''immunogenic fragment" is a polypeptide or ohgopeptide fragment of NTRAN which is capable of ehciting an immune response when introduced into a living organism, for example, a mammal. The term "immunogenic fragment" also includes any polypeptide or ohgopeptide fragment of NTRAN which is useful in any of the antibody production methods disclosed herein or known in the art.

The term "microarray" refers to an arrangement of a plurality of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate. The terms "element" and "anay 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 NTRAN. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of NTRAN. 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 ' preferentiaUy bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the ceU.

"Post-translational modification" of an NTRAN may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur syntheticaUy or biochemicaUy. Biochemical modifications wiU vary by ceU type depending on the enzymatic milieu of NTRAN.

"Probe" refers to nucleic acids encoding NTRAN, their complements, or fragments thereof, which are used to detect identical, aUelic or related nucleic acids. Probes are isolated ohgonucleotides 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, usuaUy DNA ohgonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid, e.g., by the polymerase chain reaction (PCR).

Probes and primers as used in the present invention typicaUy 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, maybe used. Methods for preparing and using probes and primers are described in, for example, Sambrook,

J. and D.W. RusseU (2001; Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor Press, Cold Spring Harbor NY), Ausubel, F.M. et al. (1999; Short Protocols in Molecular Biology, 4^th 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). Ohgonucleotides 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 ohgonucleotides 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, DaUas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope. The Primer3 primer selection program (available to the pubhc from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) aUows 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 ohgonucleotides 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 pubhc from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby aUowing selection of primers that hybridize to either the most conserved or least conserved regions of ahgned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved ohgonucleotides and polynucleotide fragments. The ohgonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microaπay elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of ohgonucleotide selection are not limited to those described above.

A "recombinant nucleic acid" is a nucleic acid that is not naturaUy 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 accomphshed 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 and RusseU (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 ceU.

Alternatively, such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.

A "regulatory element" refers to a nucleic acid sequence usuaUy 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, cheπήluminescent, or chromogenic agents; substrates; cof actors; 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 aU occunences 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 NTRAN, nucleic acids encoding NTRAN, or fragments thereof may comprise a bodily fluid; an extract from a ceU, chromosome, organeUe, or membrane isolated from a ceU; a ceU; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.

The terms "specific binding" and "specificaUy binding" refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a smaU 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 wiU reduce the amount of labeled A that binds to the antibody. The term "substantiaUy 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 naturaUy 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, shdes, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as weUs, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.

A "transcript image" or "expression profile" refers to the coUective pattern of gene expression by a particular ceU type or tissue under given conditions at a given time. "Transformation" describes a process by which exogenous DNA is introduced into a recipient ceU. Transformation may occur under natural or artificial conditions according to various methods weU 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 ceU. The method for transformation is selected based on the type of host ceU being transformed and may include, but is not limited to, bacteriophage or viral infection, elecfroporation, heat shock, hpofection, and particle bombardment. The term "transformed ceUs" includes stably transformed ceUs in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as weU as transiently transformed ceUs 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 ceUs of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques weU known in the art. The nucleic acid is introduced into the ceU, directly or indirectly by introduction into a precursor of the ceU, by way of dehberate 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 term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook and RusseU (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 2.0.9 (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 "aUehc" (as defined above), "splice," "species," or "polymorphic" variant. A splice variant may have significant identity to a reference molecule, but wifl generaUy have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The conesponding 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 wiU generaUy 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 or sequence similarity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2.0.9 (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 or sequence similarity over a certain defined length of one of the polypeptides.

THE INVENTION

Various embodiments of the invention include new human neurotransmission-associated proteins (NTRAN), the polynucleotides encoding NTRAN, and the use of these compositions for the diagnosis, treatment, or prevention of autoimmune/inflammatory, cardiovascular, neurological, developmental, ceU prohferative, transport, psychiatric, metabohc, and endocrine disorders. Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide embodiments of the invention. Each polynucleotide and its conesponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ID). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ JD NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide JD) 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, fuU length clones conesponding 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 polypeptide embodiments 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 ID NO:) and the conesponding 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 ID NO:) of the nearest PROTEOME database homologs. Column 4 shows the probabihty 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, aU of which are expressly incorporated by reference herein.

Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and

2 show the polypeptide sequence identification number (SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide JD) for each polypeptide of the invention. Column

3 shows the number of amino acid residues in each polypeptide. Column 4 shows amino acid residues comprising signature sequences, domains, motifs, potential phosphorylation sites, and potential glycosylation sites. Column 5 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 neurotransmission-associated proteins. For example, For example, SEQ ID NO:5 is 68% identical, from residue E43 to residue D287, and is 60% identical, from residue Ml to Gl 18 to murine gliacolin (GenBank ID gl4278927) as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.) The BLAST probabihty scores are 3.0e-86 and 5.6e-30, which indicate the probabilities of obtaining the observed polypeptide sequence alignments by chance. As determined by BLAST analysis using the PROTEOME database, SEQ JD NO:5 also has homology to murine and human Clq-related factors which have a coUagenous region and a globular domain and have similarity to the Clq signature domain. Both murine and human Clq-related factors are highly expressed in brain areas that are involved in motor function

(PROTEOME ID 429678|Clqrf and 567880|CRF). SEQ ID NO:5 also contains a Clq domain and a coUagen triple helix repeat (20 copies) domain as detennined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, PROFILESCAN, and additional BLAST analyses provide further conoborative evidence that SEQ ID NO:5 is a Clq-related factor.

In an alternative example, SEQ ID NO:9 is 86% identical, from residue Ml to residue F1308, to Mus musculus ceU recognition molecule CASPR4 (GenBank ID g 12330704) as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.) The BLAST probabihty score is 0.0, which indicates the probabihty of obtaining the observed polypeptide sequence ahgnment by chance. SEQ JD NO:9 also has homology to Neurexin 4 (contactin associated protein 1), a neuronal paranodal transmembrane receptor containing epidermal growth factor-like and neurexin motifs, as determined by BLAST analysis using the PROTEOME database. SEQ JD NO:9 also contains an EGF-like domain and a F5/8 type C domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS and MOTIFS analyses provide further conoborative evidence that SEQ JD NO:9 is a ceU recognition molecule.

In an alternative example, SEQ ID NO:13 is 100% identical, from residue Ml to residue S573, to human myelin-associated glycoprotein precursor (GenBank JD g307156) as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.) The BLAST probabihty score is 0.0, which indicates the probabihty of obtaining the observed polypeptide sequence ahgnment by chance. SEQ ID NO:13 also has homology to proteins that are locahzed to the nervous system, are members of the sialoadhesin subgroup of immunoglobulin superfamily lectins, and are ceU adhesion molecules, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO:13 also contains an I- type IG domain, an Immunoglobulin domain, an Ig superfamily from SCOP domain and an Immunoglobulin C-2 type domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM, INCY and SMART databases of conserved protein famihes/domains. (See Table 3.) Data from BLIMPS, MOTIFS, and additional BLAST analyses provide further corroborative evidence that SEQ JD NO: 13 is a myelin ceU adhesion molecule.

In an alternative example, SEQ ID NO:16 is 93% identical, from residue A32 to residue S412, to human ubiquilin (GenBank ID gl8254511) as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.) The BLAST probabihty score is 4.6e-185, which indicates the probabihty of obtaining the observed polypeptide sequence ahgnment by chance. SEQ ID NO: 16 also has homology to proteins that are locahzed to the endoplasmic reticulum, nucleus, and cytoplasm, is associated with the 26S proteasome, binds to and promotes the accumulation of presenilin 1 and presenilin 2, and are locahzed to neurofibriUary tangles and Lewy bodies in brains affected by Alzheimer's disease and Parkinson's disease, and are annotated as ubiquilin 1 proteins, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO: 16 also contains UBA/TS-N and ubiquitin associated domains as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM and SMART database of conserved protein famihes/domains. (See Table 3.) Data from BLAST analyses against the PRODOM database, provide further corroborative evidence that SEQ JD NO: 16 is a ubiquilin protein. In an alternative example, SEQ ID NO:19 is 89% identical, from residue Ml to residue L621, to Mus musculus punc (g3068592) as determined by the Basic Local Ahgnment Search Tool (BLAST). The BLAST probabihty score is 0.0, which indicates the probabihty of obtaining the observed polypeptide sequence ahgnment by chance. SEQ ID NO: 19 also has homology to proteins that are locahzed to the plasma membrane, are members of the immunoglobulin superfamily, and are neuronal ceU adhesion proteins, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO: 19 also contains fibronectin type 3 and immunoglobulin domains as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM, INCY, and SMART databases of conserved protein famihes/domains. (See Table 3.) Data from BLAST analyses against the PRODOM and DOMO databases, provide further corroborative evidence that SEQ JD NO: 19 is a neuronal ceU adhesion protein.

SEQ ID NO:l-4, SEQ ID NO:6-8, SEQ ID NO-.10-12, SEQ JD NO:14-15, SEQ ID NO:17- 18, and SEQ JD NO:20-22 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ JD NO:l-22 are described in Table 7.

As shown in Table 4, the fuU 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 hsts the polynucleotide sequence identification number (Polynucleotide SEQ JD NO:), the conesponding Incyte polynucleotide consensus sequence number (Incyte ID) 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 JD NO:23-44 or that distinguish between SEQ JD NO:23-44 and related polynucleotides. The polynucleotide fragments described in Column 2 of Table 4 may refer specificaUy, 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 fuU length polynucleotides. In addition, the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The S anger Centre, Cambridge, UK) database (Le., 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 (Le., those sequences including the designation "NM" or "NT") or the NCBI RefSeq Protein Sequence Records (Le., those sequences including the designation "NP"). Alternatively, the polynucleotide fragments described in column 2 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm. For example, a polynucleotide sequence identified as FL_ZXXXXXJV_i_N₂_JTΥlT_N₃_N represents a "stitched" sequence in which XXXXXX is the identification number of the cluster of sequences to which the algorithm was applied, and JTTITis the number of the prediction generated by the algorithm, and N_lι2ι3„_, if present, represent specific exons that may have been manuaUy edited during analysis (See Example V). Alternatively, the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-sfretching" algorithm. For example, a polynucleotide sequence identified as ΩΩΩOΩ __gAAAAA_gBBBBB_l_Nls a "stretched" sequence, with XXXXXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the "exon-sfretching" algorithm was apphed, gBBBBB being the GenBank identification number or ΝCBI RefSeq identification number of the nearest GenBank protein homolog, and N referring to specific exons (See Example V). In instances where a RefSeq sequence was used as a protein homolog for the "exon-stoetching" algorithm, a RefSeq identifier (denoted by "ΝM," "ΝP," or "NT") may be used in place of the GenBank identifier (i. e. , gBBBBB).

Alternatively, a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods. The foUowing Table hsts examples of component sequence prefixes and conesponding sequence analysis methods associated with the prefixes (see Example JN and Example V).

In some cases, Incyte cDΝA coverage redundant with the sequence coverage shown in

Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDΝA identification numbers are not shown.

Table 5 shows the representative cDΝA libraries for those fuU length polynucleotides which were assembled using Incyte cDΝA sequences. The representative cDΝA hbrary is the Incyte cDΝA hbrary which is most frequently represented by the Incyte cDΝA sequences which were used to assemble and confirm the above polynucleotides. The tissues and vectors which were used to construct the cDΝA libraries shown in Table 5 are described in Table 6.

Table 8 shows single nucleotide polymorphisms (SΝPs) found in polynucleotide sequences of the invention, along with aUele frequencies in different human populations. Columns 1 and 2 show the polynucleotide sequence identification number (SEQ ID NO:) and the conesponding Incyte project identification number (PDD) for polynucleotides of the invention. Column 3 shows the Incyte identification number for the EST in which the SNP was detected (EST JD), and column 4 shows the identification number for the SNP (SNP JD). 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 fuU- length polynucleotide sequence (CBl SNP). Column 7 shows the aUele found in the EST sequence. Columns 8 and 9 show the two aUeles found at the SNP site. Column 10 shows the amino acid encoded by the codon including the SNP site, based upon the aUele found in the EST. Columns 11-14 show the frequency of aUele 1 in four different human populations. An entry of n/d (not detected) indicates that the frequency of aUele 1 in the population was too low to be detected, while n/a (not available) indicates that the aUele frequency was not determined for the population.

The invention also encompasses NTRAN variants. Various embodiments of NTRAN variants can have at least about 80%, at least about 90%, or at least about 95% amino acid sequence identity to the NTRAN amino acid sequence, and can contain at least one functional or structural characteristic of NTRAN.

Various embodiments also encompass polynucleotides which encode NTRAN. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:23-44, which encodes NTRAN. The polynucleotide sequences of SEQ JD NO:23-44, as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occunences 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 NTRAN. In particular, such a variant polynucleotide wiUhave at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a polynucleotide encoding NTRAN. A particular aspect of the invention encompasses a variant of a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO:23-44 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:23-44. Any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of NTRAN.

In addition, or in the alternative, a polynucleotide variant of the invention is a splice variant of a polynucleotide encoding NTRAN. A splice variant may have portions which have significant sequence identity to a polynucleotide encoding NTRAN, but wiU generaUy 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 sphce 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 NTRAN over its entire length; however, portions of the sphce variant wiUhave 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 NTRAN. For example, a polynucleotide comprising a sequence of SEQ JD NO:33 and a polynucleotide comprising a sequence of SEQ ID NO:34 are sphce variants of each other. Any one of the sphce variants described above can encode a polypeptide which contains at least one functional or structural characteristic of NTRAN.

It wiU be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of polynucleoti.de sequences encoding NTRAN, some bearing minimal similarity to the polynucleotide sequences of any known and naturaUy occurring gene, maybe 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 apphed to the polynucleotide sequence of naturaUy occuning NTRAN, and aU such variations are to be considered as being specificaUy disclosed. Although polynucleotides which encode NTRAN and its variants are generaUy capable of hybridizing to polynucleotides encoding naturaUy occurring NTRAN under appropriately selected conditions of stringency, it maybe advantageous to produce polynucleotides encoding NTRAN or its derivatives possessing a substantiaUy different codon usage, e.g., inclusion of non-naturaUy 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 substantiaUy altering the nucleotide sequence encoding NTRAN 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 naturaUy occurring sequence. The invention also encompasses production of polynucleotides which encode NTRAN and

NTRAN 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 ceU systems using reagents weU known in the art. Moreover, synthetic chemistry maybe used to introduce mutations into a polynucleotide encoding NTRAN 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 in SEQ ID NO:23-44 and fragments thereof, under various conditions of stringency (Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol. 152:507-511). Hybridization conditions, including annealing and wash conditions, are described in "Definitions." Methods for DNA sequencing are weU 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 (Apphed Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Invitrogen, Carlsbad CA). Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Apphed Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Apphed 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 weU known in the art (Ausubel et al., supra, ch. 7; Meyers, RA. (1995) Molecular Biology and Biotechnology. Wiley VCH, New York NY, pp. 856-853).

The nucleic acids encoding NTRAN maybe extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements. For example, one method which maybe 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 Apphc. 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 sunounding 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 inhuman and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Apphc. 1:111-119). In this method, multiple restriction enzyme digestions and ligations maybe 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). AdditionaUy, one may use PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in finding inteon/exon junctions. For aU PCR-based methods, primers maybe designed using commerciaUy 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 ohgo d(T) hbrary does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5 'non-transcribed regulatory regions.

