EP1368473A2 - Neurotransmission-associated proteins - Google Patents

Neurotransmission-associated proteins

Info

Publication number
EP1368473A2
EP1368473A2 EP02718986A EP02718986A EP1368473A2 EP 1368473 A2 EP1368473 A2 EP 1368473A2 EP 02718986 A EP02718986 A EP 02718986A EP 02718986 A EP02718986 A EP 02718986A EP 1368473 A2 EP1368473 A2 EP 1368473A2
Authority
EP
European Patent Office
Prior art keywords
polynucleotide
polypeptide
seq
amino acid
ntran
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02718986A
Other languages
German (de)
English (en)
French (fr)
Inventor
Brendan M. Duggan
Cynthia D. Honchell
Craig H. Ison
Kavitha Thangavelu
Dyung Aina M. Lu
Mariah R. Baughn
Preeti G. Lal
Henry Yue
Y. Tom Tang
Bridget A. Warren
Ernestine A. Lee
Jennifer A. Griffin
Ian J. Forsythe
Narinder K. Chawla
Xin Jiang
Alan A. Jackson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Incyte Corp
Original Assignee
Incyte Genomics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Incyte Genomics Inc filed Critical Incyte Genomics Inc
Publication of EP1368473A2 publication Critical patent/EP1368473A2/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/20—Hypnotics; Sedatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/14—Drugs for disorders of the endocrine system of the thyroid hormones, e.g. T3, T4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to nucleic acid and amino acid sequences of neurotransmission- associated proteins and to the use of these sequences in the diagnosis, treatment, and prevention of autoimmune/inflammatory, cardiovascular, neurological, developmental, cell proliferative, including cancer, transport, psychiatric, metabolic, and endocrine disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of neurotransmission-associated proteins.
  • 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 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 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 millimeter 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 rnyelin sheath made up of membranes from either an oligodendrocyte cell (CNS) or a Schwann cell (PNS).
  • CNS oligodendrocyte cell
  • PNS Schwann cell
  • 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.
  • 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.
  • neural membrane protein 35 NMP35
  • SY synaptophysin
  • the chromosomal location of SY inhuman and mouse is on the X chromosome in subbands Xpll.22- pll.23. This region has been 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.) Peripherin or retinal degeneration slow protein (rds) is an integral membrane glycoprotein that is present in the rims of photoreceptor outer segment disks.
  • Rds 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.)
  • Semaphorin3A and the Semaphorin3A receptor proteins neuropilin-1 and plexin-Al include Semaphorin3A and the Semaphorin3A receptor proteins neuropilin-1 and plexin-Al (Pasterkamp, RJ. and Nerhaagen, J.(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 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).
  • ECM extracellular matrix
  • 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 (Reichaxdt, L.F. et al. (1991) Ann.
  • 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).
  • 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.
  • Nejurin 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 Milbrandt, J. (1996) Neuron 17:353-361).
  • Ninjurin2 is expressed in mature sensory and enteric neurons and promotes neurite outgrowth. Ninjurin2 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 Milbrandt, J. (2000) J. Neurosci. 20:187-195).
  • Neurexin IV is essential for axonal insulation in the PNS 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) domain-containing protein which may be involved in cortical neurogenesis and cell fate decisions in the mammalian nervous system.
  • PTB phosphotyrosine-binding
  • LNX 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-cont- ⁇ iing 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).
  • 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.
  • 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
  • synapse 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 subcehular compartments of the synapse.
  • Synaptic terminals also contain many other proteins involved in calcium transport, neurot ansmission, signaling, growth, and plasticity.
  • the axon terminal from one neuron 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.
  • 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.
  • CSPs cysteine-string proteins
  • 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.
  • NRGs Neuregulins
  • 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-HI (LII-III) of both alpha IB and alpha 1A subunits of N-type and P/Q-type Ca2+ channels bind to syntaxin, SNAP-25, and synaptotagmin.
  • LAI-III intracellular loop II-HI
  • 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. N Y 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-IX). Ca(2+)-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), rab3 A, synaptophysin, synaptotagmin ( ⁇ 65) 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 Scheller, R.H.(1993) J. Cell Sci. Suppl.17:75-79).
  • 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 (Kedzierski, W.J. et al. (1999) Neurochem. 72:430-438).
  • 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 II, and subunits c and Acl 15 of the VO sector of the V-ATPase.
  • In situ hybridization in rat brain reveals a largely neuronal distribution of Ac39/physophilin mRNA which correlates spatio-temporally with those of subunit c and synaptophysin.
  • the plasma membrane dopamine transporter is essential for the reuptake of released dop-unine 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) Natare 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).
  • RAPS YN acetylcholine receptor-associated 43 KD protein
  • RAPSYN is involved in membrane association and may link the nicotinic acetylcholine receptor to the underlying postsynaptic cytoskeleton.
  • 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 microtabule-associated protein 5 (MAP5).
  • MAP5 microtabule-associated protein 5
  • 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-microtabule interactions and is expressed throughout the rodent CNS. (Rienitz, A. et al. (1989) EMBO J. 8:2879-2888.)
  • 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-an-iinobutyric acid (GABA), glutamate, and aspartate, and neuropeptides such as endorphins and enkephalins.
  • GABA gamma-an-iinobutyric acid
  • neuropeptides such as endorphins and enkephalins.
  • GABA is the major inhibitory neurotransmitter in the CNS
  • 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, Y. et al. (1999) Trends Cell Biol. 9:356-363).
  • GABA is the predominant inhibitory neurotransmitter and is widely distributed in the mammalian nervous system.
  • GABA is cleared from the synaptic cleft by specific, high-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.
  • GABA transporters At least four GABA transporters (GAT1-GAT4) have been cloned (Liu, Q.-R. et al. (1993) J. Biol. Chem. 268:2106-2112).
  • GABA transporters exhibit differences in substrate affinity and specificity, distinct blocker pharmacologies, and different tissue localization.
  • the K tract. values of GABA uptake of the expressed GAT1 to GAT4 are 6, 79, 18, and 0.8 mM, respectively.
  • GAT2 also transports betaine
  • GAT3 and GAT4 also transport b-alanine and taurine.
  • Pharmacological studies revealed that GABA transport by GAT1 and GAT4 is more sensitive to 2,4-diaminobutyric acid and guavicine than that by GAT2 and GAT3. In sita hybridization showed that GAT1 and GAT4 expression is brain specific.
  • GAT2 and GAT3 mRNAs were detected in tissues such as liver and kidney (Schloss supra; Borden, L.A. (1996) Neurochem. Int. 29:335-356; Nelson, N. (1998) J. Neurochem. 71:1785-1803).
  • Diazepam binding inhibitor also known as endozepine and acyl-Coenzyme (CoA)- binding protein
  • DBI 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 (OM ); 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) Natare 325:529-531).
  • NMD A N-methyl-D-aspartate
  • GLYT 1 GLYT 1
  • GLYT2 Variants of GLYT1 (GLYT1 a/b) are generated by alternative splicing (Liu, Q.-R. et al.
  • 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 supra). GLYT2 is expressed mainly in brainstem and spinal cord (Schloss supra).
  • the second identified glycine transporter differs from GLYTla/b by its extended intracellular amino teiminus.
  • the predominant localization of its mRNA in brainstem and spinal cord and its insensitivity to N-memyl-ai-ninoacetic acid suggests that GLYT2 terminates signal transduction at the sfrychnine-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).
  • 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, pharnyx, vertebral discs, jaw, and nasal epithelium.
  • 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, staminergic, 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 transme branes seven and eight (Liu, Q.-R. et al. (1993) EEBS Lett.
  • Glutamate is a major excitatory neurotransmitter in the mammahan central nervous system. Electrogenic (Na + /K + )-coupled glutamate transporters, located in the plasma membranes of nerve terminals 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) Natare 383:634-637).
  • the membrane topology of the glutamate transporters reveals six membrane-spanning helices in the N-te ⁇ ninal part of the proteins (Slotboom, DJ. 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).
  • VMAT vesicular monoamine transporters
  • VMAT vesicular monoamine transporters
  • 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 diseases caused by defects in neurotransmitter transporters include schizophrenia, Tourette's syndrome, Parkinson's disease, brain ischemia, amyotrophic lateral scerlosis, depression, and epilepsy.
  • 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).
  • ALS amyotrophic lateral sclerosis
  • 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.
  • Amphet-imines enhance transporter mediated efflux in concert with a depletion of vesicular amine stores (Barker, EX. 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).
  • CTLl proteins Another family of molecules that appear to be important for neurotransmission is the choline- 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. 97:1835-40).
  • 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.
  • 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).
  • 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 Bixby, J.L. (1998) Trends Neurosci. 21:22-27). Neurological protein domains
  • CNS-associated proteins can be phosphoproteins.
  • ARPP-21 cyclic AMP-regulated phosphoprotein
  • ARPP-21 mRNA is a cytosolic neuronal phosphoprotein that is highly enriched in the striatam and in other dopaminoceptive regions of the brain.
  • the steady-state level of ARPP-21 mRNA is developmentally regulated. But, in the neonatal and matare animal, ARPP-21 mRNA is not altered following 6-hydroxydopamine lesions of the substantia nigra or by pharmacologic treatments thatupregulate the Dl- 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 dysfrophin-associated proteins.
  • NOS neuronal nitric oxide synthase
  • 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.
  • EGF epidermal growth factor
  • 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 glp-1 proteins act as receptors in specific developmental cell interactions which maybe involved in certain embyronic defects (Tax, F. E. et al. (1994) Natare 368:150-154).
  • Pecanex a maternal-effect neurogenic locus of D.
  • melanogaster is believed to encode a large transmembrane protein.
  • an embryo develops severe hyperneuralization similar to that characteristic of Notch mutant embryos (LaBonne, S. G. et al. (1989) Dev. Biol. 136:1-116).
  • 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.
  • FGF fibroblast growth factor
  • EHF polypeptides fibroblast growth factor homologous factors
  • Members of the FHF family of polypeptides are structurally distinct from prototypic FGFs, consistent with the unusual role of these FGF-related proteins (Smallwood, P.M. et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:9850-9857 and Hartang, H. et al. (1997) Mech. Dev. 64:31-39).
  • AD Alzheimer's disease
  • ⁇ JJ is a degenerative disorder of the CNS which causes progressive memory loss and cognitive decline during mid to late adult life.
  • ⁇ JJ is characterized by a wide range of neuropathologic features including amyloid deposits and intra-neuronal neurofibrillary tangles.
  • 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.
  • Familial British dementia 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 FBD patients maybe a contributive factor of this disease (El-Agnaf, O.M.A. et al. (2001) Biochemistry 40:3449-3457).
  • Astrocyto as, and the more malignant glioblastomas are the most common primary tamors of the brain, accounting for over 65 % of primary brain tamors. These tamors 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).
  • 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.
  • 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).
  • Trk family receptors Signaling initiated by the Trk family receptors plays a dynamic role in neurogenic tamors.
  • the proto-oncogene Trks encode the high-affinity receptor tyrosine kinases for nerve growth factor ( ⁇ GF) neurotrophins.
  • ⁇ GF nerve growth factor
  • 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 tamors of neuronal origin, such as neuroblastoma and meduUoblastoma (Nakagawara, A. (2001) Cancer Lett.l69:107-114).
  • Neuronal thread proteins 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.
  • 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).
  • APP Alzheimer beta-amyloid precursor protein
  • 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).
  • Amyotrophic lateral sclerosis 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 Leigh, P.N.(2000) Curr. Opin. Neurol. 13:397-405).
  • NFs neurofilaments
  • 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 (Juhen, J.P. and Beaulieu, J.M. (2000) J. Neurol. Sci.l80:7-14).
  • Miller-Dieker syndrome 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 LIS 1 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, R.J. 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).
  • 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.
  • genes encoding the 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).
  • 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 Terry, R.(1993) Brain Pathol. 3:77-85).
  • Synaptophysin a major integral membrane protein of small synaptic vesicles, is on the X chromosome in subbands Xpll.22-p 11.23, a region implicated in several inherited diseases including Wiskott-Aldrich syndrome, three forms ofX-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).
  • Associated changes in the phosphorylation of selected protein substrates in subcellular compartments including presynaptic terminals and microtabules 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).
  • 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 . 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 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 K.J. et al. (2001) FASEB J.15: 1575-1576).
  • 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 (DENB9). 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 IU (Britton'S. et al. (2000) Genomics 68:313-321).
  • 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, ruptare and, most lethally, induction of occlusive vessel thrombus.
  • LDL cholesterol-rich low-density lipoprotein
  • MM- LDL oxidized LDL
  • Ox-LDL oxidized LDL
  • scavenger 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) Natare 343:531-535; Acton et al. (1994) J Biol Chem 269:21003-21009; Endemann et al.
  • 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 (Gushing et al. (1990) Proc Natl Acad Sci 87:5134-5138; Yoshida et al.
  • cholesterol content is tightly controlled by feedback regulation of LDL receptors and biosynthetic enzymes (Brown and Goldstein (1986) Science 232:34- 47).
  • 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 lipid droplets producing a "foam cell” phenotype.
  • 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.
  • Receptors and Membrane-Associated Proteins Signal transduction is the general process by which cells respond to extracellular signals. Signal transduction across the plasma membrane begins with the binding of a signal molecule, e.g., a hormone, neurotransmitter, or growth factor, to a cell membrane receptor. The receptor, thus activated, triggers an intracellular biochemical cascade that ends with the activation of an intracellular target molecule, such as a transcription factor. This process of signal transduction regulates all types of cell functions including cell proliferation, differentiation, and gene transcription.
  • a signal molecule e.g., a hormone, neurotransmitter, or growth factor
  • Membranes surround organelles, vesicles, and the cell itself.
  • Membranes are highly selective permeability barriers made up of lipid bilayer sheets composed of phosphoglycerides, fatty acids, cholesterol, phospholipids, glycolipids, proteoglycans, and proteins.
  • Membranes contain ion pumps, ion channels, and specific receptors for external stimuli which transmit biochemical signals across the membranes. These membranes also contain second messenger proteins which interact with these pumps, channels, and receptors to amplify and regulate transmission of these signals.
  • Plasma membrane proteins are divided into two groups based upon methods of protein extraction from the membrane. Extrinsic or peripheral membrane proteins can be released using extremes of ionic strength or pH, urea, or other disrupters of protein interactions. Intrinsic or integral membrane proteins are released only when the lipid bilayer of the membrane is dissolved by detergent.
  • TM proteins transmembrane proteins
  • TM domains are typically comprised of 15 to 25 hydrophobic amino acids which are predicted to adopt an ⁇ -helical conformation.
  • TM proteins are classified as bitopic (Types I and II) and polytopic (Types OI and IV) (Singer, S.J. (1990) Annu. Rev. Cell Biol. 6:247-96).
  • Bitopic proteins span the membrane once while polytopic proteins contain multiple membrane-spanning segments.
  • TM proteins carry out a variety of important cellular functions, including acting as cell-surface receptor proteins involved in signal transduction.
  • TM proteins also act as transporters of ions or metabolites, such as gap junction channels (connexins), and ion channels, and as cell anchoring proteins, such as lectins, integrins, and fibronectins.
  • TM proteins are found in vesicle organehe-forming molecules, such as caveolins; or cell recognition molecules, such as cluster of differentiation (CD) antigens, glycoproteins, and mucins.
  • MPs contain amino acid sequence motifs that serve to localize proteins to specific subcellular sites. Examples of these motifs include PDZ domains, KDEL, RGD, NGR, and GSL sequence motifs, von Willebrand factor A (vWFA) domains, and EGF-like domains. RGD, NGR, and GSL motif-containing peptides have been used as drug delivery agents in targeted cancer treatment of tamor vasculatare (Arap, W. et al. (1998) Science, 279:377-380). Furthermore, MPs may also contain amino acid sequence motifs that serve to interact with extracellular or intracellular molecules, such as carbohydrate recognition domains (CRD).
  • CCD carbohydrate recognition domains
  • Chemical modification of amino acid residue side chains alters the manner in which MPs interact with other molecules, for example, phospholipid membranes.
  • Examples of such chemical modifications to amino acid residue side chains are covalent bond formation with glycosaminoglycans, oligosaccharides, phospholipids, acetyl and palmitoyl moieties, ADP-ribose, phosphate, and sulphate groups.
  • RNA encoding membrane proteins may have alternative splice sites which give rise to proteins encoded by the same gene but with different messenger RNA and amino acid sequences. Splice variant membrane proteins may interact with other ligand and protein isoforms.
