EP1481081A2 - Molecules associees a des lipides - Google Patents

Molecules associees a des lipides

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Publication number
EP1481081A2
EP1481081A2 EP02736944A EP02736944A EP1481081A2 EP 1481081 A2 EP1481081 A2 EP 1481081A2 EP 02736944 A EP02736944 A EP 02736944A EP 02736944 A EP02736944 A EP 02736944A EP 1481081 A2 EP1481081 A2 EP 1481081A2
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EP
European Patent Office
Prior art keywords
polynucleotide
polypeptide
seq
amino acid
sequence
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Withdrawn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
EP02736944A
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German (de)
English (en)
Inventor
Y. Tom Tang
Henry Yue
Yalda Azimzai
Mariah R. Baughn
Neil Burford
Roopa Reddy
Narinder K. Walia
Debopriya Das
Danniel B. Nguyen
Monique G. Yao
Chandra S. Arvizu
Yan Lu
Ameena R. Gandhi
Jennifer A. Griffin
Vicki S. Elliott
Jayalaxmi Ramkumar
Preeti G. Lal
Dyung Aina M. Lu
Ernestine A. Lee
Soo Yeun Lee
Huibin Yue
Junming Yang
Catherine M. Tribouley
Amy E. Kable
Anita Swarnakar
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
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Incyte Genomics Inc
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Publication date
Application filed by Incyte Genomics Inc filed Critical Incyte Genomics Inc
Publication of EP1481081A2 publication Critical patent/EP1481081A2/fr
Withdrawn legal-status Critical Current

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    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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Definitions

  • This invention relates to nucleic acid and amino acid sequences of lipid-associated molecules and to the use of these sequences in the diagnosis, treatment, and prevention of cancer, neurological, autoimmune/inflammatory, gastrointestinal, and cardiovascular disorders, and disorders of lipid metabolism, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of lipid-associated molecules.
  • Lipids are water-insoluble, oily or greasy substances that are soluble in nonpolar solvents such as chloroform or ether.
  • Neutral fats triacylglycerols
  • Fatty acids are long-chain organic acids with a single carboxyl group and a long non-polar hydrocarbon tail.
  • Long-chain fatty acids are essential components of glycolipids, phospholipids, and cholesterol, which are building blocks for biological membranes, and of triglycerides, which are biological fuel molecules.
  • Lipids, such as phospholipids, sphingolipids, glycolipids, and cholesterol, are key structural components of cell membranes. Lipids and proteins are associated in a variety of ways.
  • Glycolipids form vesicles that carry proteins within cells and cell membranes. Interactions between lipids and proteins function in targeting proteins and glycolipids involved in a variety of processes, such as cell signaling and cell proliferation, to specific membrane and intracellular locations. Various proteins are associated with the biosynthesis, transport, and uptake of lipids. In addition, key proteins involved in signal transduction and protein targeting have lipid-derived groups added to them post-translationally (Stryer, L. (1995) Biochemistry. W.H. Freeman and Co., New York NY, pp. 264-267, 934; Lehninger, A. (1982) Principles of Biochemistry, Worth Publishers, Inc. New York NY; and ExPASy "Biochemical Pathways" index of Boehringer Mannheim World Wide Web site, "http://www.expasy.ch/cgi-bin/search-biochem-index".)
  • a major class of phospholipids are the phosphoglycerides, which are composed of a glycerol backbone, two fatty acid chains, and a phosphorylated alcohol.
  • Phosphoglycerides are components of cell membranes. Principal phosphoglycerides are phosphatidyl choline, phosphatidyl e anolamine, phosphatidyl serine, phosphatidyl inositol, and diphosphatidyl glycerol. Many enzymes involved in phosphoglyceride synthesis are associated with membranes (Meyers, R.A. (1995) Molecular Biology and Biotechnology. VCH Publishers Inc., New York NY, pp. 494-501).
  • Phosphatidate is converted to CDP-diacylglycerol by the enzyme phosphatidate cytidylyltransferase (ExPASy ENZYME EC 2.7.7.41). Transfer of the diacylglycerol group from CDP-diacylglycerol to serine to yield phosphatidyl serine, or to inositol to yield phosphatidyl inositol, is catalyzed by the enzymes CDP- diacylglycerol-serine O-phosphatidyltransferase and CDP-diacylglycerol-inositol 3- phosphatidyltransferase, respectively (ExPASy ENZYME EC 2.7.8.8; ExPASy ENZYME EC 2.7.8.11).
  • the enzyme phosphatidyl serine decarboxylase catalyzes the conversion of phosphatidyl serine to phosphatidyl emanolamine, using a pyruvate cofactor (Voelker, D.R. (1997) Biochim. Biophys. Acta 1348:236-244).
  • Phosphatidyl choline is formed using diet-derived choline by the reaction of CDP-choline with 1,2-diacylglycerol, catalyzed by diacylglycerol cholinephosphotransferase (ExPASy ENZYME 2.7.8.2).
  • Phosphatidylinositol transfer protein is a ubiquitous cytosolic protein, thought to be involved in transport of phospholipids from their site of synthesis in the endoplasmic reticulum and Golgi to other cell membranes. More recently, PITP has been shown to be an essential component of the polyphosphoinositide synthesis machinery and is hence required for proper signaling by epidermal growth factor and f-Met-Leu-Phe ⁇ as well as for exocytosis. The role of PITP in polyphosphoinositide synthesis may also explain its involvement in intracellular vesicular traffic (Liscovitch, M. et al. (1995) Cell 81:659-662). The copines are phospholipid-binding proteins believed to function in membrane trafficking.
  • the phosphatidylinositol-transfer protein Secl4 which catalyses exchange of phosphatidylinositol and phosphatidylcholine between membrane bilayers in vitro, is essential for vesicle budding from the Golgi complex.
  • Secl4 includes a carboxy-ter inal domain that forms a hydrophobic pocket which represents the phospholipid-binding domain. (Sha, B. et al. (1998) Nature 391 :506-510).
  • Secl4 is a member of the cellular retinaldehyde-binding protein (CRAL)/Triple function domain (TRIO) family (TnterPro Entry IPR001251, http://www.ebi.ac.uk/interpro).
  • CRAL retinaldehyde-binding protein
  • TEO Triple function domain
  • Glycolipids are also important components of the plasma membranes of animal cells.
  • the most simple glycolipid is cerebroside which comprises only a single glucose or galactose sugar residue in addition to the lipid component.
  • Gangliosides are glycosphingolipid plasma membrane components that are abundant in the nervous systems of .vertebrates. ' Gangliosides are the most complex glycolipids and comprise ceramide (acylated sphingosine) attached to an oligosaccharide moiety containing at least one acidic sugar residue, (sialic acid), namely N-acetylneuraminate or N- glycolytoeuraminate.
  • the sugar residues are added sequentially to ceramide via UDP-glucose, UDP- galactose, N-acetylgalactosamine, and CMP-N-acetylneuraminate donors.
  • Over 15 gangliosides have been identified with G m and G ⁇ being the best characterized (Stryer, L (1988) Biochemistry, W.H Freeman and Co., Inc. New York. pp. 552-554).
  • Gangliosides are thought to play important roles in cell surface interactions, cell differentiation, neuritogenesis, the triggering and modulation of transmembrane signaling, mediatiosynaptic function, neural repair, neurite outgrowth, arid neuronal death (Hasegawa, T. et al. (2000) J. Biol. Chem. 275:8007-8015). While the presence of gangliosides in the plasma membrane is important for orchestrating these events, the subsequent removal of carbohydrate groups (desialylation) by sialidases also appears to be important for regulating neuronal differentiation.
  • Vtila has been colocalized with Golgi markers while Vtilb has been colocalized with Golgi and the trans-Golgi network of endosomal markers in fibroblast cell lines.
  • a brain-specific splice variant of Vtila is enriched in small synaptic vesicles and clathrin-coated vesicles isolated from nerve terminals.
  • Vtila-beta and synaptobrevin are integral parts of synaptic vesicles throughout their life cycle. Vtila-beta functions in a SNARE complex during recycling or biogenesis of synaptic vesicles (Antonin, W. et al. (2000) J. Neurosci. 20:5724-5732).
  • Sialidases catalyze the first step in glycosphingolipid degradation, removing carbohydrate moieties from gangliosides. These enzymes are present in the cytosol, lysosomal matrix, lysosomal membrane, and plasma membrane (Hasegawa, T. et al. (2000) J. Biol. Chem. 275:8007-8015). Hallmark features of sialidases include a transmembrane domain, an Arg-De-Pro domain, and three Asp-box sequences (Wada, T. (1999) Biochem. Biophys.Res. Commun. 261:21-27).
  • pyramidal neurons of the cerebral cortex participate in a single burst of dendritic sprouting immediately following nerve cell migration to the cortical mantle.
  • Cells undergoing dendritogenesis are characterized by increased expression of G m ganglioside which decreases following dentritic maturation.
  • Evidence suggests that no new primary dendrites are initiated following the initial burst.
  • Cholesterol composed of four fused hydrocarbon rings with an alcohol at one end, moderates the fluidity of membranes in which it is incorporated: sin addition, cholesterol is used in the synthesis of steroid hormones such as cortisol, progesterone, estrogen, and.testosterone. Bile salts derived from cholesterol facilitate the digestion of lipids. Cholesterol in. the skin forms a barrier that prevents excess water evaporation from the body. Farnesyl and geranylgeranyl groups, which are derived from cholesterol biosynthesis intermediates, are post-translationally added to signal transduction proteins such as Ras and protein-targeting proteins such as Rab. These modifications are important for the activities of these proteins (Guyton, A.C.
  • Mammals obtain cholesterol derived from both de novo biosynthesis and the diet.
  • the liver is the major site of cholesterol biosynthesis in mammals.
  • Biosynthesis is accomplished via a series of enzymatic steps known as the mevalonate pathway.
  • the rate-limiting step is the conversion of hydroxymethylglutaryl-Coenzyme A (HMG-CoA) to mevalonate by HMG-CoA reductase.
  • HMG-CoA hydroxymethylglutaryl-Coenzyme A
  • the drug lovastatin a potent inhibitor of HMG-CoA reductase, is given to patients to reduce their serum cholesterol levels.
  • Cholesterol derived from de novo biosynthesis or from the diet is transported in the body fluids in the form of lipoprotein particles. These particles also transport triacylglycerols.
  • the particles consist of a core of hydrophobic lipids surrounded by a shell of polar lipids and apolipoproteins.
  • the protein components serve in the solubilization of hydrophobic lipids and also contain cell-targeting signals.