CapiUary electrophoresis systems which are commerciaUy available maybe used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output/hght intensity maybe converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Apphed Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controUed. CapiUary electrophoresis is especiaUy preferable for sequencing smaU 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 NTRAN may be cloned in recombinant DNA molecules that direct expression of NTRAN, or fragments or functional equivalents thereof, in appropriate host ceUs. Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantiaUy the same or a functionaUy equivalent polypeptides may be produced and used to express NTRAN.

The polynucleotides of the invention can be engineered using methods generaUy known in the art in order to alter NTRAN-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 ohgonucleotides maybe used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis maybe used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce sphce 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. Bioteclmol. 17:793-797; Christians, F.C. et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Bioteclmol. 14:315-319) to alter or improve the biological properties of NTRAN, 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 hbrary of gene variants is produced using PCR-mediated recombination of gene fragments. The hbrary is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These prefened 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 maybe recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturaUy occuning genes in a directed and confroUable manner.

In another embodiment, polynucleotides encoding NTRAN maybe synthesized, in whole or in part, using one or more chemical methods weU 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, NTRAN 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 sohd-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 431 A peptide synthesizer (Apphed Biosystems).

AdditionaUy, the amino acid sequence of NTRAN, or any part thereof, maybe 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 naturaUy occurring polypeptide. The peptide may be substantiaUy purified by preparative high performance hquid chromatography (Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421). The composition of the synthetic peptides maybe confirmed by amino acid analysis or by sequencing (Creighton, supra, pp. 28-53).

In order to express a biologicaUy active NTRAN, the polynucleotides encoding NTRAN 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 NTRAN. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of polynucleotides encoding NTRAN. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where a polynucleotide sequence encoding NTRAN and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals maybe needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational confrol signals including an in-frame ATG initiation codon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host ceU system used (Scharf, D. et al. (1994) Results Probl. CeU Differ. 20:125-162).

Methods which are weU known to those skilled in the art may be used to construct expression vectors containing polynucleotides encoding NTRAN and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination (Sambrook and RusseU, supra, ch. 1-4, and 8; Ausubel et al., supra, ch. 1, 3, and 15).

A variety of expression vector/host systems may be utihzed to contain and express polynucleotides encoding NTRAN. 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 ceU systems infected with viral expression vectors (e.g., baculovirus); plant ceU 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 ceU systems (Sambrook and RusseU, supra; Ausubel et al., supra; VanHeeke, 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 Hffl Yearbook of Science and Technology (1992) McGraw Hffl, 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 dehvery of polynucleotides to the targeted organ, tissue, or ceU 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; BuUer, R.M. et al. (1985) Nature 317:813-815; McGregor, D.P. et al. (1994) Mol. Immunol. 31:219-226; Verma, I.M. and N. Somia (1997) Nature 389:239-242). The invention is not limited by the host ceU employed. In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotides encoding NTRAN. For example, routine cloning, subcloning, and propagation of polynucleotides encoding NTRAN can be achieved using a multifunctional E. coli vector such as PBLUΕSCRIPT (Stratagene, La JoUa CA) or PSPORT1 plasmid (Invitrogen). Ligation of polynucleotides encoding NTRAN into the vector's multiple cloning site disrupts the lacZ gene, aUowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, these vectors maybe useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence (Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509). When large quantities of NTRAN are needed, e.g. for the production of antibodies, vectors which direct high level expression of NTRAN may be used. For example, vectors containing the strong, inducible SP6 or T7 bacteriophage promoter maybe used. Yeast expression systems may be used for production of NTRAN. 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 intraceUular 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, CA. et al. (1994) Bio/Technology 12:181-184).

Plant systems may also be used for expression of NTRAN. Transcription of polynucleotides encoding NTRAN 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 smaU subunit of RUBISCO or heat shock promoters maybe used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broghe, R. et al. (1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. CeU Differ. 17:85-105). These constructs can be introduced into plant ceUs by direct DNA transformation or pathogen-mediated transfection ("The McGraw Hffl Yearbook of Science and Technology (1992) McGraw Hffl, New York NY, pp. 191-196).

In mammalian ceUs, a number of viral-based expression systems may be utilized, lh cases where an adenovirus is used as an expression vector, polynucleotides encoding NTRAN maybe hgated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses NTRAN in host ceUs (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 ceUs. SV40 or EB V- based vectors may also be used for high-level protein expression.

Human artificial chromosomes (HACs) may also be employed to dehver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are constructed and dehvered via conventional dehvery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes (Harrington, JJ. et al. (1997) Nat. Genet. 15:345-355).

For long term production of recombinant proteins in mammalian systems, stable expression of NTRAN in ceU lines is preferred. For example, polynucleotides encoding NTRAN can be transformed into ceU lines using expression vectors which may contain viral origins of rephcation and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. FoUowing the introduction of the vector, ceUs may be aUowed 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 aUows growth and recovery of ceUs which successfully express the introduced sequences. Resistant clones of stably transformed ceUs may be propagated using tissue culture techniques appropriate to the ceU type. Any number of selection systems may be used to recover transformed ceU lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosylfransferase genes, for use in tk and apr ceUs, respectively (Wigler, M. et al. (1977) CeU 11:223-232; Lowy, I. et al. (1980) CeU 22:817-823). Also, antimetabohte, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate; neo confers resistance to the aminoglycosides neomycin and G-418; and als and pat confer resistance to chlorsulfuron and phosphinofricin 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., tτ-pB and hisD, which alter ceUular requirements for metabohtes (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), β- glucuronidase and its substrate β-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, CA. (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 NTRAN is inserted within a marker gene sequence, transformed ceUs containing polynucleotides encoding NTRAN can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding NTRAN under the control of a single promoter. Expression of the marker gene in response to induction or selection usuaUy indicates expression of the tandem gene as weU. In general, host ceUs that contain the polynucleotide encoding NTRAN and that express

NTRAN may be identified by a variety of procedures known to those of skffl in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amphfication, 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 NTRAN using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RTAs), and fluorescence activated ceU sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on NTRAN is preferred, but a competitive binding assay may be employed. These and other assays are weU known in the art

(Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul MN, Sect. IN; Coligan, J.E. et al. (1997) Current Protocols in Immunology, Greene Pub. Associates and Wiley- Interscience, New York NY; Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa NJ). A wide variety of labels and conjugation techniques are known by those skilled in the art and maybe used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding NTRAN include ohgolabeling, nick translation, end-labeling, or PCR amphfication using a labeled nucleotide. Alternatively, polynucleotides encoding NTRAN, or any fragments thereof, maybe cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commerciaUy 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 maybe conducted using a variety of commerciaUy available kits, such as those provided by Amersham Biosciences, Promega (Madison WI), and US Biochemical. Suitable reporter molecules or labels which maybe used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as weU as substrates, cofactors, inhibitors, magnetic particles, and the like. Host ceUs transformed with polynucleotides encoding NTRAN maybe cultured under conditions suitable for the expression and recovery of the protein from ceU culture. The protein produced by a transformed ceU maybe secreted or retained intraceUularly depending on the sequence and/or the vector used. As wiU be understood by those of skill in the art, expression vectors containing polynucleotides which encode NTRAN may be designed to contain signal sequences which direct secretion of NTRAN through a prokaryotic or eukaryotic ceU membrane.

In addition, a host ceU strain may be chosen for its abihty 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 ceUs which have specific ceUular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture CoUection (ATCC, Manassas VA) and maybe chosen to ensure the correct modification and processing of the foreign protein. In another embodiment of the invention, natural, modified, or recombinant polynucleotides encoding NTRAN may be hgated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems. For example, a chimeric NTRAN protein containing a heterologous moiety that can be recognized by a commerciaUy available antibody may , facihtate the screening of peptide libraries for inhibitors of NTRAN activity. Heterologous protein and peptide moieties may also facihtate purification of fusion proteins using commerciaUy available affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable immiunoaffinity purification of fusion proteins using commerciaUy available monoclonal and polyclonal antibodies that specificaUy recognize these epitope tags. A fusion protein may also be engineered to contain a proteolytic cleavage site located between the NTRAN encoding sequence and the heterologous protein sequence, so that NTRAN maybe cleaved away from the heterologous moiety foUowing purification. Methods for fusion protein expression and purification are discussed in Ausubel et al. (supra, ch. 10 and 16). A variety of commerciaUy available kits may also be used to facihtate expression and purification of fusion proteins. In another embodiment, synthesis of radiolabeled NTRAN maybe achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, ³⁵S-methionine.

NTRAN, fragments of NTRAN, or variants of NTRAN maybe used to screen for compounds that specificaUy bind to NTRAN. One or more test compounds may be screened for specific binding to NTRAN. In various embodiments, 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened for specific binding to NTRAN. Examples of test compounds can include antibodies, anticahns, ohgonucleotides, proteins (e.g., hgands or receptors), or smaU molecules. In related embodiments, variants of NTRAN can be used to screen for binding of test compounds, such as antibodies, to NTRAN, a variant of NTRAN, or a combination of NTRAN and/or one or more variants NTRAN. In an embodiment, a variant of NTRAN can be used to screen for compounds that bind to a variant of NTRAN, but not to NTRAN having the exact sequence of a sequence of SEQ ID NO:l-22. NTRAN variants used to perform such screening can have a range of about 50% to about 99% sequence identity to NTRAN, with various embodiments having 60%, 70%, 75%, 80%, 85%, 90%, and 95% sequence identity.

In an embodiment, a compound identified in a screen for specific binding to NTRAN can be , closely related to the natural ligand of NTRAN, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner (Cohgan, J.E. et al. ( 1991) Cunent Protocols in Immunology l(2):Chapter 5). In another embodiment, the compound thus identified can be a natural ligand of a receptor NTRAN (Howard, AD. 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 NTRAN can be closely related to the natural receptor to which NTRAN binds, at least a fragment of the receptor, or a fragment of the receptor including aU or a portion of the ligand binding site or binding pocket. For example, the compound maybe a receptor for NTRAN which is capable of propagating a signal, or a decoy receptor for NTRAN which is not capable of propagating a signal (Ashkenazi, A. and V.M. Divit (1999) Cun. Opin. CeU Biol. 11:255-260; Mantovani, A. et al. (2001) Trends Immunol. 22:328- 336). The compound can be rationaUy 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 lgG₁ (Taylor, P.C. et al. (2001) Curr. Opin. Immunol. 13:611-616).

In one embodiment, two or more antibodies having similar or, alternatively, different specificities can be screened for specific binding to NTRAN, fragments of NTRAN, or variants of NTRAN. The binding specificity of the antibodies thus screened can thereby be selected to identify particular fragments or variants of NTRAN. In one embodiment, an antibody can be selected such that its binding specificity aUows for preferential identification of specific fragments or variants of NTRAN. In another embodiment, an antibody can be selected such that its binding specificity aUows for preferential diagnosis of a specific disease or condition having increased, decreased, or otherwise abnormal production of NTRAN.

In an embodiment, anticahns can be screened for specific binding to NTRAN, fragments of NTRAN, or variants of NTRAN. Anticahns are hgand-binding proteins that have been constructed based on a hpocalin scaffold (Weiss, G.A. and H.B. Lowman (2000) Chem. Biol. 7:R177-R184; Skena, A. (2001) J. Biotechnol. 74:257-275). The protein architecture of hpocalins can include a beta-banel having eight antiparaUel beta-strands, which supports four loops at its open end. These loops form the natural hgand-binding site of the hpocalins, 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 specificaUy bind to, stimulate, or inhibit NTRAN involves producing appropriate ceUs which express NTRAN, either as a secreted protein or on the ceU membrane. Prefened ceUs can include ceUs from mammals, yeast, Drosophila, or E. coli. CeUs expressing NTRAN or ceU membrane fractions which contain NTRAN are then contacted with a test compound and binding, stimulation, or inhibition of activity of either NTRAN 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 NTRAN, either in solution or affixed to a sohd support, and detecting the binding of NTRAN to the compound. Alternatively, the assay may detect or measure binding of a test compound in the presence of a labeled competitor. AdditionaUy, the assay may be carried out using ceU-free preparations, chemical libraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a sohd support.

An assay can be used to assess the abihty of a compound to bind to its natural ligand and/or to inhibit the binding of its natural ligand to its natural receptors. Examples of such assays include radio- labeling assays such as those described in U.S. Patent No. 5,914,236 and U.S. Patent No. 6,372,724. In a related embodiment, one or more amino acid substitutions can be introduced into a polypeptide compound (such as a receptor) to improve or alter its abihty to bind to its natural ligands (Matthews, D.J. and J.A. WeUs. (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 abihty to bind to its natural receptors (Cunningham, B.C. and J.A. WeUs (1991) Proc. Natl. Acad. Sci. USA 88:3407-3411; Lowman, H.B. et al. (1991) J. Biol. Chem. 266:10982-10988).

NTRAN, fragments of NTRAN, or variants of NTRAN may be used to screen for compounds that modulate the activity of NTRAN. Such compounds may include agonists, antagonists, or partial or inverse agonists. In one embodiment, an assay is performed under conditions permissive for NTRAN activity, wherein NTRAN is combined with at least one test compound, and the activity of NTRAN in the presence of a test compound is compared with the activity of NTRAN in the absence of the test compound. A change in the activity of NTRAN in the presence of the test compound is indicative of a compound that modulates the activity of NTRAN. Alternatively, a test compound is combined with an in vitro or ceU-free system comprising NTRAN under conditions suitable for NTRAN activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of NTRAN 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 maybe screened.

In another embodiment, polynucleotides encoding NTRAN or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) ceUs. Such techniques are weU known in the art and are useful for the generation of animal models of human disease (see, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No. 5,767,337). For example, mouse ES ceUs, such as the mouse 129/SvJ ceU line, are derived from the early mouse embryo and grown in culture. The ES ceUs are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphofransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292). The vector integrates into the conesponding region of the host genome by homologous recombination. Alternatively, homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330). Transformed ES ceUs are identified and microinjected into mouse ceUblastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgicaUy transfened 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 NTRAN may also be manipulated in vitro in ES ceUs derived from human blastocysts. Human ES ceUs have the potential to differentiate into at least eight separate ceU lineages including endoderm, mesoderm, and ectodermal ceU types. These ceU lineages differentiate into, for example, neural ceUs, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147).

Polynucleotides encoding NTRAN 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 NTRAN is injected into animal ES ceUs, and the injected sequence integrates into the animal ceU genome. Transformed ceUs 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 NTRAN, e.g., by secreting NTRAN in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74). THERAPEUTICS Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of NTRAN and neurotransmission-associated proteins. In addition, examples of tissues expressing NTRAN can be found in Table 6 and can also be found in Example XI. Therefore, NTRAN appears to play a role in autoimmune/inflammatory, cardiovascular, neurological, developmental, ceU prohferative, transport, psychiatric, metabohc, and endocrine disorders. In the treatment of disorders associated with increased NTRAN expression or activity, it is desirable to decrease the expression or activity of NTRAN. In the treatment of disorders associated with decreased NTRAN expression or activity, it is desirable to increase the expression or activity of NTRAN.