  • Receptors The term receptor describes proteins that specifically recognize other molecules. The category is broad and includes proteins with a variety of functions. The bulk of receptors are cell surface proteins which bind extracellular ligands and produce cellular responses in the areas of growth, differentiation, endocytosis, and immune response. Other receptors facilitate the selective transport of proteins out of the endoplasmic reticulum and localize enzymes to particular locations in the cell. The term may also be applied to proteins which act as receptors for Egands with known or unknown chemical composition and which interact with other cellular components. For example, the steroid hormone receptors bind to and regulate transcription of DNA.
  • Cell surface receptors are typically integral plasma membrane proteins. These receptors recognize hormones such as catecholamines; peptide hormones; growth and differentiation factors; small peptide factors such as thyrotropin-releasing hormone; galanin, somatostatin, and tachykinins; and circulatory system-borne signaling molecules.
  • Cell surface receptors on immune system cells recognize antigens, antibodies, and major histocompatibility complex (MHC)-bound peptides. Other cell surface receptors bind ligands to be internalized by the cell.
  • MHC major histocompatibility complex
  • LDL low density lipoproteins
  • transferrin glucose- or mannose-terminal glycoproteins
  • galactose-te ⁇ -ninal glycoproteins immunoglobulins
  • phosphovitellogenins fibrin
  • proteinase-inhibitor complexes proteinase-inhibitor complexes
  • plasminogen activators plasminogen activators
  • thrombospondin Receptor Protein Kinases
  • growth factor receptors including receptors for epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, as well as the growth modulator ⁇ -thrombin, contain intrinsic protein kinase activities. When growth factor binds to the receptor, it triggers the autophosphorylation of a serine, threonine, or tyrosine residue on the receptor. These phosphorylated sites are recognition sites for the binding of other cytoplasmic signaling proteins. These proteins participate in signaling pathways that eventually link the initial receptor activation at the cell surface to the activation of a specific intracellular target molecule. In the case of tyrosine residue autophosphorylation, these signaling proteins contain a common domain referred to as a Src homology (SH) domain.
  • SH Src homology
  • SH2 domains and SH3 domains are found in phospholipase C- ⁇ , PI-3-K p85 regulatory subunit, Ras-GTPase activating protein, and p ⁇ 60 C SIC (Lowenstein, E.J. et al. (1992) Cell 70:431-442).
  • the cytokine family of receptors share a different common binding domain and include transmembrane receptors for growth hormone (GH), interleukins, erythropoietin, and prolactin.
  • GPCRs The G-protein coupled receptors (GPCRs), encoded by one of the largest families of genes yet identified, play a central role in the transduction of extracellular signals across the plasma membrane. GPCRs have a proven history of being successful therapeutic targets.
  • GPCRs are integral membrane proteins characterized by the presence of seven hydrophobic transmembrane domains which together form a bundle of antiparallel alpha ( ⁇ ) helices. GPCRs range in size from under 400 to over 1000 amino acids (Strosberg, A.D. (1991) Eur. J. Biochem. 196:1-10; Coughlin, S.R. (1994) Curr. Opin. Cell Biol. 6:191-197).
  • the amino-terminus of a GPCRis extracellular is of variable length, and is often glycosylated. The carboxy-terminus is cytoplasmic and generally phosphorylated. Extracellular loops alternate with intracellular loops and link the transmembrane domains.
  • Cysteine disulfide bridges linking the second and third extracellular loops may interact with agonists and antagonists.
  • the most conserved domains of GPCRs are the transmembrane domains and the first two cytoplasmic loops.
  • the transmembrane domains account, in part, for structural and functional features of the receptor. In most cases, the bundle of ⁇ hehces forms a ligand-binding pocket.
  • the extracellular N-terminal segment, or one or more of the three extracellular loops, may also participate in ligand binding. Ligand binding activates the receptor by inducing a conformational change in intracellular portions of the receptor.
  • the large, third intracellular loop of the activated receptor interacts with a heterotrimeric guanine nucleotide binding (G) protein complex which mediates further intracellular signaling activities, including the activation of second messengers such as cyclic AMP (cAMP), phospholipase C, and inositol triphosphate, and the interaction of the activated GPCR with ion channel proteins.
  • G heterotrimeric guanine nucleotide binding
  • GPCRs include receptors for sensory signal mediators (e.g., light and olfactory stimulatory molecules); adenosine, ⁇ -aminobutyric acid (GABA), hepatocyte growth factor, melanocortins, neuropeptide Y, opioid peptides, opsins, somatostatin, tachykinins, vasoactive intestinal polypeptide family, and vasopressin; biogenic amines (e.g., dopamine, epinephrine and norepinephrine, Mstamine, glutamate (metabotropic effect), acetylcholine (muscarinic effect), and serotonin); chemokines; lipid mediators of inflammation (e.g., prostaglandins and prostanoids, platelet activating factor, and leukotrienes); and peptide hormones (e.g., bombesin,
  • GABA ⁇ -aminobutyric acid
  • GPCRs which act as receptors for stimuli that have yet to be identified are known as orphan receptors.
  • GPCR mutations which may cause loss of function or constitutive activation, have been associated with numerous human diseases (Coughlin, supra). For instance, retinitis pigmentosa may arise from mutations in the rhodopsin gene. Furthermore, somatic activating mutations in the thyrotropin receptor have been reported to cause hyperfunctioning thyroid adenomas, suggesting that certain GPCRs susceptible to constitutive activation may behave as protooncogenes (Parma, J. et al. (1993) Natare 365:649-651).
  • GPCR receptors for the following ligands also contain mutations associated with human disease: luteinizing hormone (precocious puberty); vasopressin V 2 (X-linked nephrogenic diabetes); glucagon (diabetes and hypertension); calcium (hyperparathyroidism, hypocalcuria, hypercalcemia); parathyroid hormone (short limbed dwarfism); b 3 -adrenoceptor (obesity, non-insulin-dependent diabetes mellitus); growth hormone releasing hormone (dwarfism); and adrenocorticotropin (glucocorticoid deficiency) (Wilson, S. et al. (1998) Br. J. Pharmocol.
  • GPCRs are also involved in depression, schizophrenia, sleeplessness, hypertension, anxiety, stress, renal failure, and several cardiovascular disorders (Horn, F. and G. Vriend (1998) J. Mol. Med. 76:464-468).
  • cardiovascular, gastrointestinal, and central nervous system disorders as well as cancer, osteoporosis and endometriosis (Wilson, supra; Stadel, supra).
  • the dopamine agonist L-dopa is used to treat Parkinson's disease, while a dopamine antagonist is used to treat schizophrenia and the early stages of Huntington's disease.
  • Agonists and antagonists of adrenoceptors have been used for the treatment of asthma, high blood pressure, other cardiovascular disorders, and anxiety; muscarinic agonists are used in the treatment of glaucoma and tachycardia; serotonin 5HT1D antagonists are used against migraine; and histamine HI antagonists are used against allergic and anaphylactic reactions, hay fever, itching, and motion sickness (Horn, supra).
  • Nuclear Receptors are used to treat allergic and anaphylactic reactions, hay fever, itching, and motion sickness (Horn, supra).
  • Nuclear receptors bind small molecules such as hormones or second messengers, leading to increased receptor-binding affinity to specific chromosomal DNA elements. In addition the affinity for other nuclear proteins may also be altered. Such binding and protein-protein interactions may regulate and modulate gene expression. Examples of such receptors include the steroid hormone receptors family, the retinoic acid receptors family, and the thyroid hormone receptors family. Ligand-Gated Receptor Ion Channels
  • Ligand-gated receptor ion channels fall into two categories.
  • the first category extracellular ligand-gated receptor ion channels (ELGs), rapidly transduce neurotransmitter-binding events into electrical signals, such as fast synaptic neurotransmission. ELG function is regulated by post- translational modification.
  • the second category intracellular ligand-gated receptor ion channels (ILGs), are activated by many intracellular second messengers and do not require post-translational modification(s) to effect a channel-opening response.
  • ELGs depolarize excitable cells to the threshold of action potential generation. In non- excitable cells, ELGs permit a limited calcium ion-influx during the presence of agonist.
  • ELGs include channels directly gated by neurotransmitters such as acetylcholine, L-glutamate, glycine, ATP, serotonin, GABA, and -Mstamine.
  • ELG genes encode proteins having strong structural and functional similarities.
  • ILGs are encoded by distinct and unrelated gene families and include receptors for cAMP, cGMP, calcium ions, ATP, and metabolites of arachidonic acid. Macrophage Scavenger Receptors
  • Macrophage scavenger receptors with broad ligand specificity may participate in the binding of low density lipoproteins (LDL) and foreign antigens.
  • Scavenger receptors types I and II are trimeric membrane proteins with each subunit containing a small N-terminal intracellular domain, a transmembrane domain, a large extracellular domain, and a C-terminal cysteine-rich domain.
  • the extracellular domain contains a short spacer domain, an ⁇ -helical coiled-coil domain, and a triple helical collagenous domain.
  • T-Cell Receptors have been shown to bind a spectrum of ligands, including chemically modified lipoproteins and albumin, polyribonucleotides, polysaccharides, phospholipids, and asbestos (Matsumoto, A. et al. (1990) Proc. Natl. Acad. Sci. USA 87:9133-9137; Elomaa, O. et al. (1995) Cell 80:603-609).
  • the scavenger receptors are thought to play a key role in atherogenesis by mediating uptake of modified LDL in arterial walls, and in host defense by binding bacterial endotoxins, bacteria, and protozoa.
  • T cells play a dual role in the immune system as effectors and regulators, coupling antigen recognition with the transmission of signals that induce cell death in infected cells and stimulate proliferation of other immune cells.
  • TCR T cell receptor
  • MHC major histocompatibility molecule
  • Both TCR subunits have an extracellular domain containing both variable and constant regions, a transmembrane domain that traverses the membrane once, and a short intracellular domain (Saito, H. et al. (1984) Nature 309 -.757-762).
  • the genes for the TCR subunits are constructed through somatic rearrangement of different gene segments. Interaction of antigen in the proper MHC context with the TCR initiates signaling cascades that induce the proliferation, maturation, and function of cellular components of the immune system (Weiss, A. (1991) Annu. Rev. Genet. 25: 487-510).
  • the netrins are a family of molecules that function as diffusible attractants and repellants to guide migrating cells and axons to their targets within the developing nervous system.
  • the netrin receptors include the C. elegans protein UNC-5, as well as homologues recently identified in vertebrates (Leonardo, E.D. et al. (1997) Natare 386:833-838). These receptors are members of the immunoglobulin superfamily, and also contain a characteristic domain called the ZU5 domain. Mutations in the mouse member of the netrin receptor family, Rcm (rostral cerebellar malformation) result in cerebellar and midbrain defects as an apparent result of abnormal neuronal migration (Ackerman, SX. et al. (1997) Natare 386:838-842). VPS 10 Domain Containing Receptors
  • VPS 10 domain containing receptor family all contain a domain with homology to the yeast vacuolar sorting protein 10 (VPS 10) receptor.
  • This family includes the mosaic receptor SorLA, the neurotensin receptor sortilin, and SorCS, which is expressed during mouse embryonal and early postnatal nervous system development (Hermey, G. et al. (1999) Biochem. Biophys. Res. Commun. 266:347-351; Hermey, G. et al. (2001) Neuroreport 12:29-32).
  • SorCS2 A recently identified member of this family, SorCS2, is highly expressed in the developing and matare mouse central nervous system.
  • TM4SF transmembrane 4 superfamily
  • TM4SF transmembrane 4 superfamily
  • TM4SF TM4SF
  • melanoma-associated antigens include platelet and endothelial cell membrane proteins, melanoma-associated antigens, leukocyte surface glycoproteins, colonal carcinoma antigens, tamor- associated antigens, and surface proteins of the schistosome parasites (Jankowski, S.A. (1994)
  • TM4SF Tumor Antigens
  • Tumor antigens are surface molecules that are differentially expressed in tumor cells relative to normal cells. Tumor antigens distinguish tamor cells immunologically from normal cells and provide diagnostic and therapeutic targets for human cancers (Takagi, S. et al. (1995) Int. J. Cancer 61: 706- 715; Liu, E. et al. (1992) Oncogene 7: 1027-1032). Ion Channels
  • Ion channels are found in the plasma membranes of virtually every cell in the body.
  • chloride channels mediate a variety of cellular functions including regulation of membrane potentials and absorption and secretion of ions across epithehal membranes.
  • chloride channels When present in intracellular membranes of the Golgi apparatus and endocytic vesicles, chloride channels also regulate organelle pH.
  • organelle pH See, e.g., Greger, R. (1988) Annu. Rev. Physiol. 50:111-122.
  • Electrophysiological and pharmacological properties of chloride channels including ion conductance, current-voltage relationships, and sensitivity to modulators, suggest that different chloride channels exist in muscles, neurons, fibroblasts, epithelial cells, and lymphocytes.
  • Many channels have sites for phosphorylation by one or more protein kinases including protein kinase A, protein kinase C, tyrosine kinase, and casein kinase II, all of which regulate ion channel activity in cells.
  • Inappropriate phosphorylation of proteins in cells has been linked to changes in cell cycle progression and cell differentiation. Changes in the cell cycle have been linked to induction of apoptosis or cancer. Changes in cell differentiation have been linked to diseases and disorders of the reproductive system, immune system, and skeletal muscle.
  • KCR1 Cerebellar granule neurons possess a non-inactivating potassium current which modulates firing frequency upon receptor stimulation by neurotransmitters and controls the resting membrane potential.
  • Potassium channels that exhibit non-inactivating currents include the ether a go-go (EAG) channel.
  • a membrane protein designated KCR1 specifically binds to rat EAG by means of its C- terminal region and regulates the cerebellar non-inactivating potassium current.
  • KCR1 is predicted to contain 12 transmembrane domains, with intracellular amino and carboxyl termini. Structural characteristics of these transmembrane regions appear to be similar to those of the transporter superfamily, but no homology between KCR1 and known transporters was found, suggesting that KCR1 belongs to a novel class of transporters.
  • KCR1 appears to be the regulatory component of non-inactivating potassium channels (Hoshi, N. et al. (1998) J. Biol. Chem. 273:23080-23085). ABC Transporters
  • ABC ATP-binding cassette
  • ABC transporter genes are associated with various disorders, such as hyperbilirubinemia ⁇ /Dubin- Johnson syndrome, recessive Stargardt's disease, X-linked adrenoleukodystrophy, multidrug resistance, celiac disease, and cystic fibrosis.
  • Intercellular communication is essential for the development and survival of multicellular organisms.
  • Cells communicate with one another through the secretion and uptake of protein signaling molecules.
  • the uptake of proteins into the cell is achieved by endocytosis, in which the interaction of signaling molecules with the plasma membrane surface, often via binding to specific receptors, results in the formation of plasma membrane-derived vesicles that enclose and transport the molecules into the cytosol.
  • the secretion of proteins from the cell is achieved by exocytosis, in which molecules inside of the cell are packaged into membrane-bound transport vesicles derived from the trans Golgi network. These vesicles fuse with the plasma membrane and release their contents into the surrounding extracellular space. Endocytosis and exocytosis result in the removal and addition of plasma membrane components, and the recycling of these components is essential to maintain the integrity, identity, and functionality of both the plasma membrane and internal membrane-bound compartments.
  • 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 glycophosphatidylinositol-linked proteins from axonal surfaces renders neurons insensitive to Nogo-66, facilitating potential recovery from CNS damage ( founder, A.E. et al. (2001) Nature 409:341-346). Lysosomes are the site of degradation of intracellular material during autophagy and of extracellular molecules following endocytosis. Lysosomal enzymes are packaged into vesicles which bud from the tr ⁇ ns-Golgi network.
  • vesicles fuse with endosomes to form the matare lysosome in which hydrolytic digestion of endocytosed material occurs.
  • Lysosomes can fuse with autophagosomes to form a unique compartment in which the degradation of organelles and other intracellular components occurs.
  • Protein sorting by transport vesicles has important consequences for a variety of physiological processes including cell surface growth, the biogenesis of distinct intracellular organelles, endocytosis, and the controlled secretion of hormones and neurotransmitters (Rothman, J.E. and Wieland, F.T. (1996) Science 272:227-234).
  • neurodegenerative disorders and other neuronal pathologies are associated with biochemical flaws during endosomal protein sorting or endosomal biogenesis (Mayer R.J. et al. (1996) Adv. Exp. Med. Biol. 389:261-269).
  • Peroxisomes are organelles independent from the secretory pathway. They are the site of many peroxide-generating oxidative reactions in the cell. Peroxisomes are unique among eukaryotic organelles in that their size, number, and enzyme content vary depending upon organism, cell type, and metabohc needs (Waterham, H.R. and Cregg, J.M. (1997) BioEssays 19:57-66).
  • TGFbeta Transforming growth factor beta signal transduction is mediated by two receptor Ser/Thr kinases acting in series, type ⁇ TGFbeta receptor and (TbetaR-II) phosphorylating type I TGFbeta receptor (TbetaR-I).
  • TbetaR-I-associated protein-1 TbetaR-I-associated protein-1 (TRECAP-1), which distinguishes between quiescent and activated forms of the type I fransforming growth factor beta receptor, has been associated with TGFbeta signaling (Charng, M.J et al. (1998) J. Biol. Chem. 273:9365-9368).
  • Retinoic acid receptor alpha mediates retinoic-acid induced maturation and has been implicated in myeloid development.
  • Genes induced by retinoic acid during granulocytic differentiation include E3, a hematopoietic-specific gene that is an immediate target for the activated RAR alpha during myelopoiesis (Scott, L.M. et al. (1996) Blood 88:2517-2530).
  • the ⁇ -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.
  • MOR-1 MOR-1
  • G protein-coupled receptors including somatostatin 2, dopamine D2, prostaglandinEP3, and serotonin receptor subtypes 5-hydroxyti ⁇ ptamine4 and 5-hydroxytryptamine7 (Pan, Y.X. et al. (1999) Mol. Pharm. 56:396-403).
  • Peripheral and Anchored Membrane Proteins include somatostatin 2, dopamine D2, prostaglandinEP3, and serotonin receptor subtypes 5-hydroxyti ⁇ ptamine4 and 5-hydroxytryptamine7 (Pan, Y.X. et al. (1999) Mol. Pharm. 56:396-403).
  • membrane proteins are not membrane-spanning but are attached to the plasma membrane via membrane anchors or interactions with integral membrane proteins.
  • Membrane anchors are covalently joined to a protein post-translationally and include such moieties as prenyl, myristyl, and glycosylphosphatidyl inositol groups.
  • Membrane localization of peripheral and anchored proteins is important for their function in processes such as receptor-mediated signal transduction. For example, prenylation of Ras is required for its localization to the plasma membrane and for its normal and oncogenic functions in signal transduction.
  • 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.
  • arrays are employed to detect the expression of a specific gene or its variants.