  • Lipoproteins include chylomicrons, chylomicron remnants, very-low- density lipoproteins (VLDL), intermediate-density lipoproteins (DDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL) (Meyers, supra; Stryer, supra, pp. 691-702).
  • VLDL very-low- density lipoprotein
  • DDL intermediate-density lipoproteins
  • LDL low-density lipoproteins
  • HDL high-density lipoproteins
  • LDL receptors bind LDL particles which are then internalized by endocytosis (Meyers, supra). Absence of the LDL receptor, the cause of the disease familial hypercholesterolemia, leads to increased plasma cholesterol levels and ultimately to atherosclerosis (Stryer, supra, pp. 691-702).
  • SREBP sterol regulatory element binding protein
  • OSBP oxysterol-binding protein
  • Squalene epoxidase a membrane-associated enzyme that converts squalene to squalene 2,3-oxide, plays an important role in the maintenance of cholesterol homeostasis.
  • SPF belongs to a family of cytosolic lipid-binding/transfer proteins such as alpha-tocopherol transfer protein, cellular retinal binding protein, yeast phosphatidylinositol transfer protein (Secl4p), and squid retinal binding protein (Shibata, N. et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98:2244-2249).
  • fatty acid synthase Long-chain fatty acids are also substrates for eicosanoid production, and are important in the functional modification of certain complex carbohydrates and proteins. 16-carbon and 18-carbon fatty acids are the most common. Fatty acid synthesis occurs in the cytoplasm. In the first step, acetyl- Coenzyme A (CoA) carboxylase (ACC) synthesizes malonyl-CoA from acetyl-CoA and bicarbonate. The enzymes which catalyze the remaining reactions are covalently linked into a single polypeptide chain, referred to as the multifunctional enzyme fatty acid synthase (FAS).
  • CoA acetyl- Coenzyme A
  • ACC acetyl- Coenzyme A carboxylase
  • FAS catalyzes the synthesis of palmitate from acetyl-CoA and malonyl-CoA.
  • FAS contains acetyl transferase, malonyl transferase, ⁇ -ketoacetyl synthase, acyl carrier protein, ⁇ -ketoacyl reductase, dehydratase, enoyl reductase, and thioesterase activities.
  • the final product of the FAS reaction is the 16-carbon fatty acid palmitate.
  • Further elongation, as well as unsaturation, of palmitate by accessory enzymes of the ER produces the variety of long chain fatty acids required by the individual cell. These enzymes include a NADH-cytochromeb 5 reductase, cytochrome b 5 , and a desaturase.
  • Diazepam binding inhibitor also known as endozepine and acyl CoA-binding protein, is an endogenous ⁇ -aminobutyric acid (GABA) receptor ligand which is thought to down-regulatbthe 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 (OMTM * 125950 Diazepam Binding Inhibitor; DBI; PROSITE PDOC00686 Acyl-CoA-binding protein signature).
  • Fat stored in liver and adipose triglycerides may be released by hydrolysis and transported in the blood. Free fatty acids are transported in the blood by albumin.
  • Triacylglycerols also known as triglycerides and neutral fats, are major energy stores in animals. Triacylglycerols are esters of glycerol with three fatty acid chains.
  • Glycerol-3-phosphate is produced from dihydroxyacetone phosphate by the enzyme glycerol phosphate dehydrogenase or from glycerol by glycerol kinase.
  • Fatty acid-CoAs are produced from fatty acids by fatty acyl-CoA synthetases.
  • Glyercol-3-phosphate is acylated with two fatty acyl-CoAs by the enzyme glycerol phosphate acyltransferase to give phosphatidate.
  • Phosphatidate phosphatase converts phosphatidate to diacylglycerol, which is subsequently acylated to a triacylglyercol by the enzyme diglyceride acyltransferase.
  • Phosphatidate phosphatase and diglyceride acyltransferase form a triacylglyerol synthetase complex bound to the ER membrane.
  • Mitochondrial and peroxisomal beta-oxidation enzymes degrade saturated and unsaturated fatty acids by sequential removal of two-carbon units from CoA-activated fatty acids.
  • the main beta- oxidation pathway degrades both saturated and unsaturated fatty acids while the auxiliary pathway performs additional steps required for the degradation of unsaturated fatty acids.
  • the pathways of mitochondria! and peroxisomal beta-oxidation use similar enzymes, but have different substrate specificities and functions.
  • Mitochondria oxidize short-, medium-, and long-chain fatty acids to produce energy for cells.
  • Mitochondrial beta-oxidation is a major energy source for cardiac and skeletal muscle.
  • peroxisomes oxidize medium-, long-, and very-long-chain fatty acids, dicarboxylic fatty acids, branched fatty acids, prostaglandins, xeuobiotics, and bile acid intermediates.
  • the chief roles of peroxisomal beta-oxidation are to shorten toxic lipophilic carboxylic acids to facilitate their excretion and to shorten very-long-chain fatty acids prior to mitochondrial beta-oxidation (Mannaerts, G.P. and P.P.
  • Enzymes involved in beta-oxidation include acyl CoA synthetase, carnitine acyltransferase, acyl CoA dehydrogenases, enoyl CoA hydratases, L-3- hydroxyacyl CoA dehydrogenase, ⁇ -ketothiolase, 2,4-dienoyl CoA reductase, and isomerase.
  • lipid metabolism enzymes Three classes of lipid metabolism enzymes are discussed in further detail. The three classes are lipases, phospholipases and lipoxygenases.
  • Triglycerides are hydrolyzed to fatty acids and.glycerol by lipases.
  • Adipocytes contain lipases that break down stored triacylglycerols, releasing fatty acids for export to other tissues where they are required as fuel. Lipases are widely distributed in animals, plants, and prokaryotes.
  • Triglyceride lipases (ExPASy ENZYME EC 3.1.1.3), also known as triacylglycerol lipases and tributyrases, hydrolyze the ester bond of triglycerides. In higher vertebrates there are at least three tissue-specific isozymes including gastric, hepatic, and pancreatic lipases.
  • lipases are structurally closely related to each other as well as to lipoprotein lipase.
  • the most conserved region in gastric, hepatic, and pancreatic lipases is centered around a serine residue which is also present in lipases of prokaryotic origin. Mutation in the serine residue renders the enzymes inactive.
  • Gastric, hepatic, and pancreatic lipases hydrolyze lipoprotein triglycerides and phospholipids.
  • Gastric lipases in the intestine aid in the digestion and absorption of dietary fats.
  • Hepatic lipases are bound to and act at the endothelial surfaces of hepatic tissues. Hepatic lipases also play a major role in the regulation of plasma lipids.
  • Lipoprotein lipases (ExPASy ENZYME EC 3.1.1.34), also known as clearing factor lipases, diglyceride lipases, or diacylglycerol lipases, hydrolyze triglycerides and phospholipids present in circulating plasma lipoproteins, including chylomicrons, very low and intermediate density lipoproteins, and high-density lipoproteins (HDL). Together with pancreatic and hepatic lipases, lipoprotein lipases (LPL) share a high degree of primary sequence homology.
  • LPL lipoprotein lipases
  • LPLs are primarily synthesized by adipocytes, muscle cells, and macrophages. Catalytic activities of LPLs are activated by apolipoprotein C-H and are inhibited by high ionic strength conditions such as 1 M NaCI.
  • LPL deficiencies in humans contribute to metabolic diseases such as hypertriglyceridemia, HDL2 deficiency, and obesity (Jackson, R.L. (1983) in The Enzymes (Boyer, P.D., ed.) Vol. XVI, pp. 141-186, Academic Press, New York NY; Eckel, R.H. (1989) New Engl. J. Med. 320:1060-1068).
  • Phospholipases a group of enzymes that catalyze the hydrolysis of membrane phospholipids, are classified according to the bond cleaved in a phospholipid. They are classified into PLA1, PLA2, PLB, PLC, and PLD families. Phospholipases are involved in many inflarnmatory reactions by making arachidonate available for eicosanoid biosynthesis. More specifically, arachidonic acid is processed into bioactive lipid mediators of inflammation such as lyso-platelet-activating factor and eicosanoids.
  • arachidonic acid from membrane phospholipids is the rate-limiting step in the biosynthesis of the four major classes of eicosanoids (prostaglandins, prostacyclins, thromboxanes and leukotrienes), whcih are 20-carbon molecules derived from fatty acids.
  • Eicosanoids are signaling molecules which have roles in pain, fever, and inflammation.
  • the precursor of all eicosanoids is arachidonate, which is generated from phospholipids by phospholipase A 2 and from diacylglycerols by diacylglycerol lipase.
  • Leukotrienes are produced from arachidonate by the action of lipoxygenases (Kaiser, E. et al. (1990) Clin. Biochem. 23:349-370).
  • leukotriene-B4 is known to function in a feedback loop which further increases PLA2 activity (Wijkander, J. et al. (1995) J. Biol. Chem. 270:26543-26549).
  • the secretory phospholipase A 2 (PLA2) superfamily comprises a number of heterogeneous enzymes whose common feature is to hydrolyze the sn-2 fatty acid acyl ester bond of phosphoglycerides. Hydrolysis of the glycerophospholipids releases free fatty acids and lysophospholipids. PLA2 activity generates precursors for the biosynthesis of biologically active lipids, hydroxy fatty acids, and platelet-activating factor. PLA2s were first described as components of snake venoms, and were later characterized in numerous species. PLA2s have traditionally been classified into several major groups and subgroups based on their amino acid sequences, divalent cation requirements, and location of disulfide bonds.
  • the PLA2s of Groups I, II, and m consist of low molecular weight, secreted, Ca 2+ -dependent proteins.
  • Group IV PLA2s are primarily 85-kDa, Ca 2+ -dependent cytosolic phospholipases.
  • Ca 2+ -independent PLA2s have been described, which comprise Group V (Davidson, F.F. and E.A. Dennis (1990) J. Mol. Evol. 31:228-238; and Dennis, E.F. (1994) J. Biol Chem. 269:13057-13060).
  • the first PLA2s to be extensively characterized were the Group I, ⁇ , and HJ PLA2s found in snake and bee venoms. These venom PLA2s share many features with mammalian PLA2s including a common catalytic mechanism, the same Ca 2+ requirement, and conserved primary and tertiary structures. In addition to their role in the digestion of prey, the venom PLA2s display neurotoxic, myotoxic, anticoagulant, and proinflammatory effects in mammalian tissues. This diversity of pathophysiological effects is due to the presence of specific, high affinity receptors for these enzymes on various cells and tissues (Lambeau, G. et al. (1995) J. Biol. Chem. 270:5534-5540). •.