Therefore, in one embodiment, NTRAN 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 NTRAN. Examples of such disorders include, but are not limited to, an autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome (ADDS), Addison's disease, adult respiratory distress syndrome, aUergies, ankylosing spondyhtis, 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 fetahs, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophiha, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thromb osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjδgren's ocytopenic purpura, ulcerative cohtis, uveitis, Werner syndrome, comphcations of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; a cardiovascular disorder such as congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitaUy bicuspid aortic valve, mitral annular calcification, mifral valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, congenital heart disease, and comphcations of cardiac transplantation, arterio venous fistula, . atherosclerosis, hypertension, vascuhtis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, and comphcations of thrombolysis, baUoon angioplasty, vascular replacement, and coronary artery bypass graft surgery; a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyofrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt- Jakob disease, and Gerstmann-Straussler-Scheihker syndrome, fatal familial insomnia, nutritional and metabohc diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebeUoret nal 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, metabohc, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; a developmental disorder such as renal tabular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormahties, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithehal dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; a ceU prohferative 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, colon, gaU bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, sahvary glands, skin, spleen, testis, thymus, thyroid, and uterus and a cancer such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gaU bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, sahvary glands, skin, spleen, testis, thymus, thyroid, and uterus; a fransport disorder such as akinesia, amyofrophic lateral sclerosis, ataxia telangiectasia, cystic fibrosis, Becker's muscular dystrophy, BeU's palsy, Charcot-Marie Tooth disease, diabetes mellitus, diabetes insipidus, diabetic neuropathy, Duchenne muscular dystrophy, hyperkalemic periodic paralysis, normokalemic periodic paralysis, Parkinson's disease, mahgnanthyperthermia, multidrug resistance, myasthenia gravis, myotonic dystrophy, catatonia, tardive dyskinesia, dystonias, peripheral neuropathy, cerebral neoplasms, prostate cancer, cardiac disorders associated with transport, e.g., angina, bradyanrythmia, tachyanythmia, hypertension, Long QT syndrome, myocarditis, cardiomyopathy, nema ne myopathy, cenfronuclear myopathy, hpid myopathy, mitochondria! myopathy, thyrotoxic myopathy, ethanol myopathy, dermatomyositis, inclusion body myositis, infectious myositis, polymyositis, neurological disorders associated with transport, e.g., Alzheimer's disease, amnesia, bipolar disorder, dementia, depression, epilepsy, Tourette's disorder, paranoid psychoses, and schizophrenia, and other disorders associated with transport, e.g., neurofibromatosis, postherpetic neuralgia, frigeminal neuropathy, sarcoidosis, sickle ceU anemia, Wilson's disease, cataracts, infertility, pulmonary artery stenosis, sensorineural autosomal deafness, hyperglycemia, hypoglycemia, Grave's disease, goiter, (lashing's disease, Addison's disease, glucose-galactose malabsorption syndrome, hypercholesterolemia, adrenoleukodystrophy, ZeUweger syndrome, Menkes disease, occipital horn syndrome, von Gierke disease, cystinuria, iminoglycinuria, Hartup disease, and Fanconi disease; a psychiatric disorder such as acute stress disorder, alcohol dependence, amphetamine dependence, anorexia nervosa, antisocial personahty disorder, attention-deficit hyperactivity disorder, autistic disorder, anxiety, avoidant personahty disorder, bipolar disorder, borderline personahty disorder, brief psychotic disorder, bulimia nervosa, cannabis dependence, cocaine dependence, conduct disorder, cyclothymic disorder, delirium, delusional disorder, dementia, dependent personahty disorder, depression, dysthymic disorder, hallucinogen dependence, histrionic personahty disorder, inhalant dependence, manic depression, multi-infarct dementia, narcissistic personahty disorder, nicotine dependence, obsessive-compulsive disorder, opioid dependence, oppositional defiant disorder, panic disorder, paranoid personahty disorder, phencychdine dependence, phobia, posttraumatic stress disorder, schizoaffective disorder, schizoid personahty disorder, schizophrenia, sedative dependence, separation anxiety disorder, and sleep disorder; a metabohc disorder such as Addison's disease, cerebrotendinous xanthomatosis, congenital adrenal hyperplasia, coumarin resistance, cystic fibrosis, fatty hepatocirrhosis, fructose- 1,6-diphosphatase deficiency, galactosemia, goiter, glucagonoma, glycogen storage diseases, hereditary fructose intolerance, hyperadrenahsm, hypoadrenahsm, hyperparathyroidism, hypoparathyroidism, hypercholesterolemia, hyperthyroidism, hypoglycemia, hypothyroidism, hyperlipidemia, hyperhpemia, hpid myopathies, hpodystrophies, lysosomal storage diseases, mannosidosis, neuraminidase deficiency, obesity, osteoporosis, phenylketonuria, pseudovitamin D- deficiency rickets, disorders of carbohydrate metabohsm such as congenital type JJ dyserythropoietic anemia, diabetes, insulin-dependent diabetes mellitus, non-insulin-dependent diabetes mellitus, galactose epimerase deficiency, glycogen storage diseases, lysosomal storage diseases, fructosuria, pentosuria, and inherited abnormahties of pyruvate metabohsm, disorders of hpid metabohsm such as fatty hver, cholestasis, primary biliary cirrhosis, carnitine deficiency, carnitine palmitoyltransferase deficiency, myoadenylate deaminase deficiency, hypertriglyceridemia, hpid storage disorders such Fabry's disease, Gaucher's disease, Niemann-Pick's disease, metachromatic leukodystrophy, adrenoleukodystrophy, GM₂ ganghosidosis, and ceroid lipofuscinosis, abetahpoproteinemia, Tangier disease, hyperhpoproteinemia, hpodysfrophy, hpomatoses, acute pannicuhtis, disseminated fat necrosis, adiposis dolorosa, hpoid adrenal hyperplasia, minimal change disease, hpomas, atherosclerosis, hypercholesterolemia, hypercholesterolemia with hypertriglyceridemia, primary hypoalphahpoproteinemia, hypothyroidism, renal disease, hver disease, lecithin:cholesterol acyltransferase deficiency, cerebrotendinous xanthomatosis, sitosterolemia, hypocholesterolemia, Tay- Sachs disease, SandhofFs disease, hyperlipidemia, hyperhpemia, and hpid myopathies, and disorders of copper metabohsm such as Menke's disease, Wilson's disease, and Ehlers-Danlos syndrome type IX diabetes; and an endocrine disorder such as a disorder of the hypothalamus and/or pituitary resulting from lesions such as a primary brain tumor, adenoma, infarction associated with pregnancy, hypophysectomy, aneurysm, vascular malformation, thrombosis, infection, immunological disorder, and complication due to head trauma, a disorder associated with hypopituitarism including hypogonadism, Sheehan syndrome, diabetes insipidus, Kallman's disease, Hand-SchuUer-Christian disease, Letterer- Siwe disease, sarcoidosis, empty seUa syndrome, and dwarfism, a disorder associated with hyperpituitarism including acromegaly, giantism, and syndrome of inappropriate antidiuretic hormone (ADH) secretion (SIADH) often caused by benign adenoma, a disorder associated with hypothyroidism including goiter, myxedema, acute thyroiditis associated with bacterial infection, subacute thyroiditis associated with viral infection, autoimmune thyroiditis (Hashimoto's disease), and cretinism, a disorder associated with hyperthyroidism including thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and Plummer's disease, a disorder associated with hyperparathyroidism including Conn disease (chronic hypercalemia), a pancreatic disorder such as Type I or Type II diabetes mellitus and associated comphcations, a disorder associated with the adrenals such as hyperplasia, carcinoma, or adenoma of the adrenal cortex, hypertension associated with alkalosis, amyloidosis, hypokalemia, Gushing' s disease, Liddle's syndrome, and Arnold-Healy-Gordon syndrome, pheochromocytoma tumors, and Addison's disease, a disorder associated with gonadal steroid hormones such as: in women, abnormal prolactin production, infertility, endometriosis, perturbation of the menstrual cycle, polycystic ovarian disease, hyperprolactinemia, isolated gonadotropin deficiency, amenonhea, galactorrhea, hermaphroditism, hirsutism and virihzation, breast cancer, and, in post-menopausal women, osteoporosis, and, in men, Leydig ceU deficiency, male climacteric phase, and germinal ceU aplasia, a hypergonadal disorder associated with Leydig ceU tumors, androgen resistance associated with absence of androgen receptors, syndrome of 5 α-reductase, and gynecomastia.

In another embodiment, a vector capable of expressing NTRAN 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 NTRAN including, but not limited to, those described above. In a further embodiment, a composition comprising a substantiaUy purified NTRAN in conjunction with a suitable pharmaceutical carrier maybe administered to a subject to treat or prevent a disorder associated with decreased expression or activity of NTRAN including, but not limited to, those provided above.

In stffl another embodiment, an agonist which modulates the activity of NTRAN maybe administered to a subject to treat or prevent a disorder associated with decreased expression or activity of NTRAN including, but not hmited to, those listed above.

In a further embodiment, an antagonist of NTRAN may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of NTRAN. Examples of such disorders include, but are not limited to, those autoinmiune/inflammatory, cardiovascular, neurological, developmental, ceU prohferative, fransport, psychiatric, metabohc, and endocrine disorders described above. In one aspect, an antibody which specificaUy binds NTRAN may be used directly as an antagonist or indirectly as a targeting or dehvery mechanism for bringing a pharmaceutical agent to ceUs or tissues which express NTRAN.

In an additional embodiment, a vector expressing the complement of the polynucleotide encoding NTRAN may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of NTRAN including, but not hmited 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 synergisticaUy to effect the treatment or prevention of the various disorders described above. Using this approach, one maybe able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

An antagonist of NTRAN may be produced using methods which are generaUy known in the art. In particular, purified NTRAN may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specificaUy bind NTRAN. Antibodies to NTRAN may also be generated using methods that are weU 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 hbrary. In an embodiment, neutralizing antibodies (i.e., those which inhibit dimer formation) can be used therapeuticaUy. Single chain antibodies (e.g., from camels or Uamas) maybe potent enzyme inhibitors and may have apphcation 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, Uamas, humans, and others may be immunized by injection with NTRAN or with any fragment or ohgopeptide 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 hmited 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 Coi nebacterium pai um are especiaUy preferable. It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to

NTRAN have an amino acid sequence consisting of at least about 5 amino acids, and generaUy wiU consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are substantiaUy identical to a portion of the amino acid sequence of the natural protein. Short stretches of NTRAN 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 NTRAN may be prepared using any technique which provides for the production of antibody molecules by continuous ceU lines in culture. These include, but are not limited to, the hybridoma technique, the human B-ceU hybridoma technique, and the EBN-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, RJ. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole, S.P. et al. (1984) Mol. CeU Biol. 62:109-120).

In addition, techniques developed for the production of "chimeric antibodies," such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (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 314:452-454). Alternatively, techniques described for the production of single chain antibodies maybe adapted, using methods known in the art, to produce NTRAN-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).

Antibody fragments which contain specific binding sites for NTRAN may also be generated. For example, such fragments include, but are not limited to, F(ab% 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 maybe constructed to aUow 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 weU known in the art. Such immunoassays typicaUy involve the measurement of complex formation between NTRAN and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering NTRAN epitopes is generaUy used, but a competitive binding assay may also be employed (Pound, supra).

Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques maybe used to assess the affinity of antibodies for NTRAN. Affinity is expressed as an association constant, K_a, which is defined as the molar concentration of NTRAN-antibody complex divided by the molar concentrations of free antigen and free antibody under equihbrium conditions. The K_a determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple NTRAN epitopes, represents the average affinity, or avidity, of the antibodies for NTRAN. The K_a determined for a preparation of monoclonal antibodies, which are monospecific for a particular NTRAN epitope, represents a true measure of affinity, ffigh-affinity antibody preparations with K_a ranging from about 10⁹ to 10¹² L/mole are prefened for use in immunoassays in which the NTRAN- antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with K_a ranging from about 10⁶ to 10⁷ L/mole are prefened for use in immunopurification and similar procedures which ultimately require dissociation of NTRAN, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL Press, Washington DC; LiddeU, 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 maybe further evaluated to determine the quality and suitabihty of such preparations for certain downstream applications. For example, a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml, is generaUy employed in procedures requiring precipitation of NTRAN-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quahty and usage in various applications, are generaUy available (Catty, supra; Cohgan et al., supra).

In another embodiment of the invention, polynucleotides encoding NTRAN, 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 ohgonucleotides) to the coding or regulatory regions of the gene encoding NTRAN. Such technology is weU known in the art, and antisense ohgonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding NTRAN (Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press, Totawa NJ).

In therapeutic use, any gene dehvery system suitable for introduction of the antisense sequences into appropriate target ceUs can be used. Antisense sequences can be dehvered intraceUularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the ceUular sequence encoding the target protein (Slater, J.E. et al. (1998) J. AUergy Clin. Immunol. 102:469-475; Scanlon, KJ. et al. (1995) 9:1288-1296). Antisense sequences can also be introduced intraceUularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (Mffler, AD. (1990) Blood 76:271; Ausubel et al, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63:323-347). Other gene dehvery mechanisms include hposome-derived systems, artificial viral envelopes, and other systems known in the art (Rossi, J J. (1995) Br. Med. BuU. 51:217-225; Boado, RJ. 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 NTRAN maybe used for somatic or germline gene therapy. Gene therapy may be performed to (i) conect a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCTD)-Xl disease characterized by X- hnked 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) CeU 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 hemophiha resulting from Factor Vm or Factor IX deficiencies (Crystal, R.G. (1995) Science 270:404-410; Verma, I.M. and N. Somia (1997) Nature 389:239-242)), (ii) express a conditionaUy lethal gene product (e.g., in the case of cancers which result from unregulated ceU proliferation), or (hi) express a protein which affords protection against intraceUular parasites (e.g., against human retroviruses, such as human immunodeficiency virus (HTV) (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 Plasmodiumfalciparum and Trypanosoma cruzi). In the case where a genetic deficiency in NTRAN expression or regulation causes disease, the expression of NTRAN from an appropriate population of transduced ceUs may aUeviate the clinical manifestations caused by the genetic deficiency.

In a further embodiment of the invention, diseases or disorders caused by deficiencies in NTRAN are treated by constructing mammalian expression vectors encoding NTRAN and introducing these vectors by mechanical means into NTRAN-deficient ceUs. Mechanical fransfer technologies for use with ceUs in vivo or ex vitro include (i) direct DNA microinjection into individual ceUs, (ii) ballistic gold particle dehvery, (hi) hposome-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; Ivies, Z. (1997) CeU 91:501-510; Boulay, J.-L. and H. Recipon (1998) Cun. Opin. Biotechnol. 9:445-450).

Expression vectors that may be effective for the expression of NTRAN include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors

(Invitrogen, Carlsbad CA), PCMV-SCPJPT, PCMV-TAG, PEGSH/PERV (Stratagene, La JoUa CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). NTRAN maybe expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or β-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) Cun. Opin. Biotechnol. 9:451-456), commerciaUy available in the T-REX plasmid (Invitrogen)); the ecdysone-inducϊble promoter (available in the plasmids PVGRXR and PUSD; Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible promoter (Rossi, F.M.V. and H.M. Blau, supra)), or (hi) a tissue-specific promoter or the native promoter of the endogenous gene encoding NTRAN from a normal individual. CommerciaUy available hposome transformation kits (e.g. , the PERFECT LJPJD

TRANSFECΉON KIT, available from Invitrogen) aUow one with ordinary skill in the art to dehver polynucleotides to target ceUs 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 elecfroporation (Neumann, E. et al. (1982) EMBO J. 1:841-845). The introduction of DNA to primary ceUs requires modification of these standardized mammalian transfection protocols.