  • 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.
  • RPMI 6666 is a B cell lymphoblast cell line derived from the peripheral blood of a 29-year-old male suffering from Hodgkin's disease. RPMI 6666 cells are immature lymphocyte-producing immunoglo-bulins and present cell-associated Epstein-Barr virus (EBV) particules. RPMI 6666 cells have been used to study signaling in human B cells and identify factors produced by those cells.
  • the outer membrane of gram-negative bacteria expresses lipopolysaccharide (LPS) complexes, designated as endotoxins, that have biological effects. Toxicity is associated with the lipid component (Lipid A) of LPS, and immunogenicity is associated with the polysaccharide components of LPS.
  • LPS lipopolysaccharide
  • LPS elicits a variety of inflammatory responses, and because it activates complement by the alternative (properdin) pathway, it is often part of the pathology of gram-negative bacterial infections.
  • Gram-negative bacteria probably release minute amounts of endotoxin while growing. For example, it is nown that small amounts of endotoxin may be released in a soluble form, especially by young cultures. For the most part, however, endotoxins remain associated with the cell wall until the bacteria disintegrate. In vivo, disintegration is the result of autolysis of the bacteria, external lysis mediated by complement and lysozyme, and phagocytic digestion of bacterial cells.
  • LPS-binding proteins proteins identified as LPS-binding proteins.
  • the LPS-binding protein complex interacts with CD 14 receptors on monocytes, macrophages, B cells, and other types of receptors on endothelial cells. Activation of human B cells with LPS results in mitogenesis as well as immunoglobulin synthesis.
  • the invention features 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,” and “NTRAN-8.”
  • the invention provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-8, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-8, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-8, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8.
  • the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:l-8.
  • the invention further provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-8, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l- 8, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-8, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8.
  • the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:l-8.
  • the polynucleotide is selected from the group consisting of SEQ ID NO:9-16.
  • the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-8, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-8, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ 3D NO:l-8
  • the invention provides a cell transformed with the recombinant polynucleotide.
  • the invention provides a transgenic organism comprising the recombinant polynucleotide.
  • the invention also provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-8, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-8.
  • the method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
  • the invention provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-8, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-8, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-8.
  • the invention further provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:9-16, b) a polynucleotide comprising a nataraUy occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:9-16, 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 polynucleotide comprises at least 60 contiguous nucleotides.
  • the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:9-16, 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:9-16, 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, and optionaUy, if present, the amount thereof.
  • the probe comprises at least 60 contiguous nucleotides.
  • the invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:9-16, b) a polynucleotide comprising a nataraUy occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:9-16, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • the method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionaUy, if present, the amount thereof.
  • the invention further provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-8, b) a polypeptide comprising a nataraUy occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8, c) a biologicaUy active fra ment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-8, and a pharmaceuticaUy acceptable excipient.
  • the composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO:l-8.
  • the invention additionaUy 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.
  • the invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-8, b) a polypeptide comprising a nataraUy occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8, c) a biologicaUy active fragment of a polypeptide having an -u-nino acid sequence selected from the group consisting of SEQ ID NO: 1-8, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-8.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample.
  • the invention provides a composition comprising an agonist compound identified by the method and a pharmaceuticaUy acceptable excipient.
  • the invention 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.
  • the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8, b) a polypeptide comprising a nataraUy occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-8, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.
  • the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceuticaUy acceptable excipient.
  • the invention 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.
  • the invention further 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 ED NO: 1-8, b) a polypeptide comprising a nataraUy occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8.
  • 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.
  • the invention further provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8, b) a polypeptide comprising a nataraUy occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-8, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8, and d) an immunogenic fragment of a polypeptide having an -imino acid sequence selected from the group consisting of SEQ ID NO.T-8.
  • the method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
  • the invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:9-16, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
  • the invention further provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:9-16, ii) a polynucleotide comprising a nataraUy occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:9-16, 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)
  • Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:9-16, ii) a polynucleotide comprising a nataraUy occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:9-16, 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).
  • the target polynucleotide comprises a fragment of a polynucleotide sequence selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
  • 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 polypeptides of the invention. The probability scores for the matches between each polypeptide and its homolog(s) are also shown.
  • Table 3 shows structural features of polypeptide sequences of the invention, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
  • Table 4 lists the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide sequences of the invention, along with selected fragments of the polynucleotide sequences.
  • Table 5 shows the representative cDNA library for polynucleotides of the invention.
  • Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
  • Table 7 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with apphcable descriptions, references, and threshold parameters.
  • 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.
  • agonist refers to a molecule which intensifies or mimics the biological activity of
  • NTRAN 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.
  • AUeHc variants are an alternative form of the gene encoding NTRAN.
  • AUeHc 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 nataraUy occurring form.
  • Common mutational changes which give rise to aUehc 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 oUgonucleotide probe of the polynucleotide encoding NTRAN, and improper or unexpected hybridization to aUelic variants, witl a locus other than the normal chromosomal locus for the polynucleotide sequence 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 substitations may be made on the basis of similarity in polarity, charge, solubility hydrophobicity, hydrophilicity, and/or the amphipathic natare of the residues, as long as the biological or immunological activity of NTRAN is retained.
  • negatively charged amino acids may include aspartic acid and glutamic acid
  • positively charged amino acids may include lysine and arginine.
  • A-mino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine.
  • A-mino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
  • amino acid and amino acid sequence refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to nataraUy occurring or synthetic molecules. Where "amino acid sequence” is recited to refer to a sequence of a nataraUy occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
  • Amplification relates to the production of additional copies of a nucleic acid sequence. AmpHfication is generaUy carried out using polymerase chain reaction (PCR) technologies weU known in the art.
  • PCR polymerase chain reaction
  • Antagonist refers to a molecule which inhibits or attenuates the biological activity of NTRAN.
  • Antagonists may include proteins such as antibodies, 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.
  • 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
  • an animal e.g., a mouse, a rat, or a rabbit
  • an animal e.g., a mouse, a rat, or a rabbit
  • 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 -inimal.
  • KLH keyhole limpet hemocyanin
  • antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to ehcit the immune response) for binding to an antibody.
  • 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 maybe 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'-NH 2 ), which may improve a desired property, e.g., resistance to nucleases or longer Ufetime 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 maybe specificaUy cross-linked to their cognate Ugands, e.g., by photo-activation of a cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13.)
  • RNA aptamer refers to an aptamer which is expressed in vivo.
  • a vaccinia vims-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).
  • 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 nataraUy occurring enzymes, which normally act on substrates containing right-handed nucleotides.
  • antisense refers to any composition capable of base-pairing with the "sense" (coding) strand of a specific nucleic acid sequence.
  • Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oUgonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oUgonucleotides 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 nataraUy 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.
  • biologicalcaUy active refers to a protein having structural, regulatory, or biochemical functions of a nataraUy occurring molecule.
  • immunologicalaUy 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 ⁇
  • composition comprising a given polynucleotide sequence and a “composition comprising a given amino acid sequence” refer broadly to any composition containing the given polynucleotide or amino acid sequence.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotide sequences encoding NTRAN or fragments of NTRAN may be employed as hybridization probes.
  • the probes maybe stored in freeze-dried form and maybe associated with a stabilizing agent such as a carbohydrate.
  • 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.).
  • salts e.g., NaCl
  • detergents e.g., sodium dodecyl sulfate; SDS
  • 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 (AppUed 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 (GCG, Madison WI) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
  • GELVTEW fragment assembly system GCG, Madison WI
  • Phrap Universality of Washington, Seattle WA
  • Constant amino acid substitations are those substitations 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 substitations.
  • the table below shows amino acids which maybe substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
  • Conservative amino acid substitations generaUy maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
  • a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • 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 the polynucleotide encoding NTRAN which is identical in sequence to but shorter in length than the parent sequence.
  • a fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue.
  • a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues.
  • a fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes 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 may be preferentiaUy selected from certain regions of a molecule.
  • 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.
  • these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
  • a fragment of SEQ ID NO:9-16 comprises a region of unique polynucleotide sequence that specificaUy identifies SEQ ID NO:9-16, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
  • a fragment of SEQ ID NO:9-16 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SE ⁇ ID NO:9-16 from related polynucleotide sequences.
  • the precise length of a fragment of SEQ ID NO:9-16 and the region of SEQ ID NO:9-16 to which the fragment corresponds 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:l-8 is encoded by a fragment of SEQ ID NO:9-16.
  • a fragment of SEQ ID NO: 1-8 comprises a region of unique amino acid sequence that specificaUy identifies SEQ ID NO: 1-8.
  • a fragment of SEQ ID NO: 1-8 is useful as an immunogenic peptide for the development of antibodies that specificaUy recognize SEQ ID NO:l-8.
  • the precise length of a fragment of SEQ ID NO:l-8 and the region of SEQ ID NO:l-8 to which the fragment corresponds are routinely determinable by one of ordinary skiU in the art based on the intended purpose for the fragment.
  • a "fuU length" polynucleotide sequence is one containing at least a translation initiation codon
  • a "fuU length" polynucleotide sequence encodes a "fuU length” polypeptide sequence.
  • Homology refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
  • percent identity and “% identity,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize aUgnment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local AUgnment Search Tool
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local AUgnment Search Tool
  • 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.
  • BLAST 2 Sequences 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 (A ⁇ ril-21-2000) set at default parameters. Such default parameters maybe, for example: Matrix: BLOSUM62 Reward for match: 1
  • Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that aU encode substantiaUy the same protein.
  • the phrases "percent identity” and "% identity,” as appUed to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aUgned using a standardized algorithm. Methods of polypeptide sequence aUgnment are weU-known. Some aUgnment methods take into account conservative amino acid substitations. Such conservative substitations, 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.
  • NCBI BLAST software suite may be used.
  • BLAST 2 Sequences Version 2.0.12 (April-21-2000) with blastp set at default parameters.
  • Such default parameters may be, for example:
  • Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity maybe measured.
  • Human artificial chromosomes 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 repUcation, segregation and maintenance.
  • 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 abiUty.
  • 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 detemrinable 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 ⁇ g/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.
  • 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%.
  • blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ⁇ g/ml.
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • 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.
  • hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex maybe formed in solution (e.g., C 0 t or Rot analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobiUzed on a soUd support (e.g., paper, membranes, filters, chips, pins or glass sUdes, or any other appropriate substrate to which ceUs or their nucleic acids have been fixed).
  • insertion and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
  • Immuno 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 eUciting an immune response when introduced into a Hving 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.
  • microarray refers to an arrangement of a pluraUty of polynucleotides, polypeptides, or other chemical compounds on a substrate.
  • element and “array element” refer to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray.
  • modulate refers to a change in the activity of NTRAN.
  • modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of NTRAN.
  • nucleic acid and nucleic acid sequence refer to a nucleotide, oUgonucleotide, 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.
  • PNA peptide nucleic acid
  • 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.
  • 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.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an oUgonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubihty to the composition.
  • PNAs preferentiaUy bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their Ufespan in the ceU.
  • Post-translational modification of an NTRAN may involve Upidation, 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 acid sequences encoding NTRAN, their complements, or fragments thereof, which are used to detect identical, aUeUc or related nucleic acid sequences. Probes are isolated oKgonucleotides or polynucleotides attached to a detectable label or reporter molecule.
  • Typical labels include radioactive isotopes, Ugands, chemiluminescent agents, and enzymes.
  • "Primers" are short nucleic acids, usuaUy DNA oUgonucleotides, which maybe 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 ampUfication (and identification) of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • 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 maybe 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 the references, for example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2 nd ed., vol.
  • 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).
  • OUgonucleotides 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 oUgonucleotides 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 pubUc 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.
  • Primer3 primer selection program (available to the pubUc from the Whitehead Institute/Mrr Center for Genome Research, Cambridge MA) aUows the user to input a "n ⁇ spriming Ubrary," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oUgonucleotides 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 pubUc from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence aUgnments, thereby aUowing selection of primers that hybridize to either the most conserved or least conserved regions of aUgned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oUgonucleotides and polynucleotide fragments.
  • oUgonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fuUy or partially complementary polynucleotides in a sample of nucleic acids. Methods of oUgonucleotide selection are not limited to those described above.
  • a "recombinant nucleic acid” is a sequence that is not nataraUy 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 accompUshed by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra.
  • the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
  • 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.
  • 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 stabihty.
  • Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuchdes; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.
  • RNA equivalent in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that aU occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