  • PLA2s from Groups I, HA, UG, and V have been described in mammalian and avian cells > . and were originally characterized by tissue distribution, although the distinction is ho longer absolute. Thus, Group I PLA2s were found in the pancreas, Group HA and UG were derived from inflammation-associated tissues (e.g., the synovium), and Group V were from cardiac tissue. The pancreatic PLA2s function in the digestion of dietary lipids and have been proposed to play a role in cell proliferation, smooth muscle contraction, and acute lung injury. The Group II inflammatory PLA2s are potent mediators of inflammatory processes and are highly expressed in serum and synovial fluids of patients with inflammatory disorders.
  • Group II PLA2s are found in most human cell types assayed and are expressed in diverse pathological processes such as septic shock, intestinal cancers, rheumatoid arthritis, and epidermal hyperplasia.
  • a Group V PLA2 has been cloned from brain tissue and is strongly expressed in heart tissue.
  • a human PLA2 was recently cloned from fetal lung, and based on its structural properties, appears to be the first member of a new group of mammalian PLA2s, referred to as Group X.
  • Other PLA2s have been cloned from various human tissues and cell lines, suggesting a large diversity of PLA2s (Chen, J. et al. (1994) J. Biol. Chem.
  • Phospholipases B (PLB) (ExPASy ENZYME EC 3.1.1.5), also known as lysophospholipase, lecithinase B, or lysolecithinase are widely distributed enzymes that metabolize intracellular Hpids, and occur in numerous isoforms. Small isoforms, approximately 15-30 kD, function as hydrolases; large isoforms, those exceeding 60 kD, function both as hydrolases and transacylases. A particular substrate for PLBs, lysophosphatidylcholine, causes lysis of cell membranes when it is formed or imported into a cell.
  • PLBs are regulated by lipid factors including acylcarnitine, arachidonic acid, and phosphatidic acid. These lipid factors are signaling molecules important in numerous pathways, including the inflammatory response (Anderson, R. et al. (1994) Toxicol. Appl. Pharmacol. 125:176- 183; Selle, H. et al. (1993); Eur. J. Biochem. 212:411-416).
  • PLC Phospholipase C
  • Phospholipase C plays an important role in transmembrane signal transduction.
  • Many extracellular signaling molecules including hormones, growth factors, neurotransmitters, and immunoglobulins bind to their respective cell surface receptors and activate PLCs.
  • the role of an activated PLC is to catalyze the hydrolysis of phosphatidyl-inositol-4, 5-bisphosphate (PIP2), a minor component of the plasma membrane, to produce diacylglycerol and inositohl, 4, 5-trisphosphate (IP3).
  • IP3 and diacylglycerol serve as second messengers and trigger a series of intracellular responses.
  • IP3 induces the release of Ca 2+ from internal cellular storage, and diacylglycerol activates protein kinase C (PKC). Both pathways are part of transmembrane signal transduction mechanisms which regulate cellular processes which include secretion, neural activity, metabolism, and proliferation.
  • PLC-beta PLC-gamma
  • PLC-delta PLC-delta
  • Subtypes are designated by adding Arabic numbers after the Greek letters, eg. PLC- ⁇ -1.
  • PLCs have a molecular mass of 62-68 kDa, and their amino acid sequences show two regions of significant similarity. The first region, designated X, has about 170 amino acids, and the second, or Y region, contains about 260 amino acids.
  • the catalytic activities of the three isoforms of PLC are dependent upon Ca 2+ . It has been suggested that the binding sites for Ca 2+ in the PLCs are located in the Y-regk , one of two conserved regions.
  • the hydrolysis of common mositol-containing phospholipids, such as phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4, 5-bisphosphate (PIP2), by any of the isoforms yields cyclic and noncyclic inositol phosphates (Rhee, S.G. and Y.S. Bae (1997) J. Biol. Chem. 272:15045-15048).
  • PH domains target PLCs to the membrane surface by interacting with either the beta/gamma subunits of G proteins or PIP2 (PROSITE PDOC50003).
  • PLD Phospholipase D
  • ExPASy ENZYME EC 3.1.4.4 Phospholipase D
  • lecithinase D lipophosphodiesterase H
  • choline phosphatase catalyzes the hydrolysis of phosphatidylcholine and other phospholipids to generate phosphatidic acid.
  • PLD plays an important role in membrane vesicle trafficking, cytoskeletal dynamics, and transmembrane signal transduction.
  • the activation of PLD is involved in cell differentiation and growth (reviewed in Liscovitch, M. (2000) Biochem. J. 345:401-415).
  • PLD is activated in mammalian cells in response to diverse stimuli that include hormones, neurotransmitters, growth factors, cytokines, activators of protein kinase C, and agonist binding to G-protein-coupled receptors.
  • PLD1 and PLD2 have been identified.
  • PLD1 is activated by protein kinase C alpha and by the small GTPases ARF and RhoA. (Houle, M.G and S. Bourgoin (1999) Biochim. Biophys. Acta 1439:135-149).
  • PLD2 can be selectively activated by unsaturated fatty acids such as oleate (Kim, J.H. (1999) FEBS Lett. 454:42-46).
  • Lipoxygenases (ExPASy ENZYME EG 1.1-3:11.12) are non-heme iron-containing enzymes that catalyze the dioxygenation of certain polyunsaturated fatty acids such as lipoproteins.
  • Oxidation of the ferric enzyme to an active form is required for catalysis (Yamamoto, S. (1992) Biochim. Biophys. Acta 1128:117-131; Brash, A.R. (1999) J. Biol. Chem. 274:23679-23682).
  • a variety of lipoxygenase inhibitors exist and are classified into five major categories according to their mechanism of inhibition. These include antioxidants, iron chelators, substrate analogues, lipoxygenase-activating protein inhibitors, and, finally, epidermal growth factor-receptor inhibitors.
  • Aloxe3 3 -Lipoxygenase, also known as e-LOX-3 or Aloxe3 has recently been cloned from murine epidermis.
  • Aloxe3 resides on mouse chromosome 11, and the deduced amino acid sequence for Aloxe3 is 54% identical to the 12-lipoxygenase sequences (Kinzig, A. (1999) Genomics 58:158-164).
  • 5-Li ⁇ oxygenase (-LOX, ExPASy ENZYME EC 1.13.11.34), also known as arachidonate.-oxygen 5-oxidoreductase, is found primarily in white blood cells, macrophages, and mast cells.
  • 5-LOX converts arachidonic acid first to 5-hydro ⁇ eroxyeicosatetraenoic acid (5-HPETE) and then to leukotriene (LTA4 (5,6-oxido-7,9,ll,14-eicosatetraenoic acid)).
  • leukotriene A4 by leukotriene A4 hydrolase yields the potent neutrophil chemoattractant leukotriene B4.
  • 5- LOX and FLAP are found to be increased in the lungs of patients with plexogenic (primary) pulmonary hypertension (Wright, L. et al. (1998) Am. J. Respir. Crit. Care Med. 157:219-229).
  • 12-Lipoxygenase (12-LOX, ExPASy ENZYME: EC 1.13.11.31) oxygenates arachidonic acid to form 12-hydroperoxyeicosatetraenoic acid (12-HPETE).
  • Mammalian 12-lipoxygenases are named after the prototypical tissues of their occurrence (hence, the leukocyte, platelet, or epidermal types). , Platelet-type 12-LOX has been found to be the predominant isoform in epidermal skin specimens and epidermoid cells. Leukocyte 12-LOX was first characterized extensively from porcine leukocytes and was found to have a rather broad distribution in mammalian tissues by immunochemical assays.
  • the leukocyte 12-LOX is distinguished from the platelet-type enzyme by its ability to form 15-HPETE, in addition to 12-HPETE, from arachidonic acid substrate.
  • Leukocyte 12- LOX is highly related to 15-lipoxgenase (15-LOX) in that both are dual specificity lipoxygenases, and they are about 85% identical in primary structure in higher mammals.
  • Leukocyte 12-LOX is found in tracheal epithelium, leukocytes, and macrophages (Conrad, D.J. (1999) Clin. Rev. Allergy Immunol.17:71-89).
  • 15-Lipoxygenase 15-Lipoxygenase
  • ExPASy ENZYME EC 1.13.11.33
  • 15-LOX has been detected in atherosclerotic lesions in mammals, specifically rabbit and man.
  • the enzyme in addition to its role in oxidative modification of lipoproteins, is important in the inflammatory reaction in atherosclerotic lesions.
  • 15-LOX has been shown to be induced in human monocytes by the cytokine IL-4, which is known to be implicated in the inflammatory process (Kuhn, H. and S. Borngraber (1999) Adv. Exp. Med. Biol. 447:5-28).
  • a variety of lipolytic enzymes with a GDSL-like motif as part of the active site have been identified. Members of this family include a lipase/acylhydrolase, thermolabile hemolysin and rabbit phospholipase (AdRab-B)(Interpro entry IPR001087, http://www.sanger.ac.uk).
  • AdRab-B rabbit phospholipase
  • a homolog of AdRab-B is guinea pig intestinal phospholipase B, a calcium-independent phospholipase that contributes to lipid digestion as an ectoenzyme by sequentially hydrolyzing the acyl ester bonds of glycerophospholipids.
  • Phospholipase B also has a role in male reproduction (Delagebeaudeuf, C. et al. (1998) J. Biol. Chem. 273:13407-13414).
  • Atherosclerosis fatty lesions form on the inside of the arterial wall. These lesions promote the loss of arterial flexibility and the formation of blood clots (Guyton, supra).
  • Absence of the LDL receptor the cause of familial hypercholesterolemia, leads to increased plasma cholesterol levels and ultimately to atherosclerosis (Stryer, supra, pp. 691- 702).
  • Oxysterols are present in human atherosclerotic plaques and are believed to play an active role in plaque development (Brown, A . (1999) Atherosclerosis 142:1-28).
  • Lipases, phospholipases, and lipoxygenases are thought to contribute to complex diseases, such as atherosclerosis, obesity, arthritis, asthma, and cancer, as well as to single gene defects, such as Wolman's disease and Type I hyperhpoproteinemia.
  • Steatosis or fatty liver
  • Steatosis is characterized by the accumulation of triglycerides in the liver and may occur in association with a variety of conditions including alcoholism, diabetes, obesity, and prolonged parenteral nutrition. Steatosis may lead to fibrosis and cirrhosis of the liver.
  • Niemann-Pick diseases types A and B are caused by accumulation of sphingomyelin (a sphingolipid) and other lipids in the central nervous system due to a defect in the enzyme sphingomyelinase, leading to neurodegeneration and lung disease.
  • Niemann-Pick disease type C results from a defect in cholesterol transport, leading to the accumulation of sphingomyelin and cholesterol in lysosomes and a secondary reduction in sphingomyelinase activity.