In another embodiment of the invention, diseases or disorders caused by genetic defects with respect to NTRAN expression are freated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding NTRAN under the control of an independent promoter or the refrovirus long tenninal repeat (LTR) promoter, (h) appropriate RNA packaging signals, and (hi) a Rev-responsive element (RRE) along with additional retrovirus m-acting RNA sequences and coding sequences required for efficient vector propagation. Retrovirus vectors (e.g., PFB and PFBNEO) are commerciaUy available (Stratagene) and are based on pubhshed data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The vector is propagated in an appropriate vector producing ceU line (VPCL) that expresses an envelope gene with a tropism for receptors on the target ceUs 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 AD. Mffler (1988) J. Virol. 62:3802-3806; DuU, 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 refrovirus packaging ceU lines producing high transducing efficiency refroviral supernatant") discloses a method for obtaining retrovirus packaging ceU lines and is hereby incorporated by reference. Propagation of refrovirus vectors, transduction of a population of ceUs (e.g., CD4⁺ T-ceUs), and the return of transduced ceUs to a patient are procedures weU known to persons skilled in the art of gene therapy and have been weU documented (Ranga, U. et al. (1997) J. Virol. 71:7020-7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-4716; Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).

In an embodiment, an adenovirus-based gene therapy dehvery system is used to dehver polynucleotides encoding NTRAN to ceUs which have one or more genetic abnormahties with respect to the expression of NTRAN. The construction and packaging of adenovirus-based vectors are weU known to those with ordinary skiU in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas f (Csete, M.E. et al. (1995) Transplantation 27:263-268). PotentiaUy 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. Nufr. 19:511-544) and Verma, I.M. and N. Somia (1997; Nature 18:389:239-242).

In another embodiment, a herpes-based, gene therapy dehvery system is used to dehver polynucleotides encoding NTRAN to target ceUs which have one or more genetic abnormahties with respect to the expression of NTRAN. The use of herpes simplex virus (HSV)-based vectors maybe especiaUy valuable for introducing NTRAN to ceUs of the central nervous system, for which HSV has a tropism. The construction and packaging of herpes-based vectors are weU known to those with ordinary skffl in the art. A rephcation-competent herpes simplex virus (HSV) type 1-based vector has been used to dehver a reporter gene to the eyes of primates (Liu, X. et al. (1999) Exp. Eye Res. 169:385-395). The construction of a HSV-1 virus vector has also been disclosed in detail in U.S. Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference. U.S. Patent No. 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be fransfened to a ceU 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 foUowing the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of ceUs with herpesvirus are techniques weU known to those of ordinary skffl in the art.

In another embodiment, an alphavirus (positive, single-stranded RNA virus) vector is used to dehver polynucleotides encoding NTRAN to target ceUs. The biology of the prototypic alphavirus, Semhki 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) Cun. Opin. Biotechnol. 9:464-469). During alphavirus RNA replication, a subgenomic RNA is generated that normaUy encodes the viral capsid proteins. This subgenomic RNA rephcates 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 NTRAN into the alphavirus genome in place of the capsid-coding region results in the production of a large number of NTRAN-coding RNAs and the synthesis of high levels of NTRAN in vector transduced ceUs. While alphavirus infection is typicaUy associated with ceU lysis within a few days, the abihty to estabhsh a persistent infection in hamster normal kidney ceUs (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 wffl aUow the introduction of NTRAN into a variety of ceU types. The specific transduction of a subset of ceUs in a population may require the sorting of ceUs prior to transduction. The methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA fransfections, and perfornήng alphavirus infections, are weU known to those with ordinary skffl in the art.

Ohgonucleotides 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 hehx base-pairing methodology. Triple hehx pairing is useful because it causes inhibition of the abihty of the double hehx 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 Immunologic Approaches, Futura Pubhshing, Mt. Kisco NY, pp. 163-177). A complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, foUowed by endonucleolytic cleavage. For example, engineered hammerhead motif ribozyme molecules may specificaUy and efficiently catalyze endonucleolytic cleavage of RNA molecules encoding NTRAN.

Specific ribozyme cleavage sites within any potential RNA target are initiaUy 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, conesponding to the region of the target gene containing the cleavage site, maybe evaluated for secondary structural features which may render the ohgonucleotide inoperable. The suitabihty of candidate targets may also be evaluated by testing accessibihty to hybridization with complementary ohgonucleotides 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 chemicaUy synthesizing ohgonucleotides such as sohd phase phosphoramidite chemical synthesis. Alternatively, RNA molecules maybe generated by in vitro and in vivo transcription of DNA molecules encoding NTRAN. 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 ceU lines, ceUs, or tissues.

RNA molecules maybe modified to increase intraceUular stabihty and half-life. Possible modifications include, but are not hmited 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 aU of these molecules by the inclusion of nonfraditional bases such as inosine, queosine, and wybutosine, as weU as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.

In other embodiments of the invention, the expression of one or more selected polynucleotides of the present invention can be altered, inhibited, decreased, or silenced using RNA interference (RNAi) or post-transcriptional gene silencing (PTGS) methods known in the art. RNAi is a post- transcriptional mode of gene silencing in which double-stranded RNA (dsRNA) introduced into a targeted ceU specificaUy suppresses the expression of the homologous gene (i.e., the gene bearing the sequence complementary to the dsRNA). This effectively knocks out or substantiaUy reduces the expression of the targeted gene. PTGS can also be accomplished by use of DNA or DNA fragments as weU. RNAi methods are described by Fire, A. et al. (1998 ; Nature 391 :806-811) and Gura, T.

(2000; Nature 404:804-808). PTGS can also be initiated by introduction of a complementary segment of DNA into the selected tissue using gene dehvery and/or viral vector dehvery methods described herein or known in the art.

RNAi can be induced in mammalian ceUs by the use of smaU interfering RNA also known as siRNA. SiRNA are shorter segments of dsRNA (typicaUy about 21 to 23 nucleotides in length) that result in vivo from cleavage of introduced dsRNA by the action of an endogenous ribonuclease. SiRNA appear to be the mediators of the RNAi effect in mammals. The most effective siRNAs appear to be 21 nucleotide dsRNAs with 2 nucleotide 3' overhangs. The use of siRNA for inducing RNAi in mammalian ceUs is described by Elbashir, S.M. et al. (2001; Nature 411:494-498).

SiRNA can either be generated indirectly by introduction of dsRNA into the targeted ceU, or directly by mammalian transfection methods and agents described herein or known in the art (such as hposome-mediated transfection, viral vector methods, or other polynucleotide dehvery/introductory methods). Suitable SiRNAs can be selected by examining a transcript of the target polynucleotide (e.g., mRNA) for nucleotide sequences downstream from the AUG start codon and recording the occunence of each nucleotide and the 3' adjacent 19 to 23 nucleotides as potential siRNA target sites, with sequences having a 21 nucleotide length being prefened. Regions to be avoided for target siRNA sites include the 5' and 3 'untranslated regions (UTRs) and regions near the start codon (within 75 bases), as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP endonuclease complex. The selected target sites for siRNA can then be compared to the appropriate genome database (e.g., human, etc.) using BLAST or other sequence comparison algorithms known in the art. Target sequences with significant homology to other coding sequences can be eliminated from consideration. The selected SiRNAs can be produced by chemical synthesis methods known in the art or by in vitro transcription using commerciaUy available methods and kits such as the SILENCER siRNA construction kit (Ambion, Austin TX).

In alternative embodiments, long-term gene silencing and/or RNAi effects can be induced in selected tissue using expression vectors that continuously express siRNA. This can be accomphshed using expression vectors that are engineered to express hairpin RNAs (shRNAs) using methods known in the art (see, e.g., Brummelkamp, T.R. et al. (2002) Science 296:550-553; and Paddison, P.J. et al. (2002) Genes Dev. 16:948-958). In these and related embodiments, shRNAs can be delivered to target ceUs using expression vectors known in the art. An example of a suitable expression vector for dehvery of siRNA is the PSILENCER1.0-U6 (circular) plasmid (Ambion). Once dehvered to the target tissue, shRNAs are processed in vivo into siRNA-like molecules capable of canying out gene- specific silencing.

In various embodiments, the expression levels of genes targeted by RNAi or PTGS methods can be determined by assays for mRNA and/or protein analysis. Expression levels of the mRNA of a targeted gene, can be determined by northern analysis methods using, for example, the

NORTHERNMAX-GLY kit (Ambion); by microanay methods; by PCR methods; by real time PCR methods; and by other RNA/polynucleotide assays known in the art or described herein. Expression levels of the protein encoded by the targeted gene can be determined by Western analysis using standard techniques known in the art.

An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding NTRAN. Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, ohgonucleotides, antisense ohgonucleotides, triple hehx-forming ohgonucleotides, 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 NTRAN expression or activity, a compound which specificaUy inhibits expression of the polynucleotide encoding NTRAN may be therapeuticaUy useful, and in the treatment of disorders associated with decreased NTRAN expression or activity, a compound which specificaUy promotes expression of the polynucleotide encoding NTRAN may be therapeuticaUy useful. In various embodiments, one or more 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, commerciaUy-available or proprietary hbrary of naturaUy-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 hbrary of chemical compounds created combinatorially or randomly. A sample comprising a polynucleotide encoding NTRAN is exposed to at least one test compound thus obtained. The sample may comprise, for example, an intact or permeabihzed ceU, or an in vitro ceU-free or reconstituted biochemical system. Alterations in the expression of a polynucleotide encoding NTRAN are assayed by any method commonly known in the art. TypicaUy, 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 NTRAN. 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 ceU line such as HeLa ceU (Clarke, M.L. et al. (2000) Biochem. Biophys. Res.

Commun. 268:8-13). A particular embodiment of the present invention involves screening a combinatorial hbrary of ohgonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified ohgonucleotides) 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 ceUs or tissues are avaUable and equaUy suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors maybe introduced into stem ceUs taken from the patient and clonaUy propagated for autologous transplant back into that same patient.

Dehvery by transfection, by hposome injections, or by polycationic amino polymers may be achieved using methods which are weU known in the art (Goldman, CK. et al. (1997) Nat. Biotechnol. 15:462-

466).

Any of the therapeutic methods described above may be apphed 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 generaUy comprises an active ingredient formulated with a pharmaceuticaUy acceptable excipient.

Excipients may include, for example, sugars, starches, ceUuloses, gums, and proteins. Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's

Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may consist of

NTRAN, antibodies to NTRAN, and mimetics, agonists, antagonists, or inhibitors of NTRAN. In various embodiments, the compositions described herein, such as pharmaceutical compositions, may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intrameduUary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means. Compositions for pulmonary administration may be prepared in hquid or dry powder form.

These compositions are generaUy aerosohzed immediately prior to inhalation by the patient. In the case of smaU molecules (e.g. traditional low molecular weight organic drugs), aerosol dehvery of fast- acting formulations is weU-known in the art. In the case of macromolecules (e.g. larger peptides and proteins), recent developments in the field of pulmonary dehvery via the alveolar region of the lung have enabled the practical dehvery of drugs such as insulin to blood circulation (see, e.g., Patton, J.S. et al., U.S. Patent No. 5,997,848). Pulmonary dehvery aUows administration without needle injection, and obviates the need for potentiaUy 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 detenriination of an effective dose is weU within the capabihty of those skilled in the art.

Specialized forms of compositions may be prepared for direct intraceUular dehvery of macromolecules comprising NTRAN or fragments thereof. For example, liposome preparations containing a ceU-impermeable macromolecule may promote ceU fusion and intraceUular dehvery of the macromolecule. Alternatively, NTRAN or a fragment thereof may be joined to a short cationic N- terminal portion from the HJV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the ceUs of aU tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).

For any compound, the therapeuticaUy effective dose can be estimated initiaUy either in ceU culture assays, e.g., of neoplastic ceUs, 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 therapeuticaUy effective dose refers to that amount of active ingredient, for example

NTRAN or fragments thereof, antibodies of NTRAN, and agonists, antagonists or inhibitors of NTRAN, which amehorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in ceU cultures or with experimental animals, such as by calculating the ED₅₀ (the dose therapeuticaUy effective in 50% of the population) or LD_S0 (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 LD₅₀ ED₅₀ ratio. Compositions which exhibit large therapeutic indices are preferred. The data obtained from ceU 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 ED₅₀ with little or no toxicity.

The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration. The exact dosage wiU be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and adminisfration 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 maybe 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 μg to 100,000 μg, up to a total dose of about 1 gram, depending upon the route of adminisfration. Guidance as to particular dosages and methods of dehvery is provided in the literature and generaUy available to practitioners in the art. Those skilled in the art wiU employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, dehvery of polynucleotides or polypeptides wffl be specific to particular ceUs, conditions, locations, etc.

DIAGNOSTICS

In another embodiment, antibodies which specificaUy bind NTRAN maybe used for the diagnosis of disorders characterized by expression of NTRAN, or in assays to monitor patients being freated with NTRAN or agonists, antagonists, or inhibitors of NTRAN. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for NTRAN include methods which utilize the antibody and a label to detect NTRAN in human body fluids or in extracts of ceUs 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 NTRAN, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of NTRAN expression. Normal or standard values for NTRAN expression are estabhshed by combining body fluids or ceU extracts taken from normal mammalian subjects, for example, human subjects, with antibodies to NTRAN under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of NTRAN expressed in subject, confrol, and disease samples frombiopsied tissues are compared with the standard values. Deviation between standard and subject values estabhshes the parameters for diagnosing disease.

In another embodiment of the invention, polynucleotides encoding NTRAN maybe used for diagnostic purposes. The polynucleotides which may be used include ohgonucleotides, 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 NTRAN may be correlated with disease. The diagnostic assay maybe used to determine absence, presence, and excess expression of NTRAN, and to monitor regulation of NTRAN levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable of detecting polynucleotides, including genomic sequences, encoding NTRAN or closely related molecules maybe used to identify nucleic acid sequences which encode NTRAN. The specificity of the probe, whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amphfication wffl determine whether the probe identifies only naturaUy occurring sequences encoding NTRAN, aUelic 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 NTRAN encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ JD NO:23-44 or from genomic sequences including promoters, enhancers, and introns of the NTRAN gene.