  • 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.
  • 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.
  • a particular structure of the protein e.g., the antigenic determinant or epitope
  • the binding molecule e.g., the binding molecule for binding the binding molecule.
  • 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.
  • substantiallyUy purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are nataraUy associated.
  • Substrate refers to any suitable rigid or semi-rigid support including membranes, filters, chips, sUdes, 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, electroporation, heat shock, Upofection, and particle bombardment.
  • transformed ceUs includes stably transformed ceUs in which the inserted DNA is capable of repUcation either as an autonomously repUcating 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 deUberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • 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 fertiUzation, but rather is directed to the introduction of a recombinant DNA molecule.
  • the transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals.
  • the isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. (1989), supra.
  • a "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 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 "aUeUc" (as defined above), “spUce,” “species,” or “polymorphic” variant.
  • a spUce variant may have significant identity to a reference molecule, but wiU generaUy have a greater or lesser number of polynucleotides due to alternate spUcing of exons during mRNA processing.
  • the corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
  • Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides 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.
  • SNPs single nucleotide polymorphisms
  • the presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
  • a "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 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 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length of one of the polypeptides.
  • the invention is based on the discovery of 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 proUferative, including cancer, transport, psychiatric, metabohc, and endocrine disorders.
  • NTRAN neurotransmission-associated proteins
  • Table 1 summarizes the nomenclature for the fuU length polynucleotide and polypeptide sequences of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ID). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide ID) as shown.
  • Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as shown.
  • Column 6 shows the Incyte ID numbers of physical, full length clones corresponding to the polypeptide and polynucleotide sequences of the invention.
  • the fuU length clones encode polypeptides which have at least 95% sequence identity to the polypeptide sequences shown in column 3.
  • Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME database.
  • Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention.
  • Column 3 shows the GenBank identification number (GenBank 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 probabiUty 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 apphcable, 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 ID) for each polypeptide of the invention.
  • Column 3 shows the number of amino acid residues in each polypeptide.
  • Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS program of the GCG sequence analysis software package (Genetics Computer Group, Madison WI).
  • Column 6 shows amino acid residues comprising signatare sequences, domains, and motifs.
  • Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were appUed.
  • SEQ ID NO:l is 98% identical, from residue Ml to residue S704, to the human choline transporter-like protein, CTL2 (GenBank ID g6996444), as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiUty score is neghg ⁇ ble, indicating the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance.
  • BLAST Basic Local AUgnment Search Tool
  • SEQ ID NO:2 is 92% identical, from residue Ml to residue A1050, to the rat Robo2 receptor (GenBank ID g6164831), as determined by BLAST analysis. The BLAST probabiUty score is also negUgible. SEQ ID NO:2 also contains domains that are consistent with the Robo receptor, 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 analysis provides further corroborative evidence that SEQ ID NO:2 is a Robo2 receptor homolog.
  • HMM hidden Markov model
  • SEQ ID NO:3 is 76% identical, from residue Y57 to residue V298, to a rat homeodomain transcription factor specificaUy expressed in sensory neurons (GenBank ID gl 144015), as determined by BLAST analysis.
  • the BLAST probabiUty score is 2.3e-93.
  • SEQ ID NO:3 also contains homeodomains as dete ⁇ nined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ ED NO:4 is 96% identical, from residue Ml to residue P233, to a human fibroblast growth factor homologous factor (FHF), associated with neuron development (GenBank ID gl563889), as determined by BLAST analysis.
  • FHF human fibroblast growth factor homologous factor
  • the BLAST probabiUty score is 2.4e- 116.
  • SEQ ED NO:4 also contains a fibroblast growth factor domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. Data from BLIMPS, PROFELESCAN, and MOTIFS analyses provide further corroborative evidence that SEQ ID NO:4 is an FHF.
  • HMM hidden Markov model
  • SEQ ED NO:5 is 68% identical, from residue N7 to residue D198, to a rat cysteine string protein, a member of the DnaJ/hsp40 (heat shock protein) chaperone family (GenBank ID g2642629), as determined by BLAST analysis.
  • the BLAST probabiUty score is 9.4e- 74.
  • SEQ ED NO:5 also contains a DnaJ domains as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ ID NO:5 is a member of the DnaJ/hsp40 chaperone family.
  • SEQ ID NO:6 is 91% identical, from residue Ml to residue P533, to Mus musculus synaptotagmin X (GenBank ED g6136792) with a BLAST probabiUty score of 7.6e-266.
  • SEQ ED NO:6 also contains a C2 domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ ID NO:6 is a synaptotagmin.
  • SEQ ID NO:7-8 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ ID NO:l-8 are described in Table 7.
  • the fuU length polynucleotide sequences of the present invention were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences.
  • Column 1 Usts the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:), the corresponding Incyte polynucleotide consensus sequence number (Incyte ED) 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 sequences of the invention, and of fragments of the polynucleotide sequences which are useful, for example, in hybridization or ampUfication technologies that identify SEQ ID NO:9-16 or that distinguisl between SEQ ED NO:9-16 and related polynucleotide sequences.
  • polynucleotide fragments described in Column 2 of Table 4 may refer specificaUy, for example, to Incyte cDNAs derived from tissue-specific cDNA Ubraries or from pooled cDNA
  • polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the fuU length polynucleotide sequences.
  • polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences including the designation "ENST").
  • the polynucleotide fragments described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences including the designation "NM” or “NT”) or the NCBI RefSeq Protein Sequence Records (i.e., those sequences including the designation "NP”).
  • 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.
  • a polynucleotide sequence identified as L XXXXJl ⁇ N ⁇ YYYYJf ⁇ represents a "stitched" sequence in which XXXXX is the identification number of the cluster of sequences to which the algorithm was appUed, and ITOTis the number of the prediction generated by the algorithm, and N 1 ⁇ 3.
  • Apply if present, represent specific exons that may have been manuaUy edited during analysis (See Example V).
  • the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-stretching" algorithm.
  • a polynucleotide sequence identified as FLXXXXX_gAAAAA_gBBBBB_AN is a "stretched" sequence, with XXXXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the "exon-sfretching" algorithm was appUed, g ⁇ BBBB being the GenBank identification number or NCBI RefSeq identification number of the nearest GenBank protein homolog, and N referring to specific exons (See Example V).
  • a RefSeq identifier (denoted by "NM,” “NP,” or “NT”) may be used in place of the GenBank identifier (i.e., g ⁇ BBBB).
  • a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods.
  • Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
  • Table 5 shows the representative cDNA Ubraries for those fuU length polynucleotide sequences which were assembled using Incyte cDNA sequences.
  • the representative cDNA Ubrary is the Incyte cDNA Ubrary which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotide sequences.
  • the tissues and vectors which were used to construct the cDNA Ubraries shown in Table 5 are described in Table 6.
  • the invention also encompasses NTRAN variants.
  • a prefe ⁇ ed NTRAN variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the NTRAN amino acid sequence, and which contains at least one functional or structural characteristic of NTRAN.
  • the invention also encompasses polynucleotides which encode NTRAN.
  • the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ED NO:9-16, which encodes NTRAN.
  • the polynucleotide sequences of SEQ ID NO:9-16 as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occu ⁇ ences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
  • the invention also encompasses a variant of a polynucleotide sequence encoding NTRAN.
  • such a variant polynucleotide sequence wiU have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding NTRAN.
  • a particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:9- 16 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:9-16. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of NTRAN.
  • a polynucleotide variant of the invention is a spUce variant of a polynucleotide sequence encoding NTRAN.
  • a spUce variant may have portions which have significant sequence identity to the polynucleotide sequence 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 spUcing of exons during mRNA processing.
  • a spUce variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50% polynucleotide sequence identity to the polynucleotide sequence encoding NTRAN over its entire length; however, portions of the spUce 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 sequence encoding NTRAN.
  • a polynucleotide comprising a sequence of SEQ ID NO: 16 is a spUce variant of a polynucleotide comprising a sequence of SEQ ID NO: 10. Any one of the spUce variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of NTRAN.
  • nucleotide sequences which encode NTRAN and its variants are generaUy capable of hybridizing to the nucleotide sequence of the nataraUy occurring NTRAN under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding NTRAN or its derivatives possessing a substantiaUy different codon usage, e.g., inclusion of non- nataraUy 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.
  • RNA transcripts having more desirable properties such as a greater half-Ufe, than transcripts produced from the nataraUy occurring sequence.
  • the invention also encompasses production of DNA sequences which encode NTRAN and NTRAN derivatives, or fragments thereof, entirely by synthetic chemistry.
  • the synthetic sequence may be inserted into any of the many available expression vectors and ceU systems using reagents weU known in the art.
  • synthetic chemistry may be used to introduce mutations into a sequence encoding NTRAN or any fragment thereof.
  • polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NO:9-16 and fragments thereof under various conditions of stringency.
  • 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 (AppUed Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE ampUfication system (Life Technologies, Gaithersburg MD).
  • 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 (AppUed Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (AppUed Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are weU known in the art. (See, e.g., Ausubel, F.M. (1997) Short Protocols in Molecular Biology, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biology and Biotechnology. Wiley VCH, New York NY, pp. 856-853.)
  • the nucleic acid sequences encoding ISTTRAN may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • restriction-site PCR uses universal and nested primers to ampUfy unknown sequence from genomic DNA within a cloning vector.
  • Another method, inverse PCR uses primers that extend in divergent directions to ampUfy unknown sequence from a circularized template.
  • the template is derived from restriction fragments comprising a known genomic locus and su ⁇ ounding sequences.
  • a third method, capture PCR involves PCR ampUfication of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
  • multiple restriction enzyme digestions an. Ugations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR. Other methods which may be used to retrieve unknown sequences are known in the art.
  • Biosciences, Beverly 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.
  • Ubraries When screening for fuU length cDNAs, it is preferable to use Ubraries that have been size-selected to include larger cDNAs. In addition, random-primed Ubraries, which often include sequences containing the 5' regions of genes, are preferable for situations in which an oUgo d(T) Ubrary does not yield a fall-length cDNA. Genomic Ubraries 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.
  • capiUary 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/Ught intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, AppUed 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.
  • polynucleotide sequences 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 DNA sequences which encode substantiaUy the same or a functionaUy equivalent amino acid sequence maybe produced and used to express NTRAN.
  • nucleotide sequences of the present 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 oUgonucleotides may be used to engineer the nucleotide sequences.
  • oUgonucleotide- mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce spUce variants, and so forth.
  • the nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDESfG (Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of NTRAN, such as its biological or enzymatic activity or its abihty to bind to other molecules or compounds.
  • MOLECULARBREEDESfG Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797
  • DNA shuffling is a process by which a Ubrary of gene variants is produced using PCR-mediated recombination of gene fragments. The Ubrary is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening.
  • 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.
  • 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 nataraUy occurring genes in a directed and controUable manner.
  • sequences encoding NTRAN maybe synthesized, in whole or in part, using chemical methods weU known in the art.
  • chemical methods See, e.g., Caruthers, M-H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232.
  • NTRAN itself or a fragment thereof may be synthesized using chemical methods.
  • peptide synthesis can be performed using various solution-phase or sohd-phase techniques.
  • Automated synthesis may be achieved using the ABI 431A peptide synthesizer (AppUed 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 nataraUy occurring polypeptide.
  • the peptide may be substantiaUy purified by preparative high performance Uquid chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421.)
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, supra, pp. 28-53.)
  • the nucleotide sequences 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 polynucleotide sequences encoding NTRAN. Such elements may vary in their strength and specificity.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding NTRAN. Such signals include the ATG initiation codon and adjacent sequences, e.g.
  • Methods which are weU known to those skilled in the art may be used to construct expression vectors containing sequences encoding NTRAN and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biology, John Wiley & Sons, New York NY, ch. 9, 13, and 16.)
  • a variety of expression vector/host systems may be utihzed to contain and express sequences 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., cauUflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal ceU systems.
  • 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.
  • Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for deUvery of nucleotide sequences to the targeted organ, tissue, or ceU population.
  • cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding NTRAN.
  • routine cloning, subcloning, and propagation of polynucleotide sequences encoding NTRAN can be achieved using a multifunctional E. coU vector such as PBLUESCRIPT (Stratagene, La JoUa CA) or PSPORT1 plasmid (Life Technologies).
  • Ligation of sequences 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.
  • these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
  • vectors which direct high level expression of NTRAN may be used.
  • 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 may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH promoters
  • such vectors direct either the secretion or intraceUular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
  • Plant systems may also be used for expression of NTRAN. Transcription of sequences encoding NTRAN maybe 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. 3:17-311). Alternatively, plant promoters such as the smaU subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; BrogUe, R. et al. (1984) Science 224:838-843; and Winter, J. et al.
  • a number of viral-based expression systems may be utilized.
  • sequences encoding NTRAN may be Ugated 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.
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammaUan host ceUs.
  • SV40 or EBV- based vectors may also be used for high-level protein expression.
  • Human artificial chromosomes (HACs) may also be employed to dehver larger fragments of
  • HACs of about 6 kb to 10 Mb are constructed and delivered via conventional deUvery methods (Uposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J. et al. (1997) Nat. Genet. 15:345- 355.) For long term production of recombinant proteins in mammaUan systems, stable expression of
  • NTRAN in ceU lines is preferred.
  • sequences encoding NTRAN can be transformed into ceU lines using expression vectors which may contain viral origins of repUcation and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector.
  • ceUs maybe 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 successfuUy 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 phosphoribosyltransferase genes, for use in tk and apr ceUs, respectively. (See, e.g., Wigler, M. et al. (1977) CeU 11:223-232; Lowy, I. et al. (1980) CeU 22:817-823.) Also, antimetaboUte, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate
  • neo confers resistance to the aminoglycosides neomycin and G-418
  • als sad pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
  • Additional selectable genes have been described, e.g., trpB and hisD, which alter ceUular requirements for metaboUtes.
  • Visible markers e.g., anmocyanins, green fluorescent proteins (GFP; Clontech), ⁇ glucuronidase and its substrate ⁇ -glucuronide, or luciferase and its subsfrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, CA. (1995) Methods Mol. Biol. 55:121-131.)
  • marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
  • sequence encoding NTRAN is inserted within a marker gene sequence, transformed ceUs cont-uning sequences encoding NTRAN can be identified by the absence of marker gene function.
  • 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.
  • host ceUs that contain the nucleic acid sequence encoding NTRAN and that express NTRAN may be identified by a variety of procedures known to those of skiU in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR ampUfication, 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.
  • ELISAs enzyme-linked immunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated ceU sorting
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding NTRAN include oUgolabeUng, nick translation, end-labeling, or PCR ampUfication using a labeled nucleotide.