  • Neurological symptoms such as grand mal seizures, ataxia, and loss of previously learned speech, manifest 1-2 years after birth.
  • NPC protein which contains a putative cholesterol-sensing domain
  • Tay-Sachs disease is an autosomal recessive, progressive neurodegenerative disorder caused by the accumulation of the G j ⁇ ganglioside in the brain (Igdoura, S. A. et al. (1999) Hum. Mol. Genet. 8:1111-6) due to a deficiency of the enzyme hexosaminidase A.
  • the disease is characterized by the onset of developmental retardation, followed by paralysis, dementia, blindness, and usually death within the second or third year of life.
  • Confirmatory evidence of Tay-Sachs disease is obtained at autopsy upon the identification of ballooned neurons in the central nervous system (Online Mendelian Inheritance in Man (OMTM). Johns Hopkins University, Baltimore, MD.
  • G j ⁇ gangliosidosis and Morquio B disease both arise from beta-galactosidase deficiency, although the diseases present with distinct phenotypes.
  • Sialidosis arises from a neuraminidase deficiency but presents with symptoms similar to gangliosidosis.
  • a likely reason for the overlapping phenotypes of sialidase deficiencies is the presence of these enzymes in a complex in lysosomes (Callahan, J.W. (1999) Biochim. Biophys. Acta. 1455:85-103).
  • PLAs are implicated in a variety of disease processes. For example, PLAs are found in the pancreas, in cardiac tissue, and in inflammation-associated tissues. Pancreatic PLAs function in the digestion of dietary lipids and have been proposed to play a role in cell proliferation, smooth muscle contraction, and acute lung injury. Inflammatory PLAs are potent mediators of inflammatory processes and are highly expressed in serum and synovial fluids of patients with inflammatory disorders. Additionally, inflammatory PLAs are found in most human cell types and are expressed in diverse pathological processes such as septic shock, intestinal cancers, rheumatoid arthritis, and epidermal hyperplasia.
  • Lipases, phospholipases, and lipoxygenases are thought to contribute to complex diseases, such as atherosclerosis, obesity, arthritis, asthma, and cancer, as well as to single gene defects, such as Wolman's disease and Type I hyperlipoproteinemia.
  • Complex diseases such as atherosclerosis, obesity, arthritis, asthma, and cancer
  • single gene defects such as Wolman's disease and Type I hyperlipoproteinemia.
  • 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.
  • LIPAM purified polypeptides, lipid-associated molecules, referred to collectively as “LIPAM” and individually as “LIPAM-1,” “LIPAM-2,” “LIPAM-3,” “LTPAM- J” “LJPAM-5,” ...
  • the invention provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-10, 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 JD NO:1-10, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-10, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO.-1-10.
  • the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1-10.
  • polynucleotide encodes a polypeptide selected from the group consisting of SEQ JD NO:1-10. In another alternative, the polynucleotide is selected from the group consisting of SEQ JD NO: 11-20.
  • 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 JD NO: 1-10, 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 JD NO: 1-10, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO:1-10, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-10.
  • 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 JD NO:1-10, 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 JD NO: 1-10, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO:1-10, and d) an immunogenic fragment of a polypeptide having an amino , acid sequence selected from the group consisting of SEQ ID NO: 1-10.
  • 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 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: 11-20, 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:11-20, 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 method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof.
  • 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 JD NO: 11 -20, 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: 11-20, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • the method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
  • the invention further provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ JD NO:1-10, 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 JD NO:1-10, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO:1-10, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-10, and a pharmaceutically acceptable excipient.
  • the composition comprises an amino acid sequence selected from the group consisting of SEQ JD NO:1-10.
  • the invention additionally provides a method of treating a disease or condition associated with decreased expression of functional LJPAM, 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 JD NO:1-10, 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 JD NO: 1-10, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO: 1-10, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-10.
  • 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 pharmaceutically acceptable excipient.
  • the invention provides a method of treating a disease or condition associated with decreased expression of functional LJPAM, 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: 1-10, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consistmg of SEQ JD NO:1-10, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO:1-10, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO: 1-10.
  • 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 pharmaceutically acceptable excipient.
  • the invention provides a method of treating a disease or condition associated with overexpression of functional LIPAM, comprising administering to a patient in need of such treatment the composition.
  • the invention further provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ JD NO:1-10, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-10, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO:1-10, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO: 1-10.
  • the method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.
  • the invention further provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ JD NO: 1-10, 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 JD NO: 1-10, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO:1-10, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-10.
  • 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 JD NO:11-20, 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: 11-20, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ JD NO:11-20, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
  • Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ JD NO: 11-20, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ JD NO:l 1-20, 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 applicable descriptions, references, and threshold parameters.
  • LIPAM refers to the amino acid sequences of substantially purified LIPAM 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 LJPAM.
  • Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of LIPAM either by directly interacting with LJPAM or by acting on components of the biological pathway in which LJPAM participates.
  • allelic variant is an alternative form of the gene encoding LJPAM. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • altered nucleic acid sequences encoding LJPAM include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as LIPAM or a polypeptide with at least one functional characteristic of LJPAM. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding LIPAM, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding LIPAM.
  • amino acid and amino acid sequence refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where “amino acid sequence” is recited to refer to a sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
  • Amplification relates to the production of additional copies of a nucleic acid sequence. Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.
  • PCR polymerase chain reaction
  • antagonists refers to a molecule which inhibits or attenuates the biological activity of LIPAM. Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of LJPAM either by directly interacting with LIPAM or by acting on components of the biological pathway in which LJPAM participates.
  • antibody refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding an epitopic determinant.
  • Antibodies that bind LIPAM polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen.
  • the polypeptide or oligopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
  • an animal e.g., a mouse, a rat, or a rabbit
  • Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
  • 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 elicit 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 lifetime in blood.
  • Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.
  • Aptamers may be specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13.)
  • RNA aptamer refers to an aptamer which is expressed in vivo.
  • a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA 96:3606-3610).
  • spiegelmer refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.
  • 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; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine.
  • Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation.
  • the designation "negative” or “minus” can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.
  • biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
  • immunologically active or “immunogenic” refers to the capability of the natural, recombinant, or synthetic LIPAM, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • Complementary describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5 -AGT-3' pairs with its complement, 3'-TCA-5'.
  • 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 LJPAM or fragments of LIPAM may be employed as hybridization probes.
  • the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
  • the probe may be deployed in an aqueous solution containing salts (e.g., NaCI), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
  • salts e.g., NaCI
  • 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 uncalled bases, extended using the XL-PCR kit (Applied Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVJEW fragment assembly system (GCG, Madison
  • Constant amino acid substitutions are those substitutions that are predicted to least interfere with the properties of the original protein, i.e. , the structure and especially the function of the protein is conserved and not significantly changed by such substitutions.
  • the table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
  • Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
  • a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • derivative refers to a chemically modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group.
  • a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
  • a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
  • a “detectable label” refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
  • “Differential expression” refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
  • Exon shuffling refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus allowing acceleration of the evolution of new protein functions.
  • a “fragment” is a unique portion of LJPAM or the polynucleotide encoding LJPAM 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 preferentially 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, maybe encompassed by the present embodiments.
  • a fragment of SEQ JD NO:11-20 comprises a region of unique polynucleotide sequence that specifically identifies SEQ JD NO: 11-20, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
  • a fragment of SEQ ID NO: 11-20 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO:l 1-20 from related polynucleotide sequences.
  • the precise length of a fragment of SEQ JD NO:11-20 and the region of SEQ JD NO:11-20 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
  • a fragment of SEQ ID NO:1-10 is encoded by a fragment of SEQ ID NO:11-20.
  • a fragment of SEQ JD NO:1-10 comprises a region of unique amino acid sequence that specifically identifies SEQ JD NO:1-10.
  • a fragment of SEQ ID NO:1-10 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ JD NO: 1-10.
  • the precise length of a fragment of SEQ ID NO:1-10 and the region of SEQ ID NO:1-10 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
  • a "full length" polynucleotide sequence is one containing at least a translation initiation codon
  • a "full length” polynucleotide sequence encodes a "full length” polypeptide sequence.
  • “Homology” refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
  • the terms “percent identity” and “% identity,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity between polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program.
  • BLAST Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410
  • NCBI National System for Mobile Science
  • BLAST 2 Sequences a tool that is used for direct pairwise comparison of two nucleotide sequences.
  • BLAST 2 Sequences can be accessed and used interactively at http://www.ncbi.nlm.n .gov/gorf l2.html.
  • the "BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed below).
  • BLAST programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) set at default parameters. Such default parameters maybe, for example:
  • Percent identity maybe measured over the length of an entire defined sequence, for example, as defined by a particular SEQ JD number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, maybe used to describe a length over which percentage identity may be measured.
  • nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
  • percent identity and % identity refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm.
  • Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge andjxydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
  • NCBI BLAST software suite maybe used.
  • BLAST 2 Sequences Version 2.0.12 (April-21-2000) withblastp set at default parameters.
  • Such default parameters maybe, for example:
  • Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ JD number, or maybe 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, maybe . used to describe a length over which percentage identity may be measured.
  • "Human artificial chromosomes" are linear microchromosomes which may contain
  • humanized antibody refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
  • Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity.
  • Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 ⁇ g/ml sheared, denatured salmon sperm DNA.
  • stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out.
  • Such wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (T j consult) 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 may be 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 will be readily apparent to those of ordinary skill in the art.
  • Hybridization particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
  • hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex may be formed in solution (e.g., C 0 t or R 0 t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
  • 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 cellular and systemic defense systems.
  • factors e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
  • an “immunogenic fragment” is a polypeptide or oligopeptide fragment of LJPAM which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal.
  • the term “immunogenic fragment” also includes any polypeptide or oligopeptide fragment of LIPAM which is useful in any of the antibody production methods disclosed herein or known in the art.
  • microarray refers to an arrangement of a plurality 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 LIPAM. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of LJPAM.
  • nucleic acid and nucleic acid sequence refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
  • operably linked refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence.
  • 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 oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
  • Post-translational modification of an LIPAM may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cell type depending on the enzymatic milieu of LJPAM.
  • Probe refers to nucleic acid sequences encoding LIPAM, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences.
  • Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
  • Primmers are short nucleic acids, usually DNA oligonucleotides, 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 amplification (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 typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers 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.
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
  • Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome- wide scope.
  • the Primer3 primer selection program (available to the public from the Whitehead Institute/MJT Center for Genome Research, Cambridge MA) allows the user to input a "mispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.)
  • the PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences.
  • this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments.