Means for producing specific hybridization probes for polynucleotides encoding NTRAN include the cloning of polynucleotides encoding NTRAN or NTRAN derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commerciaUy 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 radionuchdes 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 NTRAN may be used for the diagnosis of disorders associated with expression of NTRAN. Examples of such disorders include, but are not limited to, an autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome (AJDS), Addison's disease, adult respiratory distress syndrome, aUergies, 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 fetahs, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophiha, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thromb osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjδgren's ocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, comphcations of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; a cardiovascular disorder such as congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitaUy bicuspid aortic valve, mitral annular calcification, mitral valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, congenital heart disease, and comphcations of cardiac transplantation, arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, and comphcations of thrombolysis, baUoon angioplasty, vascular replacement, and coronary artery bypass graft surgery; a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyofrophic 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, myehtis and radiculitis, viral central nervous system disease, prion diseases including, kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabohc diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebeUoretinal hemangioblastomatosis, encephalotήgeminal 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, metabohc, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; a developmental disorder such as renal tubular acidosis, anemia, Cnshing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormahties, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithehal dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; a ceU prohferative 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, colon, gaU bladder, ganglia, gastrointestinal tract, heart, kidney, hver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, sahvary glands, skin, spleen, testis, thymus, thyroid, and uterus and a cancer such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gaU bladder, ganglia, gastrointestinal tract, heart, kidney, hver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, sahvary glands, skin, spleen, testis, thymus, thyroid, and uterus; a transport disorder such as akinesia, amyofrophic lateral sclerosis, ataxia telangiectasia, cystic fibrosis, Becker's muscular dystrophy, BeU'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, tachyanythmia, hypertension, Long QT syndrome, myocarditis, cardiomyopathy, nemahne myopathy, centronuclear myopathy, hpid myopathy, mitochondrial myopathy, thyrotoxic myopathy, ethanol myopathy, dermatomyositis, inclusion body myositis, infectious myositis, polymyositis, neurological disorders associated with fransport, 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 ceU anemia, Wilson's disease, cataracts, infertility, pulmonary artery stenosis, sensorineural autosomal deafness, hyperglycemia, hypoglycemia, Grave's disease, goiter, Cushing's disease, Addison's disease, glucose-galactose malabsorption syndrome, hypercholesterolemia, adrenoleukodystrophy, ZeUweger syndrome, Menkes disease, occipital horn syndrome, von Gierke disease, cystinuria, irmnoglycinuria, Hartup disease, and Fanconi disease; a psychiatric disorder such as acute stress disorder, alcohol dependence, amphetamine dependence, anorexia nervosa, antisocial personahty disorder, attention-deficit hyperactivity disorder, autistic disorder, anxiety, avoidant personahty disorder, bipolar disorder, borderline personahty disorder, brief psychotic disorder, bulimia nervosa, cannabis dependence, cocaine dependence, conduct disorder, cyclothymic disorder, delirium, delusional disorder, dementia, dependent personahty disorder, depression, dysthymic disorder, haUucinogen dependence, histrionic personahty disorder, inhalant dependence, manic depression, multi-infarct dementia, narcissistic personahty disorder, nicotine dependence, obsessive-compulsive disorder, opioid dependence, oppositional defiant disorder, panic disorder, paranoid personahty disorder, phencychdine dependence, phobia, posttraumatic stress disorder, schizoaffective disorder, schizoid personahty disorder, schizophrenia, sedative dependence, separation anxiety disorder, and sleep disorder; a metabohc disorder such as Addison's disease, cerebrotendinous xanthomatosis, congenital adrenal hyperplasia, coumarin resistance, cystic fibrosis, fatty hepatocirrhosis, fructose- 1,6-diphosphatase deficiency, galactosemia, goiter, glucagonoma, glycogen storage diseases, hereditary fructose intolerance, hyperadrenahsm, hypoadrenahsm, hyperparathyroidism, hypoparathyroidism, hypercholesterolemia, hyperthyroidism, hypoglycemia, hypothyroidism, hyperlipidemia, hyperhpemia, hpid myopathies, lipodystrophies, lysosomal storage diseases, mannosidosis, neuraminidase deficiency, obesity, osteoporosis, phenylketonuria, pseudovitamin D- deficiency rickets, disorders of carbohydrate metabohsm such as congenital type JJ dyserythropoietic anemia, diabetes, insulin-dependent diabetes mellitus, non-insulin-dependent diabetes mellitus, galactose epimerase deficiency, glycogen storage diseases, lysosomal storage diseases, fructosuria, pentosuria, and inherited abnormahties of pyruvate metabohsm, disorders of hpid metabohsm such as fatty hver, cholestasis, primary biliary cirrhosis, carnitine deficiency, carnitine palmitoylfransferase deficiency, myoadenylate deaminase deficiency, hypertriglyceridemia, hpid storage disorders such Fabry's disease, Gaucher's disease, Niemann-Pick's disease, metachromatic leukodystrophy, adrenoleukodystrophy, GM₂ gangliosidosis, and ceroid hpofuscinosis, abetahpoproteinemia, Tangier disease, hyperhpoproteinemia, hpodystiophy, hpomatoses, acute pannicuhtis, disseminated fat necrosis, adiposis dolorosa, lipoid adrenal hyperplasia, minimal change disease, hpomas, atherosclerosis, hypercholesterolemia, hypercholesterolemia with hypertriglyceridemia, primary hypoalphahpoproteinemia, hypothyroidism, renal disease, hver disease, lecithin:cholesterol acylttansferase deficiency, cerebrotendinous xanthomatosis, sitosterolemia, hypocholesterolemia, Tay- Sachs disease, Sandhoff s disease, hyperhpidernia, hyperhpemia, and hpid myopathies, and disorders of copper metabohsm such as Menke's disease, Wilson's disease, and Ehlers-Danlos syndrome type IX diabetes; and an endocrine disorder such as a disorder of the hypothalamus and/or pituitary resulting from lesions such as a primary brain tumor, adenoma, infarction associated with pregnancy, hypophysectomy, aneurysm, vascular malformation, thrombosis, infection, immunological disorder, and comphcation due to head trauma, a disorder associated withhypopituitarism including hypogonadism, Sheehan syndrome, diabetes insipidus, KaUman's disease, Hand-SchuUer-Christian disease, Letterer- Siwe disease, sarcoidosis, empty seUa syndrome, and dwarfism, a disorder associated with hyperpitaitarism including acromegaly, giantism, and syndrome of inappropriate antidiuretic hormone (ADH) secretion (SIADH) often caused by benign adenoma, a disorder associated with hypothyroidism including goiter, myxedema, acute thyroiditis associated with bacterial infection, subacute thyroiditis associated with viral infection, autoimmune thyroiditis (Hashimoto's disease), and cretinism, a disorder associated with hyperthyroidism including thyrotoxicosis and its various forms, Grave's disease, pretϊbial myxedema, toxic multinodular goiter, thyroid carcinoma, and Plummer's disease, a disorder associated with hyperparathyroidism including Conn disease (chronic hypercalemia), a pancreatic disorder such as Type I or Type JJ diabetes mellitus and associated comphcations, a disorder associated with the adrenals such as hyperplasia, carcinoma, or adenoma of the adrenal cortex, hypertension associated with alkalosis, amyloidosis, hypokalemia, Cushing's disease, Liddle's syndrome, and Arnold-Healy-Gordon syndrome, pheochromocytoma tumors, and Addison's disease, a disorder associated with gonadal steroid hormones such as: in women, abnormal prolactin production, infertility, endometriosis, perturbation of the menstrual cycle, polycystic ovarian disease, hyperprolactinemia, isolated gonadotropin deficiency, amenorrhea, galactonhea, hermaphroditism, hirsutism and virilization, breast cancer, and, in post-menopausal women, osteoporosis, and, in men, Leydig ceU deficiency, male climacteric phase, and germinal ceU aplasia, a hypergonadal disorder associated with Leydig ceU tumors, androgen resistance associated with absence of androgen receptors, syndrome of 5 α-reductase, and gynecomastia. Polynucleotides encoding NTRAN may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-hke assays; and in microarrays utilizing fluids or tissues from patients to detect altered NTRAN expression. Such qualitative or quantitative methods are weU known in the art.

In a particular embodiment, polynucleotides encoding NTRAN maybe used in assays that detect the presence of associated disorders, particularly those mentioned above. Polynucleotides complementary to sequences encoding NTRAN may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of polynucleotides encoding NTRAN 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 NTRAN, a normal or standard profile for expression is estabhshed. This may be accomphshed by combining body fluids or ceU extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding NTRAN, under conditions suitable for hybridization or amphfication. 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 substantiaUy 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 estabhsh the presence of a disorder. Once the presence of a disorder is estabhshed and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to detennine 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 aUow health professionals to employ preventative measures or aggressive treatment earlier, thereby preventing the development or further progression of the cancer.

Additional diagnostic uses for ohgonucleotides designed from the sequences encoding NTRAN may involve the use of PCR. These ohgomers may be chemicaUy synthesized, generated enzymaticaUy, or produced in vitro. Ohgomers wffl preferably contain a fragment of a polynucleotide encoding NTRAN, or a fragment of a polynucleotide complementary to the polynucleotide encoding NTRAN, and will be employed under optimized conditions for identification of a specific gene or condition. Ohgomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences. In a particular aspect, ohgonucleotide primers derived from polynucleotides encoding NTRAN 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, ohgonucleotide primers derived from polynucleotides encoding NTRAN 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 ohgonucleotide primers are fluorescently labeled, which aUows detection of the amplimers in high-throughput equipment such as DNA sequencing machines. AdditionaUy, sequence database analysis methods, termed in sihco 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 enors using statistical models and automated analyses of DNA sequence chromatograms. In the alternative, SNPs may be detected and characterized by mass specfrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).

SNPs maybe 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 ceU 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 utihty in pharmacogenomics, the identification of genetic variants that influence a patient's response to a drug, such as hfe-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 ALOX5 gene results in diminished clinical response to treatment with an anti-asthma drug that targets the 5-hpoxygenase pathway. Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as weU 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) Cun. Opin. Neurobiol. 11:637-641). Methods which may also be used to quantify the expression of NTRAN include radiolabeling or biotinylating nucleotides, coamphfication of a control nucleic acid, and interpolating results from standard curves (Melby, P.C. et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C et al. (1993) Anal. Biochem. 212:229-236). The speed of quantitation of multiple samples may be accelerated by ranning the assay in a high-throughput format where the ohgomer or polynucleotide of interest is presented in various dilutions and a specttophotometric or colorimetric response gives rapid quantitation.

In further embodiments, ohgonucleotides or longer fragments derived from any of the polynucleotides described herein may be used as elements on a microarray. The microanay 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 maybe 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 freatment 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, NTRAN, fragments of NTRAN, or antibodies specific for NTRAN may be used as elements on a microarray. The microanay 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 ceU type. A transcript image represents the global pattern of gene expression by a particular tissue or ceU 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 ceU 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 microanay. The resultant transcript image would provide a profile of gene activity. Transcript images maybe generated using transcripts isolated from tissues, ceU 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 ceU 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 prechnical evaluation of pharmaceuticals, as weU as toxicological testing of industrial and nataraUy-occurring environmental compounds. AU 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. IdeaUy, a genome- wide measurement of expression provides the highest quahty signature. Even genes whose expression is not altered by any tested compounds are important as weU, 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 aU 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 conesponding 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 proteome of a tissue or ceU type. The term proteome refers to the global pattern of protein expression in a particular tissue or ceU type. Each protein component of a proteome can be subjected individuaUy to further analysis. Proteome expression patterns, or profiles, are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time. A profile of a ceU's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or ceU 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, typicaUy by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains. The optical density of each protein spot is generaUy 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 freatment. The proteins in the spots are partiaUy sequenced using, for example, standard methods employing chemical or enzymatic cleavage foUowed 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 NTRAN to quantify the levels of NTRAN expression. In one embodiment, the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microanay to the sample and detecting the levels of protein bound to each anay element (Lueking, A. et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each anay element.

Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in paraUel with toxicant signatures at the transcript level. There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures maybe useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile. In addition, the analysis of transcripts in body fluids is difficult, due to rapid degradation of RNA, so proteomic profiling may be more rehable 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 conesponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.

In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. Microanays 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 WO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505; HeUer, RA. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; HeUer, M.J. et al. (1997) U.S. Patent No. 5,605,662). Various types of microanays are weU 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 NTRAN maybe used to generate hybridization probes useful in mapping the naturaUy occurring genomic sequence. Either coding or noncoding sequences maybe 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 potentiaUy cause undesired cross hybridization during chromosomal mapping. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA libraries (Harrington, J.J. et al. (1997) Nat. Genet. 15:345- 355; Price, CM. (1993) Blood Rev. 7:127-134; Trask, BJ. (1991) Trends Genet. 7:149-154). Once mapped, the nucleic acid sequences may be used to develop genetic linkage maps, for example, which conelate 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. USA 83:7353-7357).

Fluorescent in situ hybridization (FISH) maybe conelated with other physical and genetic map data (Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968). Examples of genetic map data can be found in various scientific journals or at the Online Mendehan Inheritance in Man (OMTM) World Wide Web site. Conelation between the location of the gene encoding NTRAN 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 estabhshed chromosomal markers, maybe 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 locahzed by genetic hnkage to a particular genomic region, e.g., ataxia-telangiectasia to llq22-23, 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, NTRAN, its catalytic or immunogenic fragments, or ohgopeptides 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 sohd support, borne on a ceU surface, or located intraceUularly. The formation of binding complexes between NTRAN 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 WO84/03564). In this method, large numbers of different smaU test compounds are synthesized on a sohd substrate. The test compounds are reacted with NTRAN, or fragments thereof, and washed. Bound NTRAN is then detected by methods weU known in the art. Purified NTRAN 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 sohd support. In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding NTRAN specificaUy compete with a test compound for binding NTRAN. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic detenriinants with NTRAN. In additional embodiments, the nucleotide sequences which encode NTRAN 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 skffled in the art can, using the preceding description, utihze the present invention to its fullest extent. The foUowing 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 aU patents, apphcations, and pubhcations mentioned above and below, including U.S. Ser. No. 60/340,798, U.S. Ser. No. 60/365,645, U.S. Ser. No. 60/367,662, U.S. Ser. No. 60/379,887, and U.S. Ser. No. 60/384,639, 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 ohgo 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 conesponding 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., supra, ch. 5). Reverse transcription was initiated using ohgo d(T) or random primers. Synthetic ohgonucleotide adapters were hgated 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 S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Biosciences) or preparative agarose gel electrophoresis. cDNAs were hgated into compatible restriction enzyme sites of the polyhnker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORTl plasmid (Invifrogen, Carlsbad CA), PCDNA2.1 plasmid (Invitrogen), 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 ceUs including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5α, DH10B, or ElectroMAX DH10B from Invifrogen.

II. Isolation of cDNA Clones

Plasmids obtained as described in Example I were recovered from host ceUs by in vivo excision using the UNIZAP vector system (Stratagene) or by ceU lysis. Plasmids were purified using at least one of the foUowing: 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. FoUowing precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4°C.

Alternatively, plasmid DNA was amphfied from host ceU lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host ceU lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-weU plates, and the concentration of amphfied plasmid DNA was quantified fluorometricaUy 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 foUows. Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Apphed Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) hquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Biosciences or supphed in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Apphed 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 (Apphed 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 Vffl.

The polynucleotide sequences derived from Incyte cDNAs were vahdated by removing vector, linker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic prograrnming, and dinucleotide nearest neighbor analysis. The Incyte cDNA sequences or translations thereof were then queried against a selection of pubhc 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 norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida 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 HMM-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 HMMER. The Incyte cDNA sequences were assembled to produce full length polynucleotide sequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences (see Examples TV and V) were used to extend Incyte cDNA assemblages to fuU 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 fuU length polynucleotide sequences were translated to derive the conesponding full length polypeptide sequences. Alternatively, a polypeptide may begin at any of the methionine residues of the fuU length translated polypeptide. FuU 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, bidden Markov model (HMM)-based protein family databases such as PFAM, INCY, and TIGRFAM; and HMM-based protein domain databases such as SMART. FuU 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 ahgnment program (DNASTAR), which also calculates the percent identity between ahgned sequences.

Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and full length sequences and provides apphcable 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, aU of which are incorporated by reference herein in their entirety, and the fourth column presents, where apphcable, the scores, probabihty values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probabihty 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 NO:23-44. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amphfication technologies are described in Table 4, column 2. IV. Identification and Editing of Coding Sequences from Genomic DNA Putative neurotransmission-associated proteins were initiaUy identified by running the Genscan gene identification program against pubhc 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 detennine which of these Genscan predicted cDNA sequences encode neurotransmission-associated proteins, the encoded polypeptides were analyzed by que ying against PFAM models for neurotransmission-associated proteins. Potential neurotransmission-associated proteins were also identified by homology to Incyte cDNA sequences that had been annotated as neurotransmission-associated proteins. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri pubhc databases. Where necessary, the Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to conect errors in the sequence predicted by Genscan, such as extra or omitted exons. BLAST analysis was also used to find any Incyte cDNA or pubhc cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription. When Incyte cDNA coverage was available, this information was used to conect or confirm the Genscan predicted sequence. FuU length polynucleotide sequences were obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or pubhc cDNA sequences using the assembly process described in Example m. Alternatively, fuU 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 IN. Partial cDΝAs assembled as described in Example m were mapped to genomic DΝA and parsed into clusters containing related cDΝAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic prograrnming to integrate cDΝA and genomic information, generating possible sphce 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 fransitivity. For example, if an interval was present on a cDΝA and two genomic sequences, then aU three intervals were considered to be equivalent. This process aUows unrelated but consecutive genomic sequences to be brought together, bridged by cDΝA 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 weU as sequence variants. Linkages between intervals which proceed along one type of parent sequence (cDΝA to cDΝA or genomic sequence to genomic sequence) were given preference over linkages which change parent type (cDΝA to genomic sequence). The resultant stitched sequences were translated and compared by BLAST analysis to the genpept and gbpri pubhc databases. Inconect 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 fuU length with an algorithm based on BLAST analysis. First, partial cDNAs assembled as described in Example HI were queried against pubhc 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 TV. A chimeric protein was generated by using the resultant high-scoring segment pairs (HSPs) to map the translated sequences onto the GenBank protein homolog. Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog. The GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the pubhc 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 NTRAN Encoding Polynucleotides

The sequences which were used to assemble SEQ ID NO:23-44 were compared with sequences from the Incyte LIFESEQ database and pubhc domain databases using BLAST and other implementations of the Smith- Waterman algorithm. Sequences from these databases that matched SEQ ID NO:23-44 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data available from pubhc 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 ah sequences of that cluster, including its particular SEQ JD NO:, to that map location.

Map locations are represented by ranges, or intervals, of human chromosomes. The map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p- arm. (The centiMorgan (cM) is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.) The cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters. Human genome maps and other resources available to the pubhc, such as the NCBI "GeneMap'99" World Wide Web site (http://www.ncbi.nlm.nih.gov/genemap/), can be employed to determine if previously identified disease genes map within or in proximity to the intervals indicated above. Association of a disease with a chromosomal locus can be determined by lod score. Lod score is a statistical method used to test the linkage of two or more loci within f amilies having a genetic disease. The lod score is the logarithm to base 10 of the odds in favor of linkage. Linkage is defined as the tendency of two genes located on the same chromosome to be inherited together through meiosis (Genetics in Medicine, Fifth Edition, (1991) Thompson, M.W. et al, W.B. Saunders Co. Philadelphia). A lod score of +3 or greater (1000:1 odds in favor of linkage) indicates a probabihty of 1 in 1000 that a particular marker was found solely by chance in affected individuals, which is strong evidence that two genetic loci are linked.

One such gene implicated in PD is PARK3, which maps to 2pl3 (Gasser, T. et al. (1998) Nature Genet. 18:262-265). A marker at chromosomal position D2S441 was found to have a lod score of 3.2 in the region of PARK3. This marker supported the disease association of PARK3 in the chromosomal interval from D2S134 to D2S286 (Gasser et al., supra). Markers located within chromosomal intervals D2S134 and D2S286, which map between 83.88 to 94.05 centiMorgans on the short arm of chromosome 2, were used to identify genes that map in the region between D2S 134 and D2S286. A second PD gene, implicated in early-onset recessive parkinsonism, is PARK6, located on chromosome 1 at Ip35-lp36. Several markers were obtained with lod scores greater than 3 including D1S199, D1S2732, D1S2828, D1S478, D1S2702, D1S2734, D1S2674 (Valente, E.M. et al, supra). These markers were used to determine the PD-relevant range of chromosome loci and identify sequences that map to chromosome 1 between D1S199 and D1S2885. Restriction fragment length polymorphism (RFLP) markers shown to be near regions of DNA known as sequence-tagged sites (STS), have been mapped to NT_Contigs generated by the Human Genome Project using ePCR (Schuler, GD. (1997) Genome Research 7: 541-550, and (1998) Trends Biotechnol. 16(ll):456-9). Contigs containing regions of DNA with known disease-associated markers are therefore used to identify NTRAN sequences that map to disease-associated regions of the genome.

Polynucleotides encoding NTRAN were mapped to NT_Contigs. Contigs longer than 1Mb were broken into subcontigs of 1Mb length with overlaping sections of lOOkb. A preliminary step used an algorithm, similar to MEGABLAST, to define the mRNA sequence /masked genomic DNA contig pairings. The cDNA/genomic pairings identified by the first algorithm were confirmed, and the

NTRAN polynucleotides mapped to DNA contigs, using S1M4 (Florea, L. et al. (1998) Genome Res.

8 :967-74, version May 2000) which had been optimized for high throughput processing and strand assignment confidence). The SIM4 output of the mRNA sequence/genomic contig pairs was further processed to determine the conect location of the NTRAN polynucleotides on the genomic contig, as weU as their strand identity.

SEQ ID NO:40 was mapped to NT_Contig GBI:NT_005428_001.7 from Genbank release

February 2002, covering a 9.65 Mb region of the genome that also contains PD-associated genetic markers D2S 134 and D2S286. The maximum distance between SEQ ID NO:40 and markers D2S 134 and D2S286, therefore, is 9.65 Mb. Thus, SEQ ID NO:40 is in proximity with genetic markers shown to consistently associate with PD.

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 ceU type or tissue have been bound (Sambrook and RusseU, supra, ch. 7 ; Ausubel et al., supra, ch. 4).

Analogous computer techniques applying BLAST were used to search for identical or related molecules in databases such as GenBank or LIFESEQ (Incyte Genomics). This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score, which is defined as:

BLAST Score x Percent Identity

5 x minimum {length(Seq. 1), length(Seq. 2)} The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. The product score is a normalized value between 0 and 100, and is calculated as foUows: the BLAST score is multiphed 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 ahgnment. 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 NTRAN 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 1TJ.). Each cDNA sequence is derived from a cDNA hbrary constructed from a human tissue. Each human tissue is classified into one of the foUowing organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitalia, female; genitalia, male; germ ceUs; hemic and immune system; hver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary fract. The number of libraries in each category is counted and divided by the total number of libraries across aU categories. Similarly, each human tissue is classified into one of the foUowing disease/condition categories: cancer, ceU line, developmental, inflammation, neurological, frauma, cardiovascular, pooled, and other, and the number of libraries in each category is counted and divided by the total number of libraries across aU categories. The resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding NTRAN. cDNA sequences and cDNA library/tissue information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA). VIII. Extension of NTRAN Encoding Polynucleotides

FuU length polynucleotides are produced by extension of an appropriate fragment of the full length molecule using ohgonucleotide 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 amphfication was obtained by PCR using methods weU known in the art. PCR was performed in 96-weU plates using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg²⁺, (NHASO^ and 2-mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE enzyme (Invitrogen), and Pfu DNA polymerase (Stratagene), with the foUowing 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 foUows: 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 weU was determined by dispensing 100 μl PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in IX TE and 0.5 μl of undiluted PCR product into each weU of an opaque fluorimeter plate (Corning Costar, Acton MA), aUowing 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 μl to 10 μl ahquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to detemiine which reactions were successful in extending the sequence.

The extended nucleotides were desalted and concentrated, fransfened to 384-weU plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to rehgation into pUC 18 vector (Amersham Biosciences). For shotgun sequencing, the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega). Extended clones were rehgated using T4 hgase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Biosciences), freated with Pfu DNA polymerase (Stratagene) to fffl-in restriction site overhangs, and transfected into competent E. coli ceUs. Transformed ceUs were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37 °C in 384-weU plates in LB/2x carb hquid media.

The ceUs were lysed, and DNA was amphfied by PCR using Taq DNA polymerase (Amersham Biosciences) and Pfu DNA polymerase (Stratagene) with the foUowing 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 (Apphed 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 ohgonucleotides designed for such extension, and an appropriate genomic hbrary.

IX. Identification of Single Nucleotide Polymorphisms in NTRAN Encoding Polynucleotides Common DNA sequence variants known as single nucleotide polymorphisms (SNPs) were identified in SEQ ID NO:23-44 using the LIFESEQ database (Incyte Genomics). Sequences from the same gene were clustered together and assembled as described in Example JJI, aUowing the identification of aU 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 basecaU enors by requiring a minimum Phred quahty score of 15, and removed sequence ahgnment enors and errors resulting from improper trimming of vector sequences, chimeras, and sphce variants. An automated procedure of advanced chromosome analysis analysed the original chromatogram files in the vicinity of the putative SNP. Clone error filters used statisticaUy generated algorithms to identify enors introduced during laboratory processing, such as those caused by reverse transcriptase, polymerase, or somatic mutation. Clustering enor filters used statisticaUy generated algorithms to identify enors resulting from clustering of close homologs or pseudogenes, or due to contamination by non-human sequences. A final set of filters removed duphcates and SNPs found in immunoglobulins or T-ceU receptors.

Certain SNPs were selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc.) to analyze aUele 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), aU African Americans. The Hispanic population comprised 324 individuals (162 male, 162 female), aU 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. AUele frequencies were first analyzed in the Caucasian population; in some cases those SNPs which showed no aUehc 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 NO:23-44 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of ohgonucleotides, consisting of about 20 base pahs, is specificaUy described, essentiaUy the same procedure is used with larger nucleotide fragments. Ohgonucleotides 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 μCi of [γ-³ P] adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled ohgonucleotides are substantiaUy purified using a SEPHADEX G-25 superfine size exclusion dextianbead column (Amersham Biosciences). An ahquot containing 10⁷ 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 foUowing endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu R (DuPont NEN).

The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nyfran Plus, Schleicher & SchueU, Durham NH). Hybridization is carried out for 16 hours at 40 °C. To remove nonspecific signals, blots are sequentiaUy 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 hnkage or synthesis of array elements upon a microarray can be achieved utilizing photohthography, 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 sohd with a non-porous surface (Schena, M., ed. (1999) DNA Microarrays: A Practical Approach, Oxford University Press, London). Suggested subsfrates include sihcon, sihca, glass shdes, glass chips, and sihcon wafers. Alternatively, a procedure analogous to a dot or slot blot may also be used to anange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures. A typical anay may be produced using available methods and machines weU known to those of ordinary skffl 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; MarshaU, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31). FuU length cDNAs, Expressed Sequence Tags (ESTs), or fragments or ohgomers thereof may comprise the elements of the microanay. Fragments or ohgomers suitable for hybridization can be selected using software weU 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 anay 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 microanay 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 ohgo-(dT) ceUulose method. Each poly(A)⁺ RNA sample is reverse transcribed using MMLV reverse-franscriptase, 0.05 pg/μl ohgo-(dT) primer (21mer), IX first strand buffer, 0.03 units/μl RNase inhibitor, 500 μM dATP, 500 μM dGTP, 500 μM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences). The reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A)⁺ RNA with GEMBRIGHT kits (Incyte Genomics). Specific control poly(A)⁺ RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (Clontech, 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 μl 5X SSC/0.2% SDS. Microarray Preparation

Sequences of the present invention are used to generate array elements. Each array element is amphfied from bacterial ceUs containing vectors with cloned cDNA inserts. PCR amphfication uses primers complementary to the vector sequences flanking the cDNA insert. Anay elements are amphfied in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 μg. Amphfied array elements are then purified using SEPHACRYL-400 (Amersham Biosciences). Purified array elements are immobilized on polymer-coated glass shdes. Glass microscope shdes (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distiUed water washes between and after treatments. Glass shdes are etched in 4% hydrofluoric acid (N R Scientific Products Corporation (VWR), West Chester PA), washed extensively in distiUed water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated shdes are cured in a 110°C oven.

Array elements are apphed to the coated glass substrate using a procedure described in U.S. Patent No. 5,807,522, incorporated herein by reference. 1 μl of the anay element DNA, at an average concentration of 100 ng/μl, is loaded into the open capillary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 nl of array element sample per shde.

Microanays are UV-crosslinked using a STRATALINKER TJV-crosslinker (Stratagene). Microanays are washed at room temperature once in 0.2% SDS and three times in distiUed water. Non-specific binding sites are blocked by incubation of microarrays in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60° C foUowed by washes in 0.2% SDS and distiUed water as before. Hybridization

Hybridization reactions contain 9 μl of sample mixture consisting of 0.2 μg each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer. The sample mixture is heated to 65° C for 5 minutes and is ahquoted onto the microanay surface and covered with an 1.8 cm² covershp. The arrays are fransfened to a waterproof chamber having a cavity just shghtly larger than a microscope shde. The chamber is kept at 100% humidity internaUy by the addition of 140 μl of 5X SSC in a comer of the chamber. The chamber containing the anays is incubated for about 6.5 hours at 60° C The anays are washed for 10 min at 45° C in a first wash buffer (IX 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 Jnnova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5. The excitation laser light is focused on the array using a 20X microscope objective (Nikon, Inc., Melville NY). The shde containing the array is placed on a computer-controUed 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 sequentiaUy. Emitted light is spht, based on wavelength, into two photomultipher tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) conesponding to the two fluorophores. Appropriate filters positioned between the array and the photomultipher tubes are used to filter the signals. The emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5. Each anay is typicaUy 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 typicaUy cahbrated 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, aUowing the intensity of the signal at that location to be conelated with a weight ratio of hybridizing species of 1:100,000. When two samples from different sources (e.g., representing test and control ceUs), each labeled with a different fluorophore, are hybridized to a single anay for the purpose of identifying genes that are differentiaUy 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 photomultipher tube is digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc., Norwood MA) instaUed in an IBM-compatible PC computer. The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal). The data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.

A grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid. The fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte Genomics). Array elements that exhibit at least about a two-fold change in expression, a signal-to-background ratio of at least about 2.5, and an element spot size of at least about 40%, are considered to be differentiaUy expressed. Expression

For example, SEQ ID NO:24 showed differential expression in lung squamous carcinoma tissues versus normal lung tissues as determined by microarray analysis. The expression of SEQ ID NO:24 was decreased by at least two fold in lung squamous carcinoma tissues relative to grossly uninvolved normal lung tissue from the same donors. Therefore, SEQ ID NO:24 is useful as a diagnostic marker or as a potential therapeutic target for lung cancer.

SEQ ID NO:24 also showed differential expression in ovarian adenocarcinoma tissues versus normal ovarian tissues as determined by microanay analysis. The expression of SEQ ID NO:24 was decreased by at least two fold in ovarian adenocarcinoma tissues relative to grossly uninvolved normal ovarian tissue from the same donor. Therefore, SEQ ID NO:24 is useful as a diagnostic marker or as a potential therapeutic target for ovarian cancer.