  • the sequences encoding NTRAN, or any fragments thereof maybe cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
  • T7, T3, or SP6 an appropriate RNA polymerase
  • Suitable reporter molecules or labels which may be used for ease of detection include radionucUdes, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as weU as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host ceUs transformed with nucleotide sequences 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 may be secreted or retained intraceUularly depending on the sequence and/or the vector used.
  • 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.
  • a host ceU strain maybe chosen for its abihty to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylatioi Upidation, 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.
  • ATCC American Type Culture CoUection
  • Manassas VA American Type Culture CoUection
  • natural, modified, or recombinant nucleic acid sequences encoding NTRAN may be Ugated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric NTRAN protein cont--ining a heterologous moiety that can be recognized by a commerciaUy available antibody may faciUtate the screening of peptide Ubraries for inhibitors of NTRAN activity.
  • Heterologous protein and peptide moieties may also faciUtate 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 i-mmunoaffinity 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 may be cleaved away from the heterologous moiety foUowing purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10).
  • a variety of commerciaUy available kits may also be used to faciUtate expression and purification of fusion proteins.
  • synthesis of radiolabeled NTRAN may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35 S-met-hionine.
  • NTRAN of the present invention or fragments thereof may be used to screen for compounds that specificaUy bind to NTRAN.
  • At least one and up to a pluraUty of test compounds may be screened for specific binding to NTRAN.
  • test compounds include antibodies, oUgonucleotides, proteins (e.g., receptors), or smaU molecules.
  • the compound thus identified is closely related to the natural Ugand of
  • NTRAN e.g., a Ugand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner.
  • the compound can be closely related to the natural receptor to which NTRAN binds, or to at least a fragment of the receptor, e.g., the Ugand binding site. In either case, the compound can be rationaUy designed using known techniques. In one embodiment, screening for these compounds involves producing appropriate ceUs which express NTRAN, either as a secreted protein or on the ceU membrane.
  • ceUs include ceUs from mammals, yeast, Drosophila, or K cpU. 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.
  • the assay may comprise the steps of combining at least one test compound with NTRAN, either in solution or affixed to a soUd support, and detecting the binding of NTRAN to the compound.
  • the assay may detect or measure binding of a test compound in the presence of a labeled competitor. AdditionaUy, the assay maybe carried out using ceU-free preparations, chemical Ubraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a soUd support.
  • NTRAN of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of NTRAN.
  • Such compounds may include agonists, antagonists, or partial or inverse agonists.
  • 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.
  • 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 pluraUty of test compounds maybe screened.
  • polynucleotides encoding NTRAN or their mammaUan homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) ceUs.
  • ES embryonic stem
  • 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.)
  • 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 phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292).
  • the vector integrates into the corresponding region of the host genome by homologous recombination.
  • 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 ceU blastocysts such as those from the C57BL/6 mouse strain.
  • the blastocysts are surgicaUy transfe ⁇ ed 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 'Tcnockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease.
  • pigs pigs
  • transgenic animals mice or rats
  • 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 blastalae, and the blastalae 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.
  • NTRAN overexpressing NTRAN
  • 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).
  • NTRAN neurotransmission-associated proteins
  • examples of tissues expressing NTRAN are lung tamor and B lymphoblast ceUs and also can be found in Table 6.
  • NTRAN appears to play a role in autoimmune/inflammatory, cardiovascular, neurological, developmental, ceU proUferative, including cancer, transport, psychiatric, metabohc, and endocrine disorders.
  • NTRAN appears to play a role in autoimmune/inflammatory, cardiovascular, neurological, developmental, ceU proUferative, including cancer, transport, psychiatric, metabohc, and endocrine disorders.
  • 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.
  • disorders include, but are not limited to, an autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, aUergies, ankylosing spondyUtis, 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 melUtas, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetaUs, erythema nodosum,
  • AIDS acquired immuno
  • phencycUdine dependence phobia, posttraumatic stress disorder, schizoaffective disorder, schizoid personaUty 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, hyperadrenaUsm, hypoadrenaUsm, hyperparathyroidism, hypoparathyroidism, hypercholesterolemia, hyperthyroidism, hypoglycemia, hypothyroidism, hyperUpidemia, hyperhpemia, Upid myopathies, Upodystrophies, lysosomal storage diseases, mannos
  • 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 Umited to, those described above.
  • 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.
  • an agonist which modulates the activity of NTRAN may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of NTRAN including, but not Umited to, those Usted above.
  • 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 Umited to, those autoimmune/inflammatory, cardiovascular, neurological, developmental, ceU proUferative, including cancer, transport, psychiatric, metabohc, and endocrine disorders described above.
  • an antibody which specificaUy binds NTRAN may be used directly as an antagonist or indirectly as a targeting or deUvery mechanism for bringing a pharmaceutical agent to ceUs or tissues which express NTRAN.
  • 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 -limited to, those described above.
  • any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skiU 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.
  • purified NTRAN may be used to produce antibodies or to screen Ubraries 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 Umited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression Ubrary.
  • NeutraUzing antibodies i.e., those which inhibit dimer formation
  • Single chain antibodies e.g., from camels or Uamas
  • Single chain antibodies maybe potent enzyme inhibitors and may have advantages in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S. (2001) J.
  • 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 oligopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include, but are not Umited 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.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are especiaUy preferable.
  • oUgopeptides, peptides, or fragments used to induce antibodies it is preferred that the oUgopeptides, 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 oUgopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the nataral protein. Short stretches of NTRAN amino acids maybe fused with those of another protein, such as KLH, and antibodies to the chimeric molecule maybe 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 EBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Natare 256:495-497; Kozbor, D. et al. (1985) J.
  • chimeric antibodies such as the spUcing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used.
  • spUcing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity.
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin Ubraries or panels of highly specific binding reagents as disclosed in the Uteratare. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
  • Antibody fragments which contain specific binding sites for NTRAN may also be generated.
  • such fragments include, but are not limited to, F(ab : ) 2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression Ubraries maybe constructed to aUow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.)
  • Various immunoassays may be used for screening to identify antibodies having the desired specificity.
  • K a is defined as the molar concentration of NTRAN-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions.
  • K a association constant
  • High-affinity antibody preparations with K a ranging from about 10 9 to 10 12 L/mole are preferred for use in immunoassays in which the NTRAN- antibody complex must withstand rigorous manipulations.
  • Low-affinity antibody preparations with K a ranging from about 10 6 to 10 7 L/mole are preferred 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).
  • polyclonal antibody preparations may be further evaluated to determine the quaUty and suitabiUty of such preparations for certain downstream appUcations.
  • 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 quaUty and usage in various appUcations, are generaUy available. (See, e.g., Catty, supra, and CoUgan et al. supra.)
  • the polynucleotides encoding NTRAN may be used for therapeutic purposes.
  • modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified oUgonucleotides) to the coding or regulatory regions of the gene encoding NTRAN.
  • complementary sequences or antisense molecules DNA, RNA, PNA, or modified oUgonucleotides
  • antisense oUgonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding NTRAN. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press Inc., Totawa NJ.)
  • 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(3):469-475; and Scanlon, K.J. et al.
  • Antisense sequences can also be introduced intraceUularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors.
  • viral vectors such as retrovirus and adeno-associated virus vectors.
  • Other gene dehvery mechanisms include Uposome-derived systems, artificial viral envelopes, and other systems known in the art.
  • Rossi J.J. (1995) Br. Med. BuU. 51(l):217-225; Boado, R.J. et al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al. (1997) Nucleic Acids Res. 25(14):2730-2736.
  • polynucleotides encoding NTRAN maybe used for somatic or germline gene therapy.
  • Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-Xl disease characterized by X- linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C et al.
  • SCID severe combined immunodeficiency
  • ADA adenosine deaminase
  • diseases or disorders caused by deficiencies in ⁇ TRA ⁇ are treated by constructing mammaUan expression vectors encoding ⁇ TRA ⁇ and introducing these vectors by mechanical means into ⁇ TRA ⁇ -deficient ceUs.
  • Mechanical transfer technologies for use with ceUs in vivo or ex vitro include (i) direct D A microinjection into individual ceUs, (n) ballistic gold particle deUvery, (iii) Uposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of D ⁇ A transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev. Biochem. 62:191-217; Ivies, Z.
  • Expression vectors that may be effective for the expression of ⁇ TRA ⁇ include, but are not
  • PCD ⁇ A 3.1, EPTTAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (InvitiOgen, Carlsbad CA), PCMV-SCRTPT, PCMV-TAG, PEGSH/PERV (Stratagene, La JoUa CA), and PTET-OFF, PTET-O ⁇ , PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
  • ⁇ TRA ⁇ maybe expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes), (u) an inducible promoter (e.g., the tetracycUne-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. ⁇ atl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.MN. and H.M. Blau (1998) Curr. Opin. Biotechnol.
  • a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -act
  • Uposome transformation kits e.g., the PERFECT LIPID TRANSFEC ⁇ ON KIT, available from Invitrogen
  • aUow one with ordinary skiU in the art to deUver polynucleotides to target ceUs in culture and require minimal effort to optimize experimental parameters.
  • transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J. 1:841-845).
  • the introduction of DNA to primary ceUs requires modification of these standardized mammalian transfection protocols.
  • diseases or disorders caused by genetic defects with respect to NTRAN expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding NTRAN under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (n) appropriate RNA packaging signals, and (in) a Rev-responsive element (RRE) along with additional retrovirus cz-s-acting RNA sequences and coding sequences required for efficient vector propagation.
  • Retrovirus vectors e.g., PFB and PFBNEO
  • Retrovirus vectors are commerciaUy available (Stratagene) and are based onpubUshed data (Riviere, I. et al. (1995) Proc. Natl. Acad.
  • 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 A.D. MiUer (1988) J. Virol. 62:3802-3806; DuU, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey, R.
  • VSVg vector producing ceU line
  • U.S. Patent No. 5,910,434 to Rigg discloses a method for obtaining retrovirus packaging ceU lines and is hereby incorporated by reference. Propagation of retrovirus 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 skiUed in the art of gene therapy and have been weU documented (Ranga, U. et al. (1997) J. Virol. 71:7020-7029; Bauer, G.
  • an adenovirus-based gene therapy dehvery system is used to deUver 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.
  • RepUcation defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). PotentiaUy useful adenoviral vectors are described in U.S. Patent No.
  • a herpes-based, gene therapy dehvery system is used to deUver 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 may be especiaUy valuable for introducing NTRAN to ceUs of the central nervous system, for which HSV has a tropism.
  • HSV herpes simplex virus
  • HSV herpes simplex virus
  • a repUcation-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 transferred to a ceU under the control of the appropriate promoter for purposes including human gene therapy.
  • HSV vectors see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and Xu, H et al. (1994) Dev. Biol. 163:152-161, hereby incorporated by reference.
  • herpesvirus sequences 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 herpesvims, and the infection of ceUs with herpesvirus are techniques weU known to those of ordinary skiU in the art.
  • an alphavirus (positive, single-stranded RNA virus) vector is used to deUver polynucleotides encoding NTRAN to target ceUs.
  • SFV Semliki Forest Virus
  • SFV Semliki Forest Virus
  • 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).
  • enzymatic activity e.g., protease and polymerase.
  • 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.
  • alphavirus infection is typicaUy associated with ceU lysis within a few days
  • the abihty to estabUsh a persistent infection in hamster normal kidney ceUs (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic repUcation of alphaviruses can be altered to suit the needs of the gene therapy appUcation (Dryga, S.A. et al. (1997) Virology 228:74-83).
  • the wide host range of alphaviruses wiU aU ow 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.
  • OUgonucleotides derived from the transcription initiation site may also be employed to inhibit gene expression.
  • 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. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futara PubUshing, 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.
  • engineered hammerhead motif ribozyme molecules may specificaUy and efficiently catalyze endonucleolytic cleavage of sequences encoding NTRAN.
  • RNA target Specific ribozyme cleavage sites within any potential RNA target are initiaUy identified by scanning the target molecule for ribozyme cleavage sites, including the foUowing sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibiUty to hybridization with complementary oUgonucleotides using ribonuclease protection assays.
  • RNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemicaUy synthesizing oUgonucleotides such as soUd phase phosphoramidite chemical synthesis.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding NTRAN. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6.
  • these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into ceU lines, ceUs, or tissues.
  • RNA molecules may be modified to increase intracellular stabiUty and half-life. Possible modifications include, but are not Umited 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.
  • 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 Umited to, oUgonucleotides, antisense oUgonucleotides, triple hehx-forming oUgonucleotides, 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.
  • 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.
  • At least one, and up to a pluraUty, of test compounds maybe 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 Ubrary of nataraUy-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide; and selection from a library of chemical compounds created combinatoriaUy 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 permeabiUzed 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 maybe 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.
  • 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, MX. et al. (2000) Biochem. Biophys. Res. Commun.
  • a particular embodiment of the present invention involves screening a combinatorial Ubrary of oUgonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oUgonucleotides) 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 available and equaUy suitable for use in vivo, in vitro, and ex vivo.
  • oUgonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oUgonucleotides
  • vectors may be introduced into stem ceUs taken from the patient and clonaUy propagated for autologous transplant back into that same patient. Dehvery by transfection, by Uposome injections, or by polycationic amino polymers may be achieved using methods which are weU known in the art. (See, e.g., Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:462-466.)
  • any of the therapeutic methods described above may be appUed 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 PubUshing, Easton PA).
  • Such compositions may consist of NTRAN, antibodies to NTRAN, and mimetics, agonists, antagonists, or inhibitors of NTRAN.
  • compositions utilized in this invention 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 maybe prepared in Uquid or dry powder form. These compositions are generaUy aerosoUzed 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.
  • Pulmonary dehvery has the advantage of 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 determination of an effective dose is weU within the capability of those skilled in the art.
  • SpeciaUzed forms of compositions maybe prepared for direct intraceUular deUvery of macromolecules comprising NTRAN or fragments thereof.
  • hposome preparations containing a ceU-impermeable macromolecule may promote ceU fusion and intraceUular deUvery of the macromolecule.
  • NTRAN or a fragment thereof may be joined to a short cationic N- terminal portion from the H-TV 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).
  • the therapeuticaUy effective dose can be estimated initiaUy either in ceU cultare assays, e.g., of neoplastic ceUs, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs.
  • ceU cultare assays e.g., of neoplastic ceUs
  • animal models such as mice, rats, rabbits, dogs, monkeys, or pigs.
  • An animal model may also be used to dete ⁇ nine the appropriate concentration range and route of administration. Such information can then be used to dete ⁇ nine 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 ameliorates 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 ED 50 (the dose therapeuticaUy effective in 50% of the population) or LD 50 (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 LD 50 /ED 50 ratio.