  • the oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
  • a "recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g. , by genetic engineering techniques such as those described in Sambrook, 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 cell.
  • such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
  • a “regulatory element” refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
  • Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.
  • An "RNA equivalent,” in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the nitrogenous base ihymine 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 LIPAM, nucleic acids encoding LIPAM, or fragments thereof may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
  • the terms "specific binding” and “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule.
  • substitution refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
  • Substrate refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
  • the substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
  • Transformation describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment.
  • transformed cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
  • a "transgenic organism,” as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
  • the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002) Science 295:868-872).
  • the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
  • the transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals.
  • the isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. (1989), 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%, atleast 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
  • a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
  • the corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
  • Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides will generally have significant amino acid identity relative to each other.
  • a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
  • Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base.
  • 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 lipid-associated molecules (LJPAM), the polynucleotides encoding LJPAM, and the use of these compositions for the diagnosis, treatment, or prevention of cancer, neurological, autoimmune/inflammatory, gastrointestinal, and cardiovascular disorders, and disorders of lipid metabolism.
  • LJPAM human lipid-associated molecules
  • Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project JD). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide JD) as shown.
  • Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ JD NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide JD) as shown.
  • Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME database.
  • Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ JD NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide JD) 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 JD NO:) of the nearest PROTEOME database homologs.
  • Column 4 shows the probability scores for the matches between each polypeptide and its homolog(s).
  • Column 5 shows the annotation of the GenBank and PROTEOME database homolog(s) along with relevant citations where applicable, all of which are expressly incorporated by reference herein.
  • Table 3 shows various structural features of the polypeptides of the invention.
  • Columns 1 and 2 show the polypeptide sequence identification number (SEQ 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 Wl).
  • Column 6 shows amino acid residues comprising signature sequences, domains, and motifs.
  • Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.
  • SEQ ID NO:l is 77% identical, from residue Ml to residue T1455, to Orctolagus cuniculus phospholipase (GenBank JD gl690) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
  • the BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
  • SEQ ID NO:2 is a ceramidase.
  • SEQ ID NO:6 is 36% identical, from residue L235 to residue E564 to Rattus norvegicus supernatant protein factor (GenBank ID gl3241652) as determined by the Basic Local Alignment Search Tool (BLAST).
  • the BLAST probability score is 7.5e- 48, which indicates the probability of obtaining the observed polypeptide sequence alignment hy chance.
  • SEQ JD NO:6 also contains a CRAL/TRIO domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ JD NO:7 is 60% identical, from residue S21 to residue S772, to murine oxysterol binding protein (GenBank JD gl2583596) as deteimined by the Basic Local Alignment Search Tool (BLAST).
  • BLAST probability score is 4. le-252, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
  • SEQ ID NO:7 also contains an oxysterol binding protein domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ ID NO:8 is 92% identical, from residue M285 to residue W1146, to human OSBP-related protein 7 (GenBank ID gl2382789) as determined by the Basic Local Alignment Search Tool (BLAST).
  • BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
  • SEQ JD NO: 8 also contains an oxysterol-binding protein domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ JD NO: 10 is 100% identical, from residue 168 to residue W824, to human OSBP-related protein-7 (GenBank JD gl2382789) as determined by the Basic Local Alignment Search Tool (BLAST).
  • SEQ JD NO:10 also has homology to proteins possessing plekstrin homology that mediate protein-protein and protein-lipid interactions, and are oxysterol-related binding-proteins, as determined by BLAST analysis using the PROTEOME database.
  • SEQ ID NO:10 also contains an oxysterol-binding protein domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)- based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ ID NO:10 is an oxysterol binding protein.
  • SEQ JD NO:3-5 and SEQ ID NO:9 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ ID NO:1-10 are described in Table 7.
  • the full length polynucleotide sequences of the present invention were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences.
  • Column 1 lists the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:), the corresponding Incyte polynucleotide consensus sequence number (Incyte JD) for each polynucleotide of the invention, and the length of each polynucleotide sequence inbasepairs.
  • Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences used to assemble the full length polynucleotide sequences of the invention, and of fragments of the polynucleotide sequences which are useful, for example, in hybridization or amplification technologies that identify SEQ JD NO:l 1-20 or that distinguish between SEQ JD NO:11-20 and related polynucleotide sequences.
  • the polynucleotide fragments described in Column 2 of Table 4 may refer specifically, for example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from pooled cDNA libraries.
  • the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the full length polynucleotide sequences.
  • the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (t.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 (t.e., those sequences including the designation "NM” or “NT”) or the NCBI RefSeq Protein Sequence Records (ie., 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 W ⁇ OO _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-stretching" algorithm was applied, gBBBBB being the GenBank identification number or NCBI RefSeq identification number of the nearest GenBank protein homolog, and N referring to specific exons (See Example V).
  • a RefSeq identifier (denoted by "NM,” “NP,” or “NT”) maybe used in place of the GenBank identifier (i.e., gBBBBB).
  • GenBank identifier i.e., gBBBBB.
  • a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods. The following Table lists examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example IV and Example V).
  • Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
  • Table 5 shows the representative cDNA libraries for those full length polynucleotide sequences which were assembled using Incyte cDNA sequences.
  • the representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotide sequences.
  • the tissues and vectors which were used to construct the cDNA libraries shown in Table 5 are described in Table 6.
  • the invention also encompasses LIPAM variants.
  • a preferred LJPAM 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 LJPAM amino acid sequence, and which contains at least one functional or structural characteristic of LIPAM.
  • the invention also encompasses polynucleotides which encode LJPAM.
  • the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ JD NO: 11-20, which encodes LJPAM.
  • the polynucleotide sequences of SEQ JD NO:11-20, as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
  • the invention also encompasses a variant of a polynucleotide sequence encoding LIPAM.
  • a variant polynucleotide sequence will have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding LJPAM.
  • a particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ JD NO:l 1- 20 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: 11-20. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of LIPAM.
  • a polynucleotide variant of the invention is a splice variant of a polynucleotide sequence encoding LIPAM.
  • a splice variant may have portions which have significant sequence identity to the polynucleotide sequence encoding LIPAM, but will generally have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate splicing of exons during mRNA processing.
  • Any one of the splice variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of LIPAM.
  • nucleotide sequences which encode LIPAM and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring LJPAM under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding LIPAM or its derivatives possessing a substantially different codon usage, e.g., inclusion of non- naturally occurring codons. Codons maybe 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-life, than transcripts produced from the naturally occurring sequence.
  • the invention also encompasses production of DNA sequences which encode LIPAM and LJPAM derivatives, or fragments thereof, entirely by synthetic chemistry.
  • the synthetic sequence maybe inserted into any of the many available expression vectors and cell systems using reagents well known in the art.
  • synthetic chemistry may be used to introduce mutations into a sequence encoding LJPAM or any fragment thereof.
  • polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ JD NO:11-20 and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol.
  • Hybridization conditions including annealing and wash conditions, are described in "Definitions.” Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Jnvitrogen, Carlsbad CA).
  • sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Applied Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Amersham Biosciences), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997) Short Protocols in Molecular 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 LJPAM maybe extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • restriction-site PCR uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector.
  • inverse PCR uses primers that extend in divergent directions to amplify unknown sequence from a circularized template.
  • the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences.
  • primers maybe designed using commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
  • Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
  • capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- , specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
  • Output/light intensity maybe converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled.
  • Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
  • polynucleotide sequences or fragments thereof which encode LIPAM maybe cloned in recombinant DNA molecules that direct expression of LJPAM, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express LJPAM.
  • nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter LIPAM-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences.
  • oligonucleotide- mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
  • the nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of LIPAM, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds.
  • MOLECULARBREEDING Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C.
  • DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening.
  • genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized.
  • fragments of a given gene maybe recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
  • sequences encoding LJPAM may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232.)
  • LIPAM itself or a fragment thereof may be synthesized using chemical methods.
  • peptide synthesis can be performed using various solution-phase or solid-phase techniques.
  • Automated synthesis maybe achieved using the ABI 43 IA peptide synthesizer (Applied Biosystems).
  • the amino acid sequence of LIPAM, 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 naturally occurring polypeptide.
  • the peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421.)
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, supra, pp. 28-53.)
  • the nucleotide sequences encoding LJPAM 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 LIPAM. Such elements may vary in their strength and specificity.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding LIPAM. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
  • exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector.
  • Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)
  • Methods which -are well known to those skilled in the art may be used to construct expression , vectors containing sequences encoding LIPAM 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 utilized to contain and express sequences encoding LIPAM.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with viral expression vectors (e.g., baculovirus)
  • plant cell systems transformed with viral expression vectors e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV
  • bacterial expression vectors e.g., Ti or pBR322 plasmids
  • Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population.
  • the invention is not limited by the host cell employed. In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding LJPAM.
  • routine cloning, subcloning, and propagation of polynucleotide sequences encoding LJPAM can be achieved using a multifunctional E. coli vector such as PBLUESCRTPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Invitrogen). Ligation of sequences encoding LJPAM into the vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, these vectors maybe useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested • deletions in the cloned sequence.
  • PBLUESCRTPT Stratagene, La Jolla CA
  • PSPORT1 plasmid Invitrogen
  • vectors which direct high level expression of LIPAM may be used.
  • vectors containing the strong, inducible SP6 or T7 bacteriophage promoter maybe used.
  • Yeast expression systems may be used for production of LJPAM.
  • a number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH promoters may be used in the yeast Saccharomyces cerevisiae or Pichia pasto ⁇ s.
  • such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
  • Plant systems may also be used for expression of LJPAM. Transcription of sequences encoding LIPAM may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:17-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters maybe used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl.
  • constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection.
  • pathogen-mediated transfection See, e.g., The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 191-196.
  • a number of viral-based expression systems may be utilized.
  • sequences encoding LJPAM may be ligated 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 LIPAM in host cells.
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, maybe used to increase expression in mammalian host cells.
  • SV40 or EBV- based vectors may also be used for high-level protein expression.
  • HACs Human artificial chromosomes
  • HACs may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid.
  • HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J. et al. (1997) Nat. Genet. 15:345- 355.)
  • sequences encoding LJPAM can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells maybe allowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
  • the purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
  • Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
  • selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk and apr cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate
  • neo confers resistance to the aminoglycosides neomycin and G-418
  • als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
  • Additional selectable genes have been described, e.g., tipB and hisD, which alter cellular requirements for metabolites.