Furthermore, SEQ ID NO:24 showed region-specific gene expression in the human brain as determined by microarray analysis. The expression of SEQ ID NO:24 was decreased by at least two fold in the cervical spinal cord relative to pooled brain tissues which were constituted from the major regions of the brain from two male brains; a 47 year old and a 48 year old. The tissue from the cervical spinal cord was isolated from a 47 year old male, the same 47 year old donor as in the pooled sample. Therefore, SEQ JD NO:24 se ves as a useful biomarker for human brains, specificaUy for the cervical spinal cord.

In an alternative example, the expression of SEQ ID NO:25, as determined by microarray analysis, was increased by at least two fold in sigmoid colon tissues relative to normal sigmoid colon tissues. The sigmoid colon tumor tissue which originated from a metastatic gastric sarcoma (stromal tumor) was harvested from a 48 year old female donor. The normal sigmoid colon tissue was harvested from grossly uninvolved sigmoid colon tissue of the same donor. Therefore, SEQ ID NO:25 is useful as a diagnostic marker or as a potential therapeutic target for colon cancer.

SEQ JD NO:25 also showed decreased expression in tissue affected by lung adenocarcinoma versus normal lung tissue as determined by microanay analysis. A sample of right lung tissue that showed moderately differentiated adenocarcinoma was compared to grossly uninvolved lung tissue from the same donor (Huntsman Cancer Institute, Salt Lake City, UT). Therefore, SEQ ID NO:25 is useful in diagnostic assays for and monitoring treatment of lung cancer.

Furthermore, the expression of SEQ ID NO:25, as deteraiined by microanay analysis, was increased by at least two fold in Tangier disease-derived fibroblasts relative to normal fibroblasts. Both types of ceUs were cultured in the presence of cholesterol and compared with the same ceU type in the absence of cholesterol. The human fibroblasts were obtained from skin transplants from both normal subjects and two patients with homozygous Tangier disease. CeU lines were immortalized by transfection with human papfflomavirus 16 genes E6 and E7 and a neomycin resistance selectable marker. TD derived ceUs are deficient in an assay of apoA-I mediated tritiated cholesterol efflux. Therefore, SEQ JD NO:25 is useful in diagnostic assays for and monitoring treatment of Tangier disease.

In an alternative example, SEQ ID NO:30 showed decreased expression in lung tissue affected by squamous ceU adenocarcinoma versus normal lung tissue as detennined by microarray analysis. Grossly uninvolved lung tissue with no visible abnormahties, from a 73 year-old male, was compared to lung squamous ceU adenocarcinoma tissue from the same donor (Roy Castle International Centre for Lung Cancer Research, Liverpool, UK). Ηierefore, SEQ ID NO:30 is useful in monitoring treatment of, and diagnostic assays for, lung cancer and other ceU prohferative disorders. As another example, SEQ JD NO:30 showed decreased expression in tissue affected by ovarian tumor versus normal ovary tissue as determined by microanay analysis. A normal ovary from a 79 year-old female donor was compared to an ovarian tumor from the same donor (Huntsman Cancer Institute, Salt Lake City, UT). Therefore, SEQ ID NO:30 is useful in monitoring treatment of, and diagnostic assays for, ovarian cancer and other ceU prohferative disorders. In an alternative example, SEQ ID NO:36 showed decreased expression in colon adenocarcinoma tissue versus grossly uninvolved colon tissue. Gene expression profiles were obtained by comparing normal colon tissue to colon tumor tissue from the same donor. Therefore, SEQ JD NO:36 is useful in monitoring treatment of, and diagnostic assays for, colon cancer and other ceU prohferative disorders. In an alternative example, SEQ ID NO:38 showed differential expression, as detennined by microarray analysis. Anay elements that exhibited about at least a two-fold change in expression and a signal intensity over 250 units, a signal-to-background ratio of a least 2.5, and an element spot size of at least 40% were identified as differentiaUy expressed using the GEMTOOLS program (Incyte Genomics). For example, the expression of cDNA from grossly uninvolved lung tissue from a 66 year-old male was compared to lung squamous ceU carcinoma tissue from the same donor (Roy Castle International Centre for Lung Cancer Research, Liverpool, UK). The expression of SEQ ID NO:38 showed at least a two-fold increase in expression in the lung tamor tissue when compared to normal lung tissue expression levels from the same donor. Thus, SEQ ID NO:38 is useful for monitoring progression of, and diagnostic assays for, lung cancers.

In another example, specific dissected brain regions from a normal 61 -year-old female were compared to dissected regions from the brain of a 79-year-old-female with severe Alzheimer's disease (AD). The diagnosis of normal or severe AD was estabhshed by a certified neuropathologist based on microscopic examination of multiple sections throughout the brain. The expression of SEQ JD NO:38 showed at least a two-fold decrease in expression in the amygdala and anterior hippocampus brain regions when compared to the same brain regions from the normal 61-year-old female. Therefore, SEQ ID NO:38 is also useful for monitoring progression of, and diagnostic assays for, Alzheimer's disease.

In another example, SEQ ID NO:41 showed tissue-specific expression. RNA samples isolated from a variety of normal human tissues were compared to a common reference sample. Tissues contributing to the reference sample were selected for their abihty to provide a complete distribution of RNA in the human body and include brain (4%), heart (7%), kidney (3%), lung (8%), placenta (46%), smaU intestine (9%), spleen (3%), stomach (6%), testis (9%), and uterus (5%). The normal tissues assayed were obtained from at least three different donors. RNA from each donor was separately isolated and individuaUy hybridized to the microarray. Since these hybridization experiments were conducted using a common reference sample, differential expression values are directly comparable from one tissue to another. The expression of SEQ JD NO:41 was increased by at least two-fold in brain (amygdaloid body, occipital cortex, and parietal cortex) tissues as compared to the reference sample. Therefore, SEQ JD NO:41 can be used as a marker for brain tissues.

XII. Complementary Polynucleotides

Sequences complementary to the NTRAN-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturaUy occurring NTRAN. Although use of ohgonucleotides comprising from about 15 to 30 base pairs is described, essentiahy the same procedure is used with smaUer or with larger sequence fragments. Appropriate ohgonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of NTRAN. To inhibit transcription, a complementary ohgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary ohgonucleotide is designed to prevent ribosomal binding to the NTRAN-encoding transcript.

XIII. Expression of NTRAN

Expression and purification of NTRAN is achieved using bacterial or virus-based expression systems. For expression of NTRAN in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription. Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3). Antibiotic resistant bacteria express NTRAN upon induction with isopropyl beta-D- thiogalactopyranoside (JPTG). Expression of NTRAN in eukaryotic ceUs is achieved by infecting insect or mammalian ceU lines with recombinant Autographica calif ornica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding NTRAN 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 Spodopterafrugiperda (Sf9) insect ceUs inmost cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to baculovirus (Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937- 1945).

In most expression systems, NTRAN is synthesized as a fusion protein with, e.g., glutathione S-ttansferase (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 ceU lysates. GST, a 26-kilodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Biosciences). FoUowing purification, the GST moiety can be proteolyticaUy cleaved from NTRAN at specificaUy engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffϊnity purification using commerciaUy available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel et al. (supra, ch. 10 and 16). Purified NTRAN obtained by these methods can be used directly in the assays shown in Examples XVJJ and XVJJI, where apphcable. XIV. Functional Assays

NTRAN function is assessed by expressing the sequences encoding NTRAN at physiologicaUy elevated levels in mammalian ceU 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 (Invifrogen), both of which contain the cytomegalovirus promoter. 5-10 μg of recombinant vector are transiently transfected into a human ceU line, for example, an endothelial or hematopoietic ceU line, using either liposome formulations or electroporation. 1-2 μg of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected ceUs from nonfransfected ceUs and is a rehable 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 ceUs expressing GFP or CD64-GFP and to evaluate the apoptotic state of the ceUs and other ceUular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with ceU death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in ceU 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 ceU surface and intraceUular 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 ceU surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994; Flow Cytometry, Oxford, New York NY).

The influence of NTRAN on gene expression can be assessed using highly purified populations of ceUs transfected with sequences encoding NTRAN and either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on the surface of transfected ceUs and bind to conserved regions of human immunoglobulin G (IgG). Transfected ceUs are efficiently separated from nonfransfected ceUs using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY). mRNA can be purified from the ceUs using methods weU known by those of skffl in the art. Expression of mRNA encoding NTRAN and other genes of interest can be analyzed by northern analysis or microarray techniques. XV. Production of NTRAN Specific Antibodies NTRAN substantiaUy 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 NTRAN amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of Mgh immunogenicity, and a conesponding ohgopeptide is synthesized and used to raise antibodies by means known to those of skffl in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are weU described in the art (Ausubel et al., supra, ch. 11). TypicaUy, oligopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (Apphed 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-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide and anti-NTRAN activity by, for example, binding the peptide or NTRAN 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 NTRAN Using Specific Antibodies NaturaUy occuning or recombinant NTRAN is substantiaUy purified by immunoaffinity chromatography using antibodies specific for NTRAN. An immunoaffinity column is constructed by covalently coupling anti-NTRAN 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 NTRAN are passed over the immunoaffinity column, and the column is washed under conditions that aUow the preferential absorbance of NTRAN (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/NTRAN 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 NTRAN is coUected.

XVII. Identification of Molecules Which Interact with NTRAN NTRAN, or biologicaUy active fragments thereof, are labeled with ¹²⁵I Bolton-Hunter reagent

(Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539). Candidate molecules previously arrayed in the weUs of a multi-weU plate are incubated with the labeled NTRAN, washed, and any weUs with labeled NTRAN complex are assayed. Data obtained using different concentrations of NTRAN are used to calculate values for the number, affinity, and association of NTRAN with the candidate molecules.

Alternatively, molecules interacting with NTRAN are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989; Nature 340:245-246), or using commerciaUy available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech). NTRAN 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 aU interactions between the proteins encoded by two large libraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101). XVIII. Demonstration of NTRAN Activity

An assay for NTRAN activity measures the expression of NTRAN on the ceU surface. cDNA encoding NTRAN is transfected into an appropriate mammahan ceU line. CeU surface proteins are labeled withbiotin as described (de la Fuente, M.A. et al. (1997) Blood 90:2398-2405). Lnmunoprecipitations are performed using NTRAN-specific antibodies, and immunoprecipitated samples are analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting techniques. The ratio of labeled immunoprecipitant to unlabeled immunoprecipitant is proportional to the amount of NTRAN expressed on the ceU surface. In the alternative, an assay for NTRAN activity is based on a prototypical assay for hgand/receptor-mediated modulation of ceU proliferation. This assay measures the rate of DNA synthesis in Swiss mouse 3T3 ceUs. A plasmid containing polynucleotides encoding NTRAN is added to quiescent 3T3 cultured ceUs using transfection methods weU known in the art. The transiently transfected ceUs are then incubated in the presence of [³H]thymidine, a radioactive DNA precursor molecule. Varying amounts of NTRAN ligand are then added to the cultured ceUs. Incorporation of [³H]thymidine into acid-precipitable DNA is measured over an appropriate time interval using a radioisotope counter, and the amount incorporated is directly proportional to the amount of newly synthesized DNA. A linear dose-response curve over at least a hundred-fold NTRAN ligand concentration range is indicative of receptor activity. One unit of activity per mfflihter is defined as the concentration of NTRAN producing a 50% response level, where 100% represents maximal incorporation of [³H thymidine into acid-precipitable DNA (McKay, I. and I. Leigh, eds. (1993) Growth Factors: A Practical Approach, Oxford University Press, New York NY, p. 73.)

In a further alternative, the assay for NTRAN activity is based upon the abihty of GPCR family proteins to modulate G protein-activated second messenger signal transduction pathways (e.g., cAMP; Gaudin, P. et al. (1998) J. Biol. Chem. 273:4990-4996). A plasmid encoding fuU length NTRAN is transfected into a mammahan ceU line (e.g., Chinese hamster ovary (CHO) or human embryonic kidney (HEK-293) ceU lines) using methods weU-known in the art. Transfected ceUs are grown in 12-weU trays in culture medium for 48 hours, then the culture medium is discarded, and the attached ceUs are gently washed with PBS. The ceUs are then incubated in culture medium with or without ligand for 30 minutes, then the medium is removed and ceUs lysed by treatment with 1 M perchloric acid. The cAMP levels in the lysate are measured by radioimmunoassay using methods weU-known in the art. Changes in the levels of cAMP in the lysate from ceUs exposed to ligand compared to those without ligand are proportional to the amount of NTRAN present in the transfected ceUs.

To measure changes in inositol phosphate levels, the ceUs are grown in 24-weU plates containing lxlO⁵ ceUs/weU and incubated with inositol-free media and [³H]myoinositol, 2 mCi/weU, for 48 hr. The culture medium is removed, and the ceUs washed with buffer containing 10 mM LiCl foUowed by addition of ligand. The reaction is stopped by addition of perchloric acid. Inositol phosphates are extracted and separated on Dowex AG1-X8 (Bio-Rad) anion exchange resin, and the total labeled inositol phosphates counted by liquid scintfflation. Changes in the levels of labeled inositol phosphate from ceUs exposed to ligand compared to those without ligand are proportional to the amount of NTRAN present in the transfected ceUs. In a further alternative, the ion conductance capacity of NTRAN is demonstrated using an electrophysiological assay. NTRAN is expressed by tiansforming a mammahan ceU line such as COS7, HeLa or CHO with a eukaryotic expression vector encoding NTRAN. Eukaryotic expression vectors are commerciaUy available, and the techniques to introduce them into ceUs are weU known to those skilled in the art. A smaU amount of a second plasmid, which expresses any one of a number of marker genes such as β-galactosidase, is co-transformed into the ceUs in order to aUow rapid identification of those ceUs which have taken up and expressed the foreign DNA. The ceUs are incubated for 48-72 hours after transformation under conditions appropriate for the ceU line to aUow expression and accumulation of NTRAN and β-galactosidase. Transformed ceUs expressing β- galactosidase are stained blue when a suitable colorimetric substrate is added to the culture media under conditions that are weU known in the art. Stained ceUs are tested for differences in membrane conductance due to various ions by electrophysiological techniques that are weU known in the art. Untransformed ceUs, and/or ceUs transformed with either vector sequences alone or β-galactosidase sequences alone, are used as controls and tested in paraUel. The contribution of NTRAN to cation or anion conductance can be shown by incubating the ceUs using antibodies specific for either NTRAN. The respective antibodies wiU bind to the extraceUular side of NTRAN, thereby blocking the pore in the ion channel, and the associated conductance. To study the dependence of NAP on external ions, sodium can be replaced by choline or N-methyl-D-glucamine and chloride by gluconate, NO₃, or SO₄ (Kavanaugh, M.P. et al. (1992) J. Biol. Chem. 267:22007-22009).