  • Compositions which exhibit large therapeutic indices are prefe ⁇ ed.
  • 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 ED 50 with Uttle or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient,
  • the exact dosage wiU be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting compositions maybe administered every 3 to 4 days, every week, or biweekly depending on the half-Ufe and clearance rate of the particular formulation. Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration.
  • antibodies which specificaUy bind NTRAN may be used for the diagnosis of disorders characterized by expression of NTRAN, or in assays to monitor patients being treated 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.
  • NTRAN neurotrophic factor
  • ELISAs e.g., IL-12
  • RIAs e.g., IL-12
  • FACS fluorescence-activated cell sorting
  • NTRAN expression normal or standard values for NTRAN expression are estabhshed by combining body fluids or ceU extracts taken from normal mammaUan subjects, for example, human subjects, with antibodies to NTRAN under conditions suitable for complex formation. The amount of standard complex formation maybe quantitated by various methods, such as photometric means. Quantities of NTRAN expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values estabhshes the parameters for diagnosing disease.
  • the polynucleotides encoding NTRAN may be used for diagnostic purposes.
  • the polynucleotides which may be used include ohgonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides maybe used to detect and quantify gene expression in biopsied tissues in which expression of NTRAN may be correlated with disease.
  • the diagnostic assay may be used to determine absence, presence, and excess expression of NTRAN, and to monitor regulation of NTRAN levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, 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 ampUfication wiU determine whether the probe identifies only nataraUy occurring sequences encoding NTRAN, aUeUc 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 ID NO:9-16 or from genomic sequences including promoters, enhancers, and introns of the NTRAN gene.
  • Means for producing specific hybridization probes for DNAs encoding NTRAN include the cloning of polynucleotide sequences encoding NTRAN or NTRAN derivatives into vectors for the production of mRNA probes.
  • 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 radionucUdes such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • Polynucleotide sequences encoding NTRAN may be used for the diagnosis of disorders associated with expression of NTRAN.
  • disorders include, but are not Umited to, an autoii-r-tmune/mflammatory disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, aUergies, ankylosing spondyUtis, 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 melhtas, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetaUs, erythema nodosum
  • the polynucleotide sequences 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-like assays; and in microarrays utilizing fluids or tissues from patients to detect altered NTRAN expression.
  • Southern or northern analysis dot blot, or other membrane-based technologies
  • PCR technologies in PCR technologies
  • dipstick, pin, and multiformat ELISA-like assays and in microarrays utilizing fluids or tissues from patients to detect altered NTRAN expression.
  • microarrays utilizing fluids or tissues from patients to detect altered NTRAN expression.
  • the nucleotide sequences encoding NTRAN maybe useful in assays that detect the presence of associated disorders, particularly those mentioned above.
  • the nucleotide sequences encoding NTRAN maybe labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding 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.
  • a normal or standard profile for expression is estabUshed. 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 ampUfication.
  • 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 maybe 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.
  • hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
  • the results obtained from successive assays maybe used to show the efficacy of treatment over a period ranging from several days to months.
  • 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.
  • oUgonucleotides designed from the sequences encoding NTRAN may involve the use of PCR. These ohgomers may be chemicaUy synthesized, generated enzymaticaUy, or produced in vitro.
  • OUgomers wiU preferably contain a fragment of a polynucleotide encoding NTRAN, or a fragment of a polynucleotide complementary to the polynucleotide encoding NTRAN, and wiU be employed under optimized conditions for identification of a specific gene or condition. OUgomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
  • oUgonucleotide primers derived from the polynucleotide sequences encoding NTRAN maybe used to detect single nucleotide polymorphisms (SNPs). SNPs are substitations, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans. Methods of SNP detection include, but are not Umited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods.
  • SSCP single-stranded conformation polymorphism
  • fSSCP fluorescent SSCP
  • oligonucleotide primers derived from the polynucleotide sequences encoding NTRAN are used to ampUfy DNA using the polymerase chain reaction (PCR).
  • the DNA maybe 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.
  • the oUgonucleotide primers are fluorescently labeled, which aUows detection of the ampUmers 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.
  • SNPs maybe detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
  • SNPs may be used to study the genetic basis of human disease. For example, at least 16 common SNPs have been associated with non-insulin-dependent diabetes meUitas. 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 co ⁇ elated 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 Ufe-threatening toxicity.
  • 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.
  • NTRAN NTRAN-derived neurotrophic factor receptor mediated endometrial fibroblasts
  • Methods which may also be used to quantify the expression of NTRAN include radiolabeling or biotinylating nucleotides, coampUfication of a control nucleic acid, and interpolating results from standard curves.
  • biotinylating nucleotides e.g., radiolabeling or biotinylating nucleotides, coampUfication 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.
  • the speed of quantitation of multiple samples maybe accelerated by mnning the assay in a high-throughput format where the ohgomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
  • oUgonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as elements on a microa ⁇ ay.
  • the microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below.
  • the microa ⁇ ay may also be used to identify genetic variants, mutations, and polymorphisms. This information may be used to dete ⁇ nine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease.
  • 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.
  • therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
  • NTRAN fragments of NTRAN, or antibodies specific for NTRAN may be used as elements on a microa ⁇ ay.
  • the microarray may be used to monitor or measure protein-protein interactions, drug-target interactions, and gene expression profiles, as described above.
  • a particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or 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. (See Seilhamer et al., "Comparative Gene Transcript Analysis," U.S. Patent No.
  • a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totahty of transcripts or reverse transcripts of a particular tissue or ceU type.
  • the hybridization takes place in Mgh-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a pluraUty of elements on a microa ⁇ ay.
  • the resultant transcript image would provide a profile of gene activity.
  • Transcript images may be 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 preclinical 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 signatares, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and NX. Anderson (2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein).
  • 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 signatares 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 quality signatare. 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 normaUze the rest of the expression data. The normaUzation procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant signatare aids in interpretation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatares which leads to prediction of toxicity.
  • the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound.
  • Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified.
  • the transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
  • proteome refers to the global pattern of protein expression in a particular tissue or ceU type.
  • 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.
  • 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 visuahzed 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 are compared to identify any changes in protein spot density related to the treatment.
  • 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 the present invention. In some cases, further sequence data may be obtained for definitive protein identification.
  • a proteomic profile may also be generated using antibodies specific for NTRAN to quantify the levels of NTRAN expression.
  • the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microa ⁇ ay to the sample and detecting the levels of protein bound to each a ⁇ ay element (Lueking, A. et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27.778-788).
  • Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.
  • Toxicant signatares 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 co ⁇ elation between transcript and protein abundances for some proteins in some tissues (Anderson, NX. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatares maybe useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile.
  • the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified. The amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
  • the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
  • Microarrays may be prepared, used, and analyzed using methods known in the art.
  • methods known in the art See, e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474J96; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT appUcation WO95/251116; Shalon, D. et al. (1995) PCT appUcation WO95/35505; HeUer, R.A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; and HeUer, M.J.
  • nucleic acid sequences encoding NTRAN may be used to generate hybridization probes useful in mapping the nataraUy 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.
  • 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 Ubraries.
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PI constructions or single chromosome cDNA Ubraries.
  • the nucleic acid sequences of the invention may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP).
  • RFLP restriction fragment length polymorphism
  • FISH Fluorescent in situ hybridization
  • 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.
  • NTRAN in another embodiment, can be used for screening Ubraries 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 soUd 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.
  • This method large numbers of different smaU test compounds are synthesized on a solid 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-neutrahzing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • 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 Umited to, such properties as the triplet genetic code and specific base pair interactions.
  • Incyte cDNAs were derived from cDNA Ubraries 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 denatarants, such as TR-ZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
  • 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).
  • RNA was provided with RNA and constructed the co ⁇ esponding cDNA Ubraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the
  • UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6.) Reverse transcription was initiated using oUgo d(T) or random primers. Synthetic oUgonucleotide adapters were Ugated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes.
  • the cDNA was size-selected (300- 1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis.
  • cDNAs were Ugated into compatible restriction enzyme sites of the polyUnker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE (Incyte Genomics), or pENCY (Incyte Genomics), or derivatives thereof.
  • Recombinant plasmids were transformed into competent E. coh ceUs including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Life Technologies.
  • 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.
  • Plasmids were resuspended in 0.1 ml of distiUed water and stored, with or without lyophiUzation, at 4 °C
  • plasmid DNA was ampUfied 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.
  • 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
  • cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supphed in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (AppUed Biosystems). Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (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 (reviewed in Ausubel, 1997, supra, unit 1.1). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VHI.
  • the polynucleotide sequences derived from Incyte cDNAs were validated by removing vector, linker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis.
  • the Incyte cDNA sequences or translations thereof were then queried against a selection of 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; and HMM-based protein domain databases such as SMART (Schultz et al.
  • GenBank primate rodent, mammalian, vertebrate, and eukaryote databases
  • BLOCKS, PRINTS DOMO
  • PRODOM PRODOM
  • PROTEOME databases with sequence
  • HMM is a probabilistic approach which analyzes consensus primary structures of gene families. See, for example, Eddy, S.R. (1996) Cu ⁇ . 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 fuU length polynucleotide sequences.
  • 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 pro rams based on Phred, Phrap, and Consed, and cDNA assemblages were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were translated to derive the co ⁇ esponding fuU length polypeptide sequences. Alternatively, a polypeptide of the invention 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,
  • HMM-based protein family databases such as PFAM; and HMM-based protein domain databases such as SMART.
  • FuU length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence aUgnments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aUgned 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 probabiUty value, the greater the identity between two sequences).
  • Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94, and Burge, C. and S. Karlin (1998) Cu ⁇ . 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.
  • 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.
  • the encoded polypeptides were analyzed by querying 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.
  • Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri pubhc databases.
  • Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to co ⁇ ect e ⁇ ors 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.
  • Incyte cDNA coverage was available, this information was used to correct 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 HI. Alternatively, full length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences.
  • Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example HI were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible splice variants that were subsequently confirmed, edited, or extended to create a fall length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by transitivity.
  • Inco ⁇ ect exons predicted by Genscan were co ⁇ ected 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.
  • 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.
  • HSPs high-scoring segment pairs
  • 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.
  • sequences which were used to assemble SEQ ID NO:9-16 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 ED NO:9-16 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 aU sequences of that cluster, including its particular SEQ ED NO:, to that map location.
  • 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
  • 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.
  • centiMorgan cM
  • centiMorgan 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
  • 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. (See, e.g., Sambrook, supra, ch. 7; Ausubel (1995) supra, ch. 4 and 16.)
  • 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 normaUzed value between 0 and 100, and is calculated as foUows: the BLAST score is multipUed 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 quaUty in a BLAST aUgnment. 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.
  • polynucleotide sequences 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 IE). Each cDNA sequence is derived from a cDNA Ubrary 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; genitaUa, female; genitaUa, male; germ ceUs; hemic and immune system; Uver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract.
  • the number of Ubraries in each category is counted and divided by the total number of libraries across aU categories.
  • each human tissue is classified into one of the foUowing disease/condition categories: cancer, ceU line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of Ubraries in each category is counted and divided by the total number of Ubraries across aU categories. The resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding NTRAN.
  • cDNA sequences and cDNA Ubrary/tissue information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA). VIII.
  • FuU length polynucleotide sequences were also produced by extension of an appropriate fragment of the fuU length molecule using oUgonucleotide 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.
  • the reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg 2+ , (NI ⁇ SO ⁇ and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), 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.
  • 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 ⁇ l PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in IX TE and 0.5 ⁇ l 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 H (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
  • a 5 ⁇ l to 10 ⁇ l aUquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to determine which reactions were successful in extending the sequence.
  • the extended nucleotides were desalted and concentrated, transfe ⁇ ed to 384-weU plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to reUgation into pUC 18 vector (Amersham Pharmacia Biotech).
  • CviJI cholera virus endonuclease Molecular Biology Research, Madison WI
  • sonicated or sheared prior to reUgation into pUC 18 vector
  • 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 Ugase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fiU-in restriction site overhangs, and transfected into competent E. coh 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 Uquid media. The cells were lysed, and DNA was ampUfied by PCR using Taq DNA polymerase
  • 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 reamphfied using the same conditions as described above.
  • fuU length polynucleotide sequences are verified using the above procedure or are used to obtain 5' regulatory sequences using the above procedure along with oUgonucleotides designed for such extension, and an appropriate genomic Ubrary.
  • SNPs single nucleotide polymorphisms
  • LIFESEQ database Incyte Genomics
  • Sequences from the same gene were clustered together and assembled as described in Example HI, 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 e ⁇ ors by requiring a minimum Phred quality score of 15, and removed sequence aUgnment e ⁇ ors and e ⁇ ors resulting from improper trimming of vector sequences, chimeras, and spUce variants.
  • 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 deciualan, 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 aUeUc variance in this population were not further tested in the other three populations.
  • Hybridization probes derived from SEQ ID NO:9-16 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oUgonucleotides, consisting of about 20 base pairs, is specificaUy described, essentiaUy the same procedure is used with larger nucleotide fragments. OUgonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each ohgomer, 250 Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
  • the labeled oligonucleotides are substantiaUy purified using a SEPHADEX G-25 superfine size exclusion dextranbead column (Amersham Pharmacia Biotech). An aliquot containing 10 7 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 ⁇ , Eco RI, Pst I, Xba I, or Pvu H (DuPont NEN).
  • the DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran 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 visuahzed using autoradiography or an alternative imaging means and compared. XI.
  • Microarrays The linkage or synthesis of array elements upon a microarray can be achieved utilizing photoUthography, piezoelectric printing (ink-jet printing, See, e.g., Baldeschweiler, supra.), mechanical microspotting technologies, and derivatives thereof.
  • the substrate in each of the aforementioned technologies should be uniform and soUd with a non-porous surface (Schena (1999), supra).
  • Suggested substrates include sihcon, siUca, glass sUdes, glass chips, and sihcon wafers.
  • a procedure analogous to a dot or slot blot may also be used to a ⁇ ange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
  • a typical a ⁇ ay may be produced using available methods and machines weU known to those of ordinary skiU in the art and may contain any appropriate number of elements. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; MarshaU, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)
  • FuU length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oUgomers thereof may comprise the elements of the microarray. Fragments or oligomers 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.
  • a fluorescence scanner is used to detect hybridization at each a ⁇ ay element.
  • laser desorbtion and mass spectrometry maybe used for detection of hybridization.
  • the degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microa ⁇ ay may be assessed.
  • microa ⁇ ay preparation and usage is described in detail below.
  • 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-transcriptase, 0.05 pg/ ⁇ l ohgo-(dT) primer (21mer), IX first strand buffer, 0.03 units/ ⁇ l RNase inhibitor, 500 ⁇ M dATP, 500 ⁇ M dGTP, 500 ⁇ M dTTP, 40 ⁇ M dCTP, 40 ⁇ M dCTP-Cy3 (BDS) or dCTP-Cy5 (-Amersham Pharmacia Biotech).
  • the reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A) + RNA with GEMBRIGHT kits (Incyte).
  • Specific control poly(A) + RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.
  • 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 ⁇ l 5X SSC/0.2% SDS.
  • Sequences of the present invention are used to generate a ⁇ ay elements.
  • Each array element is amplified from bacterial ceUs containing vectors with cloned cDNA inserts.
  • PCR ampUfication uses primers complementary to the vector sequences flanking the cDNA insert.
  • Array elements are ampUfied in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g.
  • AmpUfied a ⁇ ay elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech). Purified array elements are immobilized on polymer-coated glass sUdes. Glass microscope sUdes (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments. Glass slides are etched in 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester PA), washed extensively in distihed water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated sUdes are cured in a 110°C oven.
  • a ⁇ ay elements are appUed to the coated glass substrate using a procedure described in U.S.
  • Patent No. 5,807,522 incorporated herein by reference.
  • 1 ⁇ l of the a ⁇ ay element DNA, at an average concentration of 100 ng/ ⁇ l, is loaded into the open capiUary printing element by a high-speed robotic apparatus.
  • the apparatus then deposits about 5 nl of array element sample per sUde.
  • Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene).
  • Microarrays are washed at room temperature once in 0.2% SDS and three times in distilled water.
  • Non-specific binding sites are blocked by incubation of microa ⁇ ays 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%
  • PBS phosphate buffered saline
  • Hybridization reactions contain 9 ⁇ l of sample mixture consisting of 0.2 ⁇ g each of Cy3 and
  • the chamber containing the a ⁇ ays is incubated for about 6.5 hours at 60° C
  • the arrays are washed for 10 min at 45° C in a first wash buffer (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 Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5.
  • the excitation laser Ught is focused on the a ⁇ ay using a 20X microscope objective (Nikon, Inc., Melville NY).
  • the sUde 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 a ⁇ ay used in the present example is scanned with a resolution of 20 micrometers:
  • a mixed gas multiline laser excites the two fluorophores sequentiaUy. Emitted Ught is spUt, based on wavelength, into two photomultipUer tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) co ⁇ esponding to the two fluorophores. Appropriate filters positioned between the a ⁇ ay and the photomultipUer tabes are used to filter the signals.
  • the emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5.
  • Each a ⁇ ay 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 caUbrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration.
  • a specific location on the a ⁇ ay contains a complementary DNA sequence, aUowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000.
  • the caUbration is done by labeling samples of the caUbrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
  • the output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc., Norwood MA) 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 co ⁇ ected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore 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 co ⁇ esponding to the average intensity of the signal.
  • the software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
  • Expression Peripheral blood of a 29-year-old male suffering from Hodgkin's disease was obtained and used to prepare a B lymphoblast ceU line.
  • SEQ ID NO: 15 showed differential expression in a B lymphoblast (Hodgkins) ceU line treated with Upopolysaccharide complexes versus untreated, as determined by microarray analysis. Lipopolysaccharide complexes eUcit a variety of inflammatory responses. Therefore, SEQ ED NO:15 is useful in treatment for autoimmune/i-nflammatory disorders.
  • SEQ ED NO: 16 showed differential expression in lung cancer tissue, as determined by microa ⁇ ay analysis.
  • 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.
  • Lung cancers are divided into four histopathologicaUy distinct groups. Three groups, including squamous ceU carcinoma and adenocarcinoma, are classified as non-smaU ceU lung cancers, whereas the fourth group is classified as smaU ceU lung cancer. CoUectively the non-smaU ceU lung cancers account for 70% of aU cases. Pair comparisons were performed in which tamor tissue was compared to normal tissue from the same donor.
  • SEQ ED NO: 16 The expression of SEQ ED NO: 16 was decreased by at least two-fold in moderately differentiated lung adenocarcinoma tissue, as compared to grossly uninvolved lung tissue, derived from a 60-year-old patient. This experiment indicates that SEQ ID NO: 16 is useful in diagnostic and disease staging assays for lung cancer and as a potential biological marker and therapeutic agent in the treatment of lung cancer.
  • Sequences complementary to the NTRAN-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of nataraUy occurring NTRAN.
  • oUgonucleotides comprising from about 15 to 30 base pairs is described, essentiaUy the same procedure is used with smaUer or with larger sequence fragments.
  • Appropriate oUgonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of NTRAN.
  • a complementary oUgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
  • a complementary oUgonucleotide is designed to prevent ribosomal binding to the NTRAN-encoding transcript.
  • NTRAN expression and purification of NTRAN is achieved using bacterial or vims-based expression systems.
  • cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
  • promoters include, but are not Umited to, the tip-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 (IPTG).
  • NTRAN in eukaryotic ceUs is achieved by infecting insect or mammaUan ceU lines with recombinant Autographica ca fornica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus.
  • AcMNPV Autographica ca fornica nuclear polyhedrosis virus
  • 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 Spodoptera frugiperda (Sf9) insect ceUs in most cases, or human hepatocytes, in some cases.
  • NTRAN is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, pe ⁇ nitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude ceU lysates.
  • GST a 26-kilodalton enzyme from Schistosoma aponicum, enables the purification of fusion proteins on immobihzed glutathione under conditions that maintain protein activity and antigenieity (Amersham Pharmacia Biotech).
  • the GST moiety can be proteolyticaUy cleaved from NTRAN at specificaUy engineered sites.
  • FLAG an 8-amino acid peptide, enables immunoa finity 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
  • NTRAN function is assessed by expressing the sequences encoding NTRAN at physiologicaUy elevated levels in mammaUan ceU cultare systems. cDNA is subcloned into a mammaUan expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human ceU line, for example, an endotheUal or hematopoietic ceU line, using either Uposome formulations or electroporation. 1-2 ⁇ g 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 nontransfected ceUs and is a reUable 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.
  • FCM Flow cytometry
  • 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.
  • NTRAN 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 nontransfected 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 skiU in the art. Expression of mRNA encoding NTRAN and other genes of interest can be analyzed by northern analysis or microarray techniques.
  • PAGE polyacrylamide gel electrophoresis
  • the NTRAN amino acid sequence is analyzed using LASERGENE software
  • oUgopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (AppUed 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.
  • MBS N-maleimidobenzoyl-N-hydroxysuccinimide ester
  • Rabbits are immunized with the oUgopeptide-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.
  • NataraUy occurring or recombinant NTRAN is substantiaUy purified by in-munoaffinity 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 Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
  • NTRAN 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.
  • NTRAN or biologicaUy active fragments thereof, are labeled with 125 I Bolton-Hunter reagent.
  • Bolton-Hunter reagent See, e.g., Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.
  • Candidate molecules previously a ⁇ ayed 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.
  • NTRAN molecules interacting with molecules interacting with NTRAN are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989) Natare 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 PATHCALLTNG 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 Ubraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
  • NTRAN activity can be demonstrated using an electrophysiological assay for ion conductance.
  • Capped NTRAN mRNA transcribed with T7 polymerase is injected into defolliculated 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 V. 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 HiU, AustraUa).
  • choline transporter activity or choline-transporter-like CTLl protein activity of NTRAN is deteirnined 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 [ 3 H]choline.
  • the ceUs are then filtered, and washed.
  • the amount of [ 3 H]chohne present in the ceUs is proportional to the activity of NTRAN in the ceUs (O'Regan, S. supra).
  • NTRAN activity measures the expression of NTRAN on the ceU surface.
  • cDNA encoding NTRAN is transfected into an appropriate mammaUan ceU line.
  • CeU surface proteins are labeled with biotin as described (de la Fuente, M.A. et al. (1997) Blood 90:2398-2405).
  • Immunoprecipitations are performed using NTRAN-specific antibodies, and immunoprecipitated samples are analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and inmiunoblotting techniques. The ratio of labeled immunoprecipitant to unlabeled immunoprecipitant is proportional to the amount of NTRAN expressed on the ceU surf ce.
  • an assay for NTRAN activity is based on a prototypical assay for hgand/receptor-mediated modulation of ceU proUferation. This assay measures the rate of DNA synthesis in Swiss mouse 3T3 ceUs.
  • a plasmid containing polynucleotides encoding NTRAN is added to quiescent 3T3 cultared ceUs using transfection methods weU known in the art.
  • the transiently transfected ceUs are then incubated in the presence of [ 3 H]thymidine, a radioactive DNA precursor molecule.
  • Varying amounts of NTRAN Ugand are then added to the cultared ceUs.
  • Incorporation of [ 3 H]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 Ugand concentration range is indicative of receptor activity.
  • One unit of activity per miUiUter is defined as the concentration of NTRAN producing a 50% response level, where 100% represents maximal incorporation of [ 3 H]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.)
  • 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.
  • a plasmid encoding fuU length NTRAN is transfected into a mammaUan ceU line (e.g., Chinese hamster ovary (CHO) or human embryonic kidney (HEK-293) ceU lines) using methods weU-known in the art.
  • a mammaUan ceU line e.g., Chinese hamster ovary (CHO) or human embryonic kidney (HEK-293) ceU lines
  • Transfected ceUs are grown in 12-weU trays in cultare medium for 48 hours, then the cultare medium is discarded, and the attached ceUs are gently washed with PBS. The ceUs are then incubated in cultare medium with or without Ugand for 30 minutes, then the medium is removed and ceUs lysed by treatment with 1 M perchloric acid.
  • 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 Ugand compared to those without Ugand are proportional to the amount of NTRAN present in the transfected ceUs.
  • the ceUs are grown in 24-weU plates containing lxlO 5 ceUs/weU and incubated with inositol-free media and [ 3 H]myoinositol, 2 mCi/weU, foi 48 hr.
  • the culture medium is removed, and the ceUs washed with buffer containing 10 mM LiCl foUowed by addition of Ugand.
  • 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 Uquid scintiUation. Changes in the levels of labeled inositol phosphate from ceUs exposed to Ugand compared to those without Ugand 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 fr- sforming a mammaUan 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 b-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.
  • ceUs are incubated for 48-72 hours after transformation under conditions appropriate for the ceU line to aUow expression and accumulation of NTRAN and b-galactosidase.
  • Transformed ceUs expressing b- galactosidase are stained blue when a suitable colorimetric substrate is added to the cultare 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 b-galactosidase sequences alone, are used as controls and tested in paraUel.
  • NTRAN neurotrophic factor
  • 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.
  • sodium can be replaced by choline or N-methyl-D-glucamine and chloride by gluconate, NO 3 , or SO (Kavanaugh, M.P. et al. (1992) J. Biol. Chem. 267:22007-22009).
  • NTRAN transport activity is assayed by measuring uptake of labeled substrates 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 KCl, 1 mM CaCla, 1 mM MgCl ⁇ 1 mM N- ⁇ HPO ⁇ 5 mM Hepes, 3.8 mM NaOH , 50 ⁇ g/ml gentamycin, pH 7.8) to aUow expression of NTRAN protein.
  • Oocytes are then transfe ⁇ ed to standard uptake medium (100 mM NaCl, 2 mM KCl, 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
  • substrates e.g., amino acids, sugars, drugs, and neurotransmitters
  • uptake is terminated by washing the oocytes three times in Na + -free medium, measuring the incorporated 3 H, and comparing with controls.
  • NTRAN activity is proportional to the level of internaUzed 3 H substrate.
  • NTRAN protein kinase (PK) activity is measured by phosphorylation of a protein substrate using gamma-labeled [ 32 P]-ATP and quantitation of the incorporated radioactivity using a gamma radioisotope counter.
  • NTRAN is incubated with the protein substrate, [ 32 P]-ATP, and an appropriate kinase buffer.
  • the 32 P incorporated into the product is separated from free [ 32 P]-ATP by electrophoresis and the incorporated 32 P is counted.
  • the amount of 32 P 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.
  • NTRAN is expressed in a eukaryotic ceU line such as CHO (Chinese Hamster Ovary) or HEK (Human Embryonic Kidney) 293 which have a good history of GPCR expression and which contain a wide range of G-proteins ahowing for functional coupling of the expressed NTRAN to downstream effectors.
  • the transformed ceUs are assayed for activation of the expressed receptors in the presence of candidate Ugands.
  • Activity is measured by changes in intraceUular second messengers, such as cycUc AMP or Ca 2+ . These may be measured directly using standard methods weU known in the art, or by the use of reporter gene assays in which a luminescent protein (e.g.
  • firefly luciferase or green fluorescent protein is under the transcriptional control of a promoter responsive to the stimulation of protein kinase C by the activated receptor (MilUgan, G. et al. (1996) Trends Pharmacol. Sci. 17:235-237).
  • Assay technologies are available for both of these second messenger systems to aUow high throughput readout in multi-weU plate format, such as the adenylyl cyclase activation FlashPlate Assay (NEN Life Sciences Products), or fluorescent Ca 2+ indicators such as Fluo-4 AM (Molecular Probes) in combination with the FLIPR fluorimetric plate reading system (Molecular Devices).
  • NTRAN may be coexpressed with the G-proteins G al5/16 which have been demonstrated to couple to a wide range of G-proteins (Offermanns, S. and M.L Simon (1995) J. Biol. Chem. 270:15175-15180), in order to funnel the signal transduction of the NTRAN through a pathway involving phosphohpase C and Ca 2+ mobilization.
  • G-proteins Offermanns, S. and M.L Simon (1995) J. Biol. Chem. 270:15175-15180
  • NTRAN may be expressed in engineered yeast systems which lack endogenous GPCRs, thus providing the advantage of a nuU background for NTRAN activation screening.
  • yeast systems substitate a human GPCR and G a protein for the co ⁇ esponding components of the endogenous yeast pheromone receptor pathway. Downstream signaling pathways are also modified so that the normal yeast response to the signal is converted to positive growth on selective media or to reporter gene expression (Broach, J.R. and J. Thorner (1996) Natare 384 (supp.): 14-16).
  • the receptors are screened against putative Ugands including known GPCR Hgands and other nataraUy occurring bioactive molecules. Biological extracts from tissues, biological fluids and ceU supernatants are also screened.
  • ABI FACTURA A program that removes vector sequences and Applied Biosystems, Foster City, CA. masks ambiguous bases in nucleic acid sequences.
  • ABI/PARACEL FDF A Fast Data Finder useful in comparing and Applied Biosystems, Foster City, CA; Mismatch ⁇ 50% annotating amino acid or nucleic acid sequences. Paracel Inc., Pasadena, CA.
  • ABI AutoAssembler A program that assembles nucleic acid sequences. Applied Biosystems, Foster City, CA.
  • fastx score 100 or greater
  • HMM hidden Markov model
  • Phred A base-calling algorithm that examines automated Ewing, B. et al. (1998) Genome Res. sequencer traces with high sensitivity and probability. 8: 175-185; Ewing, B. and P. Green (1998) Genome Res. 8:186-194.
  • TMHMMER A program that uses a hidden Markov model (HMM) to Sonnhammer, E.L. et al. (1998) Proc. Sixth Intl. delineate transmembrane segments on protein sequences Conf. on Intelligent Systems for Mol. Biol., and determine orientation. Glasgow et al., eds., The Am. Assoc. for Artificial Intelligence Press, Menlo Park, CA, pp. 175-182.
  • HMM hidden Markov model

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Public Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Diabetes (AREA)
  • Neurology (AREA)
  • Biomedical Technology (AREA)
  • Neurosurgery (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Pulmonology (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Rheumatology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Endocrinology (AREA)
  • Urology & Nephrology (AREA)
  • Oncology (AREA)
  • Obesity (AREA)
  • Communicable Diseases (AREA)
  • Psychology (AREA)
  • Dermatology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Anesthesiology (AREA)
  • Cell Biology (AREA)
  • Toxicology (AREA)
EP02718986A 2001-02-16 2002-02-15 Neurotransmission-associated proteins Withdrawn EP1368473A2 (en)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US26974801P 2001-02-16 2001-02-16
US269748P 2001-02-16
US29052401P 2001-05-11 2001-05-11
US290524P 2001-05-11
US34374201P 2001-10-19 2001-10-19
US343742P 2001-10-19
PCT/US2002/004536 WO2002066646A2 (en) 2001-02-16 2002-02-15 Neurotransmission-associated proteins

Publications (1)

Publication Number Publication Date
EP1368473A2 true EP1368473A2 (en) 2003-12-10

Family

ID=27402213

Family Applications (1)

Application Number Title Priority Date Filing Date
EP02718986A Withdrawn EP1368473A2 (en) 2001-02-16 2002-02-15 Neurotransmission-associated proteins

Country Status (4)

Country Link
EP (1) EP1368473A2 (ja)
JP (1) JP2004523236A (ja)
CA (1) CA2438235A1 (ja)
WO (1) WO2002066646A2 (ja)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK2730277T3 (da) 2004-12-22 2020-04-06 Nitto Denko Corp Lægemiddelbærer og lægemiddelbærerkit til at inhibere fibrose
US20120269886A1 (en) 2004-12-22 2012-10-25 Nitto Denko Corporation Therapeutic agent for pulmonary fibrosis
JP5342834B2 (ja) * 2008-09-05 2013-11-13 日東電工株式会社 骨髄線維症処置剤
US9572886B2 (en) 2005-12-22 2017-02-21 Nitto Denko Corporation Agent for treating myelofibrosis
TWI407971B (zh) 2007-03-30 2013-09-11 Nitto Denko Corp Cancer cells and tumor-related fibroblasts
US8734807B1 (en) 2013-04-06 2014-05-27 Gabriel Langlois-Rahme Preventing and curing Schistosomiasis mansoni by inhibiting Trk receptors on female Schistosoma

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2365081A3 (en) * 1999-04-12 2012-07-18 Agensys, Inc. 13 Transmembrane protein expressed in prostate cancer
EP1074617A3 (en) * 1999-07-29 2004-04-21 Research Association for Biotechnology Primers for synthesising full-length cDNA and their use

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO02066646A2 *

Also Published As

Publication number Publication date
CA2438235A1 (en) 2002-08-29
JP2004523236A (ja) 2004-08-05
WO2002066646A2 (en) 2002-08-29
WO2002066646A3 (en) 2003-09-18

Similar Documents

Publication Publication Date Title
WO2002010387A2 (en) G-protein coupled receptors
WO2001092304A2 (en) Transporters and ion channels
WO2002034783A2 (en) Transmembrane proteins
WO2002094990A2 (en) Receptors and membrane-associated proteins
WO2002026825A2 (en) G-protein coupled receptors
EP1383798A2 (en) Transporters and ion channels
EP1368473A2 (en) Neurotransmission-associated proteins
EP1259611A2 (en) G-protein coupled receptors
WO2003025130A2 (en) Receptors and membrane-associated proteins
WO2002063004A2 (en) G-protein coupled receptors
US20040106125A1 (en) Neurotransmission-associated proteins
EP1409677A2 (en) Receptors and membrane-associated proteins
WO2002002610A2 (en) Secretion and trafficking molecules
EP1297014A2 (en) Transporters and ion channels
WO2004022705A2 (en) Neurotransmission-associated proteins
EP1480994A1 (en) Neurotransmission-associated proteins
WO2002063006A2 (en) Receptors and membrane-associated proteins
WO2003023009A2 (en) G-protein coupled receptors
WO2002079448A2 (en) G-protein coupled receptors
WO2004111202A2 (en) Neurotransmission-association proteins
WO2003074726A2 (en) Immune response associated proteins
WO2002097035A2 (en) Secreted proteins
EP1578902A2 (en) Neurotransmission-associated proteins
WO2003000859A2 (en) G-protein coupled receptors
WO2002029061A2 (en) G-protein coupled receptors

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20030903

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

RIN1 Information on inventor provided before grant (corrected)

Inventor name: JACKSON, ALAN, A.

Inventor name: JIANG, XIN

Inventor name: CHAWLA, NARINDER, K.

Inventor name: FORSYTHE, IAN, J.

Inventor name: GRIFFIN, JENNIFER, A.

Inventor name: LEE, ERNESTINE, A.

Inventor name: WARREN, BRIDGET, A.

Inventor name: TANG, Y., TOM

Inventor name: YUE, HENRY

Inventor name: LAL, PREETI, G.

Inventor name: BAUGHN, MARIAH, R.

Inventor name: LU, DYUNG AINA, M.

Inventor name: THANGAVELU, KAVITHA

Inventor name: ISON, CRAIG, H.

Inventor name: HONCHELL, CYNTHIA, D.

Inventor name: DUGGAN, BRENDAN, M.

RIN1 Information on inventor provided before grant (corrected)

Inventor name: JACKSON, ALAN, A.

Inventor name: JIANG, XIN

Inventor name: CHAWLA, NARINDER, K.

Inventor name: FORSYTHE, IAN, J.

Inventor name: GRIFFIN, JENNIFER, A.

Inventor name: LEE, ERNESTINE, A.

Inventor name: WARREN, BRIDGET, A.

Inventor name: TANG, Y., TOM

Inventor name: YUE, HENRY

Inventor name: LAL, PREETI, G.

Inventor name: BAUGHN, MARIAH, R.

Inventor name: LU, DYUNG AINA, M.

Inventor name: THANGAVELU, KAVITHA

Inventor name: ISON, CRAIG, H.

Inventor name: HONCHELL, CYNTHIA, D.

Inventor name: DUGGAN, BRENDAN, M.

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20050728

RIN1 Information on inventor provided before grant (corrected)

Inventor name: JACKSON, ALAN, A.

Inventor name: JIANG, XIN

Inventor name: CHAWLA, NARINDER, K.

Inventor name: FORSYTHE, IAN, J.

Inventor name: GRIFFIN, JENNIFER, A.

Inventor name: LEE, ERNESTINE, A.

Inventor name: WARREN, BRIDGET, A.

Inventor name: TANG, Y., TOM

Inventor name: YUE, HENRY

Inventor name: LAL, PREETI, G.

Inventor name: BAUGHN, MARIAH, R.

Inventor name: LU, DYUNG AINA, M.

Inventor name: THANGAVELU, KAVITHA

Inventor name: ISON, CRAIG, H.

Inventor name: HONCHELL, CYNTHIA, D.

Inventor name: DUGGAN, BRENDAN, M.