  • Visible markers e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), ⁇ glucuronidase and its substrate ⁇ -glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, 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 LIPAM is inserted within a marker gene sequence
  • transformed cells containing sequences encoding LIPAM can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding LJPAM under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
  • host cells that contain the nucleic acid sequence encoding LIPAM and .that express LIPAM may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences. Immunological methods for detecting and measuring the expression of LIPAM using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS).
  • ELISAs enzyme-linked immunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated cell sorting
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding LIPAM include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • the sequences encoding LIPAM, 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 RNA polymerase
  • reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chem ⁇ uminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with nucleotide sequences encoding LIPAM may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode LIPAM maybe designed to contain signal sequences which direct secretion of LJPAM through a prokaryotic or eukaryotic cell membrane.
  • a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a "prepro” or “pro” form of the protein may also be used to specify protein targeting, folding, and/or activity.
  • LJPAM Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC, Manassas VA) and maybe chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture Collection
  • natural, modified, or recombinant nucleic acid sequences encoding LJPAM may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric LJPAM protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of LJPAM activity.
  • Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices.
  • Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA).
  • GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags.
  • a fusion protein may also be engineered to contain a proteolytic cleavage site located between the LJPAM encoding sequence and the heterologous protein sequence, so that LJPAM maybe cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10). A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
  • LJPAM of the present invention or fragments thereof may be used to screen for compounds that specifically bind to LIPAM. At least one and up to a plurality of test compounds may be screened for specific binding to LJPAM. Examples of test compounds include antibodies, oligonucleotides, proteins (e.g., ligands or receptors), or small molecules. In one embodiment, the compound thus identified is closely related to the natural ligand of LJPAM, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner. (See, e.g., Coligan, J.E. et al.
  • the compound thus identified is a natural ligand of a receptor LJPAM.
  • LJPAM receptor LJPAM
  • the compound can be closely related to the natural receptor to which LJPAM binds, at least a fragment of the receptor, or a fragment of the receptor including all or a portion of the ligand binding site or binding pocket.
  • the compound may be a receptor for LJPAM which is capable of propagating a signal, or a decoy receptor for LIPAM which is not capable of propagating a signal (Ashkenazi, A. and V.M. Divit (1999) Curr. Opin. Cell Biol. 11:255- 260; Mantovani, A. et al. (2001) Trends Immunol. 22:328-336).
  • the compound can be rationally designed using known techniques.
  • etanercept is an engineered p75 tumor necrosis factor (TNF) receptor dimer linked to the Fc portion of human IgGi (Taylor, P.C et al. (2001) Curr. Opin. Immunol. 13:611- 616).
  • screening for compounds which specifically bind to, stimulate, or inhibit LIPAM involves producing appropriate cells which express LIPAM, either as a secreted protein or on the cell membrane.
  • Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing LIPAM or cell membrane fractions which contain LIPAM are then contacted with a test compound and binding, stimulation, or inhibition of activity of either LIPAM or the compound is analyzed.
  • An assay can be used to assess the ability of a compound to bind to its natural ligand and/or to inhibit the binding of its natural ligand to its natural receptors.
  • examples of such assays include radio- labeling assays such as those described in U.S. Patent No. 5,914,236 and U.S. Patent No. 6,372,724.
  • one or more amino acid substitutions can be introduced into a polypeptide compound (such as a receptor) to improve or alter its ability to bind to its natural ligands. (See, e.g., Matthews, D.J. and J.A. Wells. (1994) Chem. Biol.
  • one or more amino acid substitutions can be introduced into a polypeptide compound (such as a ligand) to improve or alter its ability to bind to its natural receptors.
  • a polypeptide compound such as a ligand
  • polynucleotides encoding LJPAM or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells.
  • ES embryonic stem
  • Such techniques are well known in the art and are useful for the generation of animal models of human disease.
  • mouse ES cells such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture.
  • the ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292).
  • 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, JD. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
  • Transformed ES cells are identified and microinjected into mouse cellblastocysts such as those from the C57BL/6 mouse strain.
  • the blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.
  • Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
  • Polynucleotides encoding LJPAM may also be manipulated in vitro in ES cells derived from human blastocysts.
  • Human ES cells have the potential to differentiate into at least eight separate, cell lineages including endoderm, mesoderm, and ectodermal cell types. These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147).
  • Polynucleotides encoding LJPAM can also be used to create "knockin” humanized animals (pigs) or transgenic animals (mice or rats) to model human disease.
  • knockin technology a region of a polynucleotide encoding LJPAM is injected into animal ES cells, and the injected sequence integrates into the animal cell genome.
  • Transformed cells are injected into blastulae, and the blastulae are implanted as described above.
  • Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
  • a mammal inbred to overexpress LJPAM e.g., by secreting LJPAM in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).
  • THERAPEUTICS e.g., by secreting LJPAM in its milk.
  • LJPAM or a fragment or derivative thereof maybe administered to a subject to treat or prevent a disorder associated with decreased expression or activity of LJPAM.
  • disorders include, but are not limited to, a cancer, such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; a cardiovascular disorder such as arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicose
  • a vector capable of expressing LIPAM 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 LIPAM including, but not limited to, those described above.
  • composition comprising a substantially purified LJPAM in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of LJPAM including, but not limited to, those provided above.
  • an agonist which modulates the activity of LJPAM maybe administered to a subject to treat or prevent a disorder associated with decreased expression or activity of LIPAM including, but not limited to, those listed above.
  • an antagonist of LJPAM maybe administered to a subject to treat or prevent a disorder associated with increased expression or activity of LIPAM.
  • disorders include, but are not limited to, those cancer, neurological, autoimmune/inflammatory, gastrointestinal, and cardiovascular disorders, and disorders of lipid metabolism described above.
  • an antibody which specifically binds LJPAM may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express LIPAM.
  • a vector expressing the complement of the polynucleotide encoding LJPAM maybe administered to a subject to treat or prevent a disorder associated with increased expression or activity of LJPAM 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 skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • An antagonist of LJPAM may be produced using methods which are generally known in the art.
  • purified LJPAM may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind LJPAM.
  • Antibodies to LIPAM may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are generally preferred for therapeutic use. Single chain antibodies (e.g., from camels or llamas) may be potent enzyme inhibitors and may have advantages in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S. (2001) J. Biotechnol. 74:277-302).
  • various hosts including goats, rabbits, rats, mice, camels, dromedaries, llamas, humans, and others may be immunized by injection with LJPAM 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 limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are especially preferable.
  • the oligopeptides, peptides, or fragments used to induce antibodies to LJPAM have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein. Short stretches of LIPAM amino acids maybe fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
  • Monoclonal antibodies to LJPAM may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, RJ. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
  • chinieric antibodies such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity.
  • techniques developed for the production of "chinieric antibodies” such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used.
  • techniques described for the production of single chain antibodies maybe adapted, using methods known in the art, to produce LIPAM-specific single chain antibodies.
  • Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.) Antibody fragments which contain specific binding sites for LJPAM may also be generated.
  • 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 libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.)
  • immunoassays may be used for screening to identify antibodies having the desired specificity.
  • Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art.
  • Such immunoassays typically involve the measurement of complex formation between LIPAM and its specific antibody.
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering LIPAM epitopes is generally used, but a competitive binding assay may also be employed (Pound, supra).
  • Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for LIPAM.
  • polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications.
  • a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is generally employed in procedures requiring precipitation of LJPAM-antibody complexes.
  • Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available. (See, e.g., Catty, supra, and Coligan et al. supra.)
  • Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein.
  • Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors.
  • diseases or disorders caused by deficiencies in LJPAM are treated by constructing mammalian expression vectors encoding LJPAM and introducing these vectors by mechanical means into LIPAM-deficient cells.
  • Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (ii) ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R. A. and W.F. Anderson (1993) Annu. Rev. Biochem. 62:191-217; Ivies, Z. (1997) Cell 91:501-510; Boulay, J-L. and H. Recipon (1998) Curr. Opin. Biotechnol. 9:445-450).
  • liposome transformation kits e.g., the PERFECT LTPJD TRANSFEC ⁇ ON KIT, available from Invitrogen
  • PERFECT LTPJD TRANSFEC ⁇ ON KIT available from Invitrogen
  • transformation is performed using the calcium phosphate method (Graham, F.L. and A J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J. 1:841-845).
  • the introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.
  • diseases or disorders caused by genetic defects with respect to LIPAM expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding LJPAM under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE) along with additional retrovirus cts-acting RNA sequences and coding sequences required for efficient vector propagation.
  • Retrovirus vectors e.g., PFB and PFBNEO
  • PFB and PFBNEO are commercially available (Stratagene) and are based on published data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci.
  • the vector is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al. (1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and AD. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey, R. et al. (1998) J. Virol.
  • VSVg vector producing cell line
  • adenoviral vectors for gene therapy
  • adenoviral vectors see also Antinozzi, P.A. et al. (1999) Annu. Rev. Nutr. 19:511-544 and Verma, I.M. and N. Somia (1997) Nature 18:389:239-242, both incorporated by reference herein.
  • an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding LIPAM to target cells.
  • SFV Semliki Forest Virus
  • This subgenomic RNA replicates to higher levels than the full length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase).
  • enzymatic activity e.g., protease and polymerase.
  • inserting the coding sequence for LJPAM into the alphavirus genome in place of the capsid-coding region results in the production of a large number of LTPAM-coding RNAs and the synthesis of high levels of LJPAM in vector transduced cells.
  • alphavirus infection is typically associated with cell lysis within a few days
  • the ability to establish a persistent infection in hamster normal kidney cells (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S.A. et al. (1997) Virology 228:74-83).
  • the wide host range of alphaviruses will allow the introduction of LIPAM into a variety of cell types.
  • the specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction.
  • a complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding LJPAM.
  • RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, maybe evaluated for secondary structural features which may render the oligonucleotide inoperable.
  • the suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary - oligonucleotides using ribonuclease protection assays. ...
  • RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5 ' and/or 3 ' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding LJPAM.
  • Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oligonucleotides, antisense oligonucleotides, triple heUx-forming oligonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression.
  • a compound which specifically inhibits expression of the polynucleotide encoding LJPAM may be therapeutically useful, and in the treatment of disorders associated with decreased LIPAM expression or activity, a compound which specifically promotes expression of the polynucleotide encoding LJPAM maybe therapeutically useful.
  • test compounds may be screened for effectiveness in altering expression of a specific polynucleotide.
  • a test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commercially-available or proprietary library of naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide; and selection from a library of chemical compounds created combinatorially or randomly.
  • a sample comprising a polynucleotide encoding LJPAM is exposed to at least one test compound thus obtained.
  • the sample may comprise, for example, an intact or permeabilized cell, or an in vitro cell-free or reconstituted biochemical system.
  • Alterations in the expression of a polynucleotide encoding LIPAM are assayed by any method commonly known in the art.
  • 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 LJPAM.
  • 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, GM. et al. (2000) Nucleic Acids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem. Biophys. Res. Commun.
  • a particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence
  • oligonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides
  • vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient.
  • Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C.K. et al. (1997) Nat.
  • An additional embodiment of the invention relates to the administration of a composition which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient.
  • Excipients may include, for example, sugars, starches, celluloses, gums, and proteins.
  • Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's
  • compositions may consist of LJPAM,; antibodies to LJPAM, and mimetics, agonists, antagonists, or inhibitors of LIPAM.
  • compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • compositions for pulmonary administration may be prepared in liquid or dry powder form.
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • Specialized forms of compositions may be prepared for direct intracellular delivery of macromolecules comprising LIPAM or fragments thereof.
  • liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule.
  • LJPAM or a fragment thereof maybe joined to a short cationic N- terminal portion from the HJV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs.
  • An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of active ingredient, for example LIPAM or fragments thereof, antibodies of LIPAM, and agonists, antagonists or inhibitors of LJPAM, which ameliorates the symptoms or condition.
  • Therapeutic efficacy and toxicity may be deterniined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeutically 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 preferred.
  • the data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED 50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration. The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combinations), reaction sensitivities, and response to therapy. Long-acting compositions maybe administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. DIAGNOSTICS
  • antibodies which specifically bind LJPAM maybe used for the diagnosis of disorders characterized by expression of LJPAM, or in assays to monitor patients being treated with LIPAM or agonists, antagonists, or inhibitors of LIPAM.
  • Antibodies useful for diagnostic purposes maybe prepared in the same manner as described above for therapeutics. Diagnostic assays for LIPAM include methods which utilize the antibody and a label to detect LIPAM in human body fluids or in extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
  • a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
  • LIPAM low-density lipoprotein
  • ELISAs ELISAs
  • RIAs RIAs
  • FACS fluorescence-activated cell sorting
  • normal or standard values for LIPAM expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibodies to LIPAM under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of LIPAM expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
  • the polynucleotides encoding LJPAM may be used for diagnostic purposes.
  • the polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of LIPAM may be correlated with disease.
  • the diagnostic assay may be used to determine absence, presence, and excess expression of LJPAM, and to monitor regulation of LIPAM levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding LIPAM or closely related molecules may be used to identify nucleic acid sequences which encode LIPAM.
  • the specificity of the probe whether it is made from a highly specific region, e.g., the 5'regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification will dete ⁇ nine whether the probe identifies only naturally occurring sequences encoding LIPAM, allelic variants, or related sequences.
  • Means for producing specific hybridization probes for DNAs encoding LJPAM include the cloning of polynucleotide sequences encoding LJPAM or LJPAM derivatives into vectors for the production of mRNA probes.
  • Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
  • Hybridization probes maybe labeled by a variety of reporter groups, for example, by radionuclides such as 32 P or 3S S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • Polynucleotide sequences encoding LJPAM may be used for the diagnosis of disorders associated with expression of LJPAM.
  • disorders include, but are not limited to, a cancer, such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; a cardiovascular disorder such as arteriovenous fistula, atherosclerosis, hypertension, vasculitis,, Raynaud's disease, aneurysms, arterial dissections, varicose veins, throm
  • the polynucleotide sequences encoding LIPAM 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 LIPAM expression. Such qualitative or quantitative methods are well known in the art.
  • a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding LJPAM, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
  • 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 allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
  • oligonucleotides designed from the sequences encoding LJPAM may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding LJPAM, or a fragment of a polynucleotide complementary to the polynucleotide encoding LIPAM, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
  • oligonucleotide primers derived from the polynucleotide sequences encoding LIPAM may be used to detect single nucleotide polymorphisms (SNPs).
  • SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans.
  • Methods of SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods.
  • SSCP single-stranded conformation polymorphism
  • fSSCP fluorescent SSCP
  • oligonucleotide primers derived from the polynucleotide sequences encoding LIPAM are used to amplify DNA using the polymerase chain reaction (PCR).
  • the DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
  • SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels.
  • the oligonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high-throughput equipment such as DNA sequencing machines.
  • sequence database analysis methods termed in silico SNP (isSNP) are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence.
  • SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASS ARRAY system (Sequenom, Inc., San Diego CA).
  • SNPs may be used to study the genetic basis of human disease. For example, at least 16 common SNPs have been associated with non-insulin-dependent diabetes mellitus. SNPs are also useful for examining differences in disease outcomes in monogenic disorders, such as cystic fibrosis, sickle cell anemia, or chronic granulomatous disease. For example, variants in the mannose-binding lectin, MBL2, have been shown to be correlated with deleterious pulmonary outcomes in cystic fibrosis. SNPs also have utility in pharmacogenomics, the identification of genetic variants that influence a patient's response to a drug, such as Me-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-lipoxygenase pathway.
  • Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as well as for tracing the origins of populations and their migrations.
  • oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as elements on a microarray.
  • the microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below.
  • the microarray may also be used to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor 5 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.
  • LIPAM LIPAM, fragments of LIPAM, or antibodies specific for LIPAM may be used as elements on a microarray.
  • the microarray may be used to monitor or measure protein-protein interactions, drug-target interactions, and gene expression profiles, as described above.
  • a particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or cell type.
  • a transcript image represents the global pattern of gene expression by a particular tissue or cell type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time. (See Seilhamer et al, "Comparative Gene Transcript Analysis," U.S. Patent No.
  • 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.
  • Microarrays may be prepared, used, and analyzed using methods known in the art.
  • methods known in the art See, e.g., Brennan, TM. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application WO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505; Heller, R.A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; and Heller, M J. et al. (1997) U.S. Patent No. 5,605,662.
  • Various types of microarrays are well known and thoroughly described in DNA Microarrays: A Practical Approach, M. Schena, ed. (1999) Oxford University Press, London, hereby express
  • the sequences maybe mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA libraries.
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • bacterial PI constructions or single chromosome cDNA libraries.
  • the nucleic acid sequences of the invention maybe 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
  • any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • the nucleotide sequence of the instant . invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
  • LJPAM its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques.
  • the fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between LIPAM 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 small test compounds are synthesized on a solid substrate. The test compounds are reacted with LJPAM, or fragments thereof, and washed. Bound LIPAM is then detected by methods well known in the art. Purified LJPAM can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • the nucleotide sequences which encode LJPAM may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
  • cDNA was size-selected (300- 1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Biosciences) or preparative agarose gel electrophoresis.
  • cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCPJPT plasmid (Stratagene), PSPORT1 plasmid (Invitrogen), PCDNA2.1 plasmid
  • plasmid DNA was amplified from host cell lysates using direct link PCR in a Mgh-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland). III. Sequencing and Analysis
  • Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Amersham Biosciences); the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VTJJ.
  • the polynucleotide sequences derived from Incyte cDNAs were validated by removing vector, linker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis.
  • the Incyte cDNA sequences or translations thereof were then queried against a selection of public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans.
  • HMM is a probabilistic approach which analyzes consensus primary structures of gene families. See, for example, Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.)
  • the queries were performed using programs based on BLAST, FASTA, BLJMPS, and HMMER.
  • the Incyte cDNA sequences were assembled to produce full length polynucleotide sequences.
  • GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences were used to extend Incyte cDNA assemblages to full length.
  • a polypeptide of the invention may begin at any of the methionine residues of the full length translated polypeptide.
  • Full length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, bidden Markov model (HMM)-based protein family databases such as PFAM, INCY, and ⁇ GRFAM; and HMM-based protein domain databases such as SMART.
  • GenBank protein databases Genpept
  • PROTEOME databases
  • BLOCKS BLOCKS
  • PRINTS DOMO
  • PRODOM Prosite
  • Prosite bidden Markov model
  • Prosite bidden Markov model
  • HMM-based protein family databases such as PFAM, INCY, and ⁇ GRFAM
  • HMM-based protein domain databases such as SMART.
  • Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
  • Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.
  • Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of
  • Incyte cDNA and full length sequences and provides applicable descriptions, references, and threshold parameters.
  • the first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probability value, the greater the identity between two sequences).
  • Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (See Burge, C and S. Karlin (1997) J. Mol. Biol. 268:78-94, and Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354).
  • the program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon.
  • the output of Genscan is a FASTA database of polynucleotide and polypeptide sequences.
  • Genscan The maximum range of sequence for Genscan to analyze at once was set to 30 kb. To determine which of these Genscan predicted cDNA sequences encode lipid-associated molecules, the encoded polypeptides were analyzed by querying against PFAM models for lipid-associated molecules. Potential lipid-associated molecules were also identified by homology to Incyte cDNA sequences that had been annotated as lipid-associated molecules. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri public databases. Where necessary, the Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct errors in the sequence predicted by Genscan, such as extra or omitted exons.
  • BLAST analysis was also used to find any Incyte cDNA or public cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription. When Incyte cDNA coverage was available, this information was used to correct or confirm the Genscan predicted sequence.
  • Full length polynucleotide sequences were obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or public cDNA sequences using the assembly process described in Example JU. 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 JV. 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 full length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by transitivity.
  • 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.
  • GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the public human genome databases. Partial DNA sequences were therefore "stretched” or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene.
  • sequences which were used to assemble SEQ JD NO: 11-20 were compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith- Waterman dgorithm. Sequences from these databases that matched SEQ JD NO: 11-20 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of all sequences of that cluster, including its particular SEQ JD NO:, to that map location.
  • SHGC Stanford Human Genome Center
  • WIGR Whitehead Institute for Genome Research
  • 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 public, such as the NCBI "GeneMap'99" World Wide Web site
  • the product score takes into account both the degree of similarity between two sequences and the length of the sequence match.
  • the product score is a normalized value between 0 and 100, and is calculated as follows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences).
  • the BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP (separated by gaps). If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score.
  • the product score represents a balance between fractional overlap and quality in a BLAST alignment.
  • 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 LJPAM 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 HI). Each cDNA sequence is derived from a cDNA library constructed from a human tissue.
  • Each human tissue is classified into one of the following organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitalia, female; genitalia, male; germ cells; hemic and immune system; liver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract.
  • the number of libraries in each category is counted and divided by the total number of libraries across all categories.
  • each human tissue is classified into one of the following disease/condition categories: cancer, cell line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of libraries in each category is counted and divided by the total number of libraries across all categories. The resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding LIPAM.
  • One primer was synthesized to initiate 5' extension of the known fragment, and the other primer was synthesized to initiate 3 ' extension of the known fragment.
  • the initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72°C Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
  • Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed. High fidelity amplification was obtained by PCR using methods well known in the art. PCR was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.).
  • the reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg 2+ , (N ⁇ ⁇ SO ⁇ and 2-mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE enzyme (Invitrogen), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C
  • the parameters for primer pair T7 and SK+ were as follows: Step 1: 94 °C, 3 min; Step 2: 94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 °C, 5
  • the plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
  • a 5 l to 10 ⁇ l aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to determine which reactions were successful in extending the sequence.
  • the extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison Wl), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Biosciences).
  • Step 1 94°C, 3 min
  • Step 2 94 °C, 15 sec
  • Step 3 60°C, 1 min
  • Step 4 72°C, 2 min
  • Step 5 steps 2, 3, and 4 repeated 29 times
  • Step 6 72°C, 5 min
  • Step 7 storage at 4°C DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamplified using the same conditions as described above.
  • SNPs single nucleotide polymorphisms
  • LIFESEQ database Incyte Genomics
  • Sequences from the same gene were clustered together and assembled as described in Example JH, allowing the identification of all sequence variants in the gene.
  • An algorithm consisting of a series of filters was used to distinguish SNPs from other sequence variants. Preliminary filters removed the majority of basecall errors by requiring a minimum Phred quality score of 15, and removed sequence alignment errors and errors resulting from improper trirruning of vector sequences, chimeras, and splice variants.
  • An automated procedure of advanced chromosome analysis analysed the original chromatogram files in the vicinity of the putative SNP.
  • Clone error filters used statistically generated algorithms to identify errors introduced during laboratory processing, such as those caused by reverse transcriptase, polymerase, or somatic mutation.
  • Clustering error filters used statistically generated algorithms to identify errors resulting from clustering of close homologs or pseudogenes, or due to contamination by non-human sequences.
  • a final set of filters removed duplicates and SNPs found in immunoglobulins or T-cell receptors. Certain SNPs were selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at the SNP sites in four different human populations.
  • the Caucasian population comprised 92 individuals (46 male, 46 female), including 83 from Utah, four French, three Venezuelan, and two Amish individuals.
  • the African population comprised 194 individuals (97 male, 97 female), all African Americans.
  • the Hispanic population comprised 324 individuals (162 male, 162 female), all Mexican Hispanic.
  • the Asian population comprised 126 individuals (64 male, 62 female) with a reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian. Allele frequencies were first analyzed in the Caucasian population; in some cases those SNPs which showed . no allelic variance in this population were not further tested in the other three populations. X. Labeling and Use of Individual Hybridization Probes
  • Hybridization probes derived from SEQ ID NO:11-20 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 Ci of
  • [ ⁇ - 32 P] adenosine tiiphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
  • the labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dextranbead column (Amersham Biosciences). 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 following endonucleases: Ase I, Bgl H, 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 & Schuell, Durham NH). Hybridization is carried out for 16 hours at 40 °C. To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared. XL Microarrays
  • the linkage or synthesis of array elements upon a microarray can be achieved utilizing photolithography, piezoelectric printing (ink-jet printing, See, e.g., Baldeschweiler, supra.), mechanical rmcrospotting technologies, and derivatives thereof.
  • the substrate in each of the aforementioned technologies should be uniform and solid with a non-porous surface (Schena (1999), supra). Suggested substrates include silicon, silica, glass slides, glass chips, and silicon wafers. Alternatively, a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
  • a typical array may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropriate number of elements. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)
  • Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers thereof may comprise the elements of the microarray. Fragments or oligomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR).
  • the array elements are hybridized with polynucleotides in a biological sample.
  • the polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
  • a fluorescence scanner is used to detect hybridization at each array element.
  • laser desorbtion and mass spectrometry may be used for detection of hybridization.
  • the degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microarray may be assessed.
  • microarray preparation and usage is described in detail below.
  • RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A) + RNA is purified using the oligo-(dT) cellulose method.
  • Each poly(A) + RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg ⁇ l oligo-(dT) primer (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 Biosciences).
  • the reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A) + RNA with GEMBRIGHT kits
  • 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 array elements.
  • Each array element is amphfied from bacterial cells containing vectors with cloned cDNA inserts.
  • PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert.
  • Array elements are amplified in Ihirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g.
  • Amplified array elements are then purified using SEPHACRYL-400 (Amersham Biosciences).
  • Purified array elements are immobilized on polymer-coated glass slides.
  • Glass microscope slides (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments.
  • Glass slides are etched in 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are cured in a 110°C oven.
  • Array elements are applied to the coated glass substrate using a procedure described in U.S.
  • Patent No. 5,807,522 incorporated herein by reference.
  • 1 ⁇ l of the array element DNA, at an average concentration of 100 ng/ ⁇ l, is loaded into the open capillary printing element by a high-speed robotic apparatus.
  • the apparatus then deposits about 5 nl of array element sample per slide.
  • Microarrays are UV-crosslihked using a STRATALINKER UV-crosslinker (Stratagene).
  • Microarrays are washed at room temperature once in 0.2% SDS and three times in distilled water.
  • Non-specific binding sites are blocked by incubation of microarrays in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc. , Bedford MA) for 30 minutes at 60° C followed by washes in 0.2%
  • PBS phosphate buffered saline
  • Hybridization reactions contain 9 ⁇ l of sample mixture consisting of 0.2 ⁇ g each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
  • the sample mixture is heated to 65° C for 5 minutes and is aliquoted onto the microarray surface and covered with an 1.8 cm 2 coverslip.
  • the arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber is kept at 100% humidity internally by the addition of 140 ⁇ l of 5X SSC in a corner of the chamber.
  • the chamber containing the arrays is incubated for about 6.5 hours at 60°C
  • the arrays are washed for 10 min at 45°C in a first wash buffer (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 light is focused on the array using a 20X microscope objective (Nikon, Inc., Melville NY).
  • the slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster- scanned past the objective.
  • the 1.8 cm x 1.8 cm array used in the present example is scanned with a resolution of 20 micrometers.
  • a mixed gas multiline laser excites the two fluorophores sequentially. Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultiplier tubes are used to filter the signals.
  • the emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5.
  • Each array is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
  • the sensitivity of the scans is typically calibrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration.
  • a specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000.
  • the calibration is done by labeling samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
  • the output of the photomultiplier tube is digitized using a 12-bit RTT-835H analog-to-digital (AID) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-compatible PC computer.
  • the digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
  • the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore 's emission spectrum.
  • a grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid.
  • the fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal.
  • the software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte). Array elements that exhibited at least about a two-fold change in expression, a signal-to-background ratio of at least 2.5, and an element spot size of at least 40% were identified as differentially expressed using the GEMTOOLS program (Incyte Genomics). Expression
  • microarray analysis can be used to monitor the expression of SEQ JD NO: 17 and SEQ JD NO:18, which encode the polypeptides of SEQ ID NO:7 and SEQ JD NO:8 respectively, in lymphoblast cell lines.
  • B lymphoblastic cells derived from a patient with Hodgkin's disease (i.e., RPMI 6666) and from a patient with Burkitt's lymphoma (i.e., Raji) were treated with l ⁇ g/ml lipopolysaccharide (LPS) oruntreated. Cells were harvested at 0.5, 1, 2, 4, and 8 hours and microarray analysis was performed as described above.
  • the lymphoblast cells showed a greater than two-fold decrease in the SEQ JD NO: 17 mRNA levels, primarily at the 8 hour time point and a greater than two-fold decrease in the SEQ JD NO: 18 mRNA levels, primarily at the 4 and 8 hour time points in LPS-treated versus untreated cells. These results are consistent with reduced requirements for oxysterol binding proteins as stimulated cells channel more sterol intermediates into cholesterol synthesis to support increased plasma membrane synthesis. The levels of oxysterol derivatives, normally resulting from the autoxidation of accumulated sterol derivatives or specific cytochrome P450-mediated oxidation are reduced.
  • SEQ ID NO: 17 and SEQ ID NO: 18 are individually useful for monitoring the levels of oxysterol binding protein following the LPS-induced stimulation of B lymphoblast-derived cells for the purposes of disease detection and expression profiling.
  • Sequences complementary to the LIPAM-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring LJPAM. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of LIPAM. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the LIPAM-encoding transcript. XIII.
  • LIPAM expression and purification of LIPAM is achieved using bacterial or virus-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 limited to, the p-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 LJPAM upon induction with isopropyl beta-D- thiogalactopyranoside (IPTG).
  • LIPAM in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autograpbica caHfornica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus.
  • AcMNPV Autograpbica caHfornica nuclear polyhedrosis virus
  • the nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding LIPAM by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription.
  • Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases.
  • LIPAM is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates.
  • GST glutathione S-transferase
  • a peptide epitope tag such as FLAG or 6-His
  • FLAG an 8-amino acid peptide
  • 6-His a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel (1995, supra, ch. 10 and 16). Purified LIPAM obtained by these methods can be used directly in the assays shown in Examples XVH and XVJH where applicable. XIV. Functional Assays
  • LIPAM function is assessed by expressing the sequences encoding LIPAM at physiologically elevated levels in mammalian cell culture systems.
  • cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include PCMV SPORT plasmid (Invitrogen, Carlsbad CA) and PCR3.1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation.
  • 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected.
  • Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector.
  • Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein.
  • Flow cytometiy (FCM) an automated, laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties.
  • FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometiy are discussed in Ormerod, M.G. (1994) Flow Cvtometrv, Oxford, New York NY.
  • LJPAM The influence of LJPAM on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding LIPAM and either CD64 or CD64-GFP.
  • CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG).
  • Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
  • mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding LJPAM and other genes of interest can be analyzed by northern analysis or microarray techniques.
  • LIPAM substantially purified using polyacrylamide gel electrophoresis (PAGE; see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize animals (e.g., rabbits, mice, etc.) and to produce antibodies using standard protocols.
  • PAGE polyacrylamide gel electrophoresis
  • LJPAM amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art.
  • LASERGENE software DNASTAR
  • Methods for selection of appropriate epitopes, such as those near the C-te ⁇ ninus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
  • oligopeptides of about 15 residues in length are synthesized using an ABI 43 IA peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-malein ⁇ dobenzoyl-N-hy ⁇ oxysuccinimide ester (MBS) to increase immunogenicity.
  • ABI 43 IA peptide synthesizer Applied Biosystems
  • KLH Sigma- Aldrich, St. Louis MO
  • MBS N-malein ⁇ dobenzoyl-N-hy ⁇ oxysuccinimide ester
  • Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant.
  • Resulting antisera are tested for