In a further alternative, NTRAN transport activity is assayed by measuring uptake of labeled subsfrates into Xenopus laevis oocytes. Oocytes at stages V and VI are injected with NTRAN mRNA (10 ng per oocyte) and incubated for 3 days at 18 °C in OR2 medium (82.5 mM NaCl, 2.5 mM KC1, 1 mM CaCl₂, 1 mM MgC^, 1 mM Na₂HPO₄, 5 mM Hepes, 3.8 mM NaOH , 50 μg/ml gentamycin, pH 7.8) to aUow expression of NTRAN protein. Oocytes are then transferred to standard uptake medium (100 mM NaCl, 2 mM KC1, 1 mM CaCL., 1 mM MgCL_,, 10 mM Hepes/Tris pH 7.5). Uptake of various substrates (e.g., amino acids, sugars, drugs, and neurotransmitters) is initiated by adding a ³H substrate to the oocytes. After incubating for 30 minutes, uptake is terminated by washing the oocytes three times in Na⁺-free medium, measuring the incorporated ³H, and comparing with controls. NTRAN activity is proportional to the level of internalized ³H substrate. In a further alternative, NTRAN activity can be demonstrated using an electrophysiological assay for ion conductance. Capped NTRAN mRNA transcribed with T7 polymerase is injected into defoUiculated stage V Xenopus oocytes, similar to the previously described method. Two to seven days later, transport is measured by two-electrode voltage clamp recording. Two-electrode voltage clamp recordings are performed at a holding potential of 50 mV. The data are filtered at 10 Hz and recorded with the MacLab digital-to-analog converter and software for data acquisition and analysis (AD Instruments, Castle Hffl, Australia). To study the dependence of NTRAN on external ions, sodium can be replaced by choline or N-methyl-D-glucamine and chloride by gluconate, NO₃, or SO₄ (Kavanaugh, M.P. et al. (1992) J. Biol. Chem. 267:22007-22009).

Calmodulin-binding activity of NTRAN can be demonstrated by mixing 1.5 ml of 0.2 μM calmodulin with increasing concentrations of NTRAN (30 μl in concentrations ranging from 0.5 to 3.1 μM). Analysis of binding activity is detennined by Scatchard representation. The bound NTRAN is calculated as AN/ N^^o X [total calmodulin]. The free NTRAN concentration is calculated as [total NTRAN] - [bound NTRAN]. (Bosc, C et al. (2001) J. Biol. Chem. 276:30904-30913).

Alternatively, calmodulin-binding activity of NTRAN can be determined in a binding assay using calmodulin Sepharose beads. Calmodulin Sepharose 6B beads (1 mg/ml calmodulin) are equihbrated in 20 mM Hepes-KOH, pH 7.2, 0.15 M NaCl, 5 mg/ml bovine serum albumin, 0.02% Triton X-100 (v/v) (buffer C) and either 2.5 mM CaCl^ or 5 mM EGTA. Samples of NTRAN are incubated with 5 μl of calmodulin-Sepharose 6B beads for 3 hours at 22 °C in 0.5 ml of buffer C in the presence or absence (5 mM EGTA) of 2.5 mM CaCl₂. Subsequently, beads are peUeted by centrifugation at 1000 x g and washed five times (1.5 ml each wash) with modified buffer C (Triton X-100 concentration is increased to 0.1% (v/v) and albumin is omitted), by resuspension and centrifugation at 1000 x g. Washed beads containing bound ligand are incubated in 0.1 ml of SDS gel loading buffer for 5 minutes at 95 °C. Bound NTRAN is visualized by Western immunoblotting. (Rossi, E.A. et al. (1999) J. Biol. Chem. 274:27201-27210).

Sialic acid-binding activity of NTRAN can be demonstrated in a COS ceU binding assay. COS ceUs are transfected with plasmids encoding fuU-length NTRAN using DEAE-dextran as described in Simmons D.L. (1993) Cloning cell surface molecules by transient expression in mammahan ceUs. In CeUular Interactions in Development — A Practical Approach. Edited by Hartley D.A.. Oxford: IRL Press, 93-128. On the third day after transfection, binding assays with eryfhrocytes are carried out for 1 hour at 37 °C in DMEM culture medium with 0.2 % BSA. Unbound erythrocytes are washed off and the ceUs are fixed in 0.25 % glutardialdehyde. To assess the effect of antibodies on binding, COS ceUs are pre-incubated with immunoglobulins at 20 μg/ml for 1 hour prior to the addition of erythrocytes (Kehn, S. et al. (1994) Cun. Biol. 4:965-972).

In the alternative, choline transporter activity or chohne-fransporter-like CTLl protein activity of NTRAN is determined by measuring choline uptake by yeast transformed with expression vectors harboring polynucleotides encoding NTRAN. The assay is performed in nitrogen-free medium at 30°C for 10 or 30 min in the presence of 25 nM [³HJchohne. The ceUs are then filtered, and washed. The amount of [³H]choline present in the ceUs is proportional to the activity of NTRAN in the ceUs (O'Regan, S. supra). In a further alternative, NTRAN protein kinase (PK) activity is measured by phosphorylation of a protein substrate using gamma-labeled [³²P]-ATP and quantitation of the incorporated radioactivity using a gamma radioisotope counter. NTRAN is incubated with the protein substrate, [³²P]-ATP, and an appropriate kinase buffer. The ³²P incorporated into the product is separated from free [³²P]-ATP by electrophoresis and the incorporated ³ P is counted. The amount of ³²P recovered is proportional to the PK activity of NTRAN in the assay. A determination of the specific amino acid residue phosphorylated is made by phosphoamino acid analysis of the hydrolyzed protein.

An assay for NTRAN activity measures the expression of NTRAN on the ceU surface. cDNA encoding NTRAN is fransfected into a non-leukocytic ceU line. CeU surface proteins are labeled withbiotin (de la Fuente, M.A. et al. (1997) Blood 90:2398-2405). Lmnunoprecipitations are performed using NTRAN-specific antibodies, and immunoprecipitated samples are analyzed using SDS-PAGE and immunoblotting techniques. The ratio of labeled immunoprecipitant to unlabeled immunoprecipitant is proportional to the amount of NTRAN expressed on the ceU surface.

Alternatively, an assay for NTRAN activity measures the amount of ceU aggregation induced by overexpression of NTRAN. In this assay, cultured ceUs such as NJH3T3 are transfected with cDNA encoding NTRAN contained within a suitable mammahan expression vector under confrol of a strong promoter. Cotransfection with cDNA encoding a fluorescent marker protein, such as Green Fluorescent Protein (CLONTECH), is useful for identifying stable transfectants. The amount of ceU agglutination, or clumping, associated with transfected ceUs is compared with that associated with untransfected ceUs. The amount of ceU agglutination is a direct measure of NTRAN activity.

Alternatively, an assay for NTRAN activity measures the disruption of cytoskeletal filament networks upon overexpression of NTRAN in cultured ceU lines (Rezniczek, G. A. et al. (1998) J. CeU Biol. 141:209-225). cDNA encoding NTRAN is subcloned into a mammahan expression vector that drives high levels of cDNA expression. This construct is fransfected into cultured ceUs, such as rat kangaroo PtK2 or rat bladder carcinoma 804G ceUs. Actin filaments and intermediate filaments such as keratin and vimentin are visualized by immunofluorescence microscopy using antibodies and techniques weU known in the art. The configuration and abundance of cyoskeletal filaments can be assessed and quantified using confocal imaging techniques. In particular, the bundling and coUapse of cytoskeletal filament networks is indicative of NTRAN activity.

Alternatively, ceU adhesion activity in NTRAN is measured in a 96-weU plate in which weUs are first coated with NTRAN by adding solutions of NTRAN of varying concentrations to the weUs. Excess NTRAN is washed off with saline, and the weUs incubated with a solution of 1% bovine serum albumin to block non-specific ceU binding. Ahquots of a ceU suspension of a suitable ceU type are then added to the weUs and incubated for a period of time at 37 °C. Non-adherent ceUs are washed off with saline and the ceUs stained with a suitable ceU stain such as Coomassie blue. The intensity of staining is measured using a variable wavelength multi-well plate reader and compared to a standard curve to detennine the number of ceUs adhering to the NTRAN coated plates. The degree of ceU staining is proportional to the ceU adhesion activity of NTRAN in the sample.

Various modifications and variations of the described compositions, methods, and systems of the invention wfflbe apparent to those skffled in the art without departing from the scope and spirit of the invention. It wfflbe appreciated that the invention provides novel and useful proteins, and their encoding polynucleotides, which can be used in the drug discovery process, as weU 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 foUowing claims and their equivalents.

Table 1

to

Table 2

Table 2

Table 2

ω o

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 8

Table 4

Table 4

Table 4

Table 4

Table 4

Table 4

Table 4

Table 4

Polynucleotide Sequence Fragments SEQ ID NO:/ Incyte ID/ Sequence Length

35/7510060CB1/ 1-530, 12-268, 12-640, 14-251, 14-566, 14-579, 14-632, 14-653, 14-686, 14-697, 17-572, 25-343, 26-579, 26-657, 2431 26-672, 26-693, 26-716, 27-296, 27-437, 27-685, 27-876, 27-2426, 30-320, 31-234, 33-339, 34-551, 37-599, 39-635, 41-275, 42-352, 43-587, 43-714, 43-726, 43-801, 43-840, 43-853, 43-854, 43-857, 43-867, 44-561, 47-721, 48-302, 50-479, 239-890, 381-983, 414-997, 421-680, 421-692, 465-569, 520-1196, 566-839, 569-832, 569-1146, 574-1221, 591-1202,712-1329,733-1049,733-1118,786-1062, 800-1011, 832-1373, 856-1117, 885-1400, 889-1465,928- 1148, 973-1473, 1078-1371, 1120-1477, 1152-1352, 1153-1644, 1159-1426, 1165-1760, 1168-1789, 1192-1476, 1193-1473, 1197-1657, 1210-1512, 1233-1480, 1248-1568, 1261-1506, 1331-1582, 1397-1516, 1399-1953, 1406- 1696, 1409-1700, 1416-1645, 1416-1664, 1428-2344, 1438-1681, 1455-1711, 1477-1553, 1480-1707, 1523-1781, 1544-2344, 1572-2344, 1737-1990, 1737-1991, 1760-2424, 1762-1827, 1796-2286, 1843-2357, 1950-2351, 1950- 2393, 1954-2403, 1965-2376, 2000-2431, 2013-2426, 2059-2309, 2087-2415, 2098-2415, 2099-2417, 2100-2412, 2110-2412, 2112-2413, 2177-2427, 2216-2415, 2227-2415, 2238-2415, 2287-2426, 2299-2406, 2299-2426

36/7510226CB1/ 1-442, 1-3737, 802-1028, 869-1196, 1090-1337, 1117-1318 , 1473-2148, 1478-2258 , 1478-2284, 1481-2279, 1549-

3842 2159, 1556-1777, 1631-2169, 1632-2077, 1632-2166, 1641-2091, 1643-2347, 1648-■2086, 1649-2268, 1653-2236, 1663-2374, 1664-2442, 1679-2167, 1706-2185, 1727-2327, 1734-2437, 1735-2148, 1738-2281, 1756-2330, 1756- 2333, 1757-2284, 1762-2409, 1762-2488, 1781-2405, 1791-2563, 1792-2215, 1793-■2054, 1793-2083, 1802-2112, 1804-2276, 1808-2200, 1809-2312, 1825-2504, 1825-2704, 1827-2362, 1833-2522, 1839-2125, 1856-2484, 1863- 2541, 1872-2394, 1885-2064, 1885-2224, 1936-2513, 1939-2220, 1951-2213, 1952-•2608, 1957-2220, 1963-2700, 1977-2548, 1980-2238, 1980-2450, 1989-2483, 2002-2703, 2026-2426, 2026-2738, 2029-2615, 2050-2813, 2066- 2783, 2070-2767, 2072-2312, 2107-2727, 2108-2419, 2109-2616,2119-2338,2122-2360, 2134-2394, 2142-2689, 2146-2829, 2147-2801, 2159-2322, 2167-2286, 2176-2415, 2179-2542,2182-2445, 2193-2887, 2214-2815, 2216- 2782, 2218-2468, 2219-2455, 2231-2866, 2245-2555, 2270-2567, 2275-2968, 2293-2520, 2294-2499,

Table 4

Table 4

Table 4

Table 4

Table 4

Table 4

Table 4

Table 5

VO

Table 6

Table 6

Table 6

Table 6

Table 7

Table 7

Table 7

Claims

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:l-22, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consistmg of SEQ ID NO:l-2, SEQ ID NO:5-6, SEQ ID NO:10, SEQ ID NO:15, and SEQ ID NO:17-19, c) a polypeptide comprising a naturally occurring amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:3 , d) a polypeptide comprising a naturally occurring amino acid sequence at least 96% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:9, and SEQ ID NO: 14, e) a polypeptide comprising a naturally occurring amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO:7, f) a polypeptide comprising a naturally occurring amino acid sequence at least 99% identical to an amino acid sequence selected from the group consisting of of SEQ ID NO:13, and SEQ ID NO:20, g) a polypeptide consisting essentially of a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:ll-12, SEQ ID NO:16, and SEQ JD NO:21-22, h) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consistmg of SEQ JD NO: 1-22, and i) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22.

2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-22.

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:23-44.

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 consistmg of SEQ ID NO:l-22.

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:23-44, 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:23-28, SEQ ID NO:31-34, SEQ ID NO:36-39, and SEQ ID NO:41, c) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 93% identical to the polynucleotide sequence of SEQ ID NO:40, d) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 94% identical to the polynucleotide sequence of SEQ ID NO:30, e) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 96% identical to the polynucleotide sequence of SEQ ID NO:44, f) a polynucleotide comprising a naturally occurring polynucleotide sequence at least

97% identical to the polynucleotide sequence of SEQ ID NO:35, g) a polynucleotide comprising a naturally occurring polynucleotide sequence at least

99% identical to the polynucleotide sequence of SEQ ID NO:43, h) a polynucleotide consisting essentially of a naturaUy occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:29, and SEQ ID NO:42 i) a polynucleotide complementary to a polynucleotide of a), j) a polynucleotide complementary to a polynucleotide of b), k) a polynucleotide complementary to a polynucleotide of c), 1) a polynucleotide complementary to a polynucleotide of d), m) a polynucleotide complementary to a polynucleotide of e), n) a polynucleotide complementary to a polynucleotide of f), o) a polynucleotide complementary to a polynucleotide of g), p) a polynucleotide complementary to a polynucleotide of h), and q) an RNA equivalent of a)-p).

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 JD NO: 1-22.

19. A method for treating a disease or condition associated with decreased expression of functional NTRAN, comprising admMstering 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 NTRAN, comprising adn inistering 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 NTRAN, 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 compnsmg: 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 method for a diagnostic test for a condition or disease associated with the expression of NTRAN 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 NTRAN in a subject, comprising administering to said subject an effective amount of the composition of claim

32.

34. A composition of claim 32, further comprising a label.

35. A method of diagnosing a condition or disease associated with the expression of NTRAN 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:l-22, 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: 1-22.

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) immurjizing an ariimal with a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:l-22, 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:l-22.

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 consistmg of SEQ ID NO: 1-22 in a sample, the method comprising: a) incubating the antibody of claim 11 with the 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: 1-22 in the sample.

45. A method of purifying a polypeptide comprising an amino acid sequence selected from the group consistmg of SEQ JD NO: 1-22 from a sample, the method comprising: a) incubating the antibody of claim 11 with the 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 JD NO: 1-22.

46. A microarray wherein at least one element of the microanay 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 microanay 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 anay 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 anay 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 distmct physical location on the substrate.

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

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

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

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

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

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

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

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

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

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

68. A polypeptide of claim 1, comprising the amino acid sequence of SEQ JD NO:l3.

69. A polypeptide of claim 1, comprising the amino acid sequence of SEQ JD 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 polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ JD NO:23.

79. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:24.

80. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ JD NO:25.

81. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:26.

82. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ JD 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 JD NO:29.

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

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

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

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

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

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

91. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ JD 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 JD 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 JD NO:43.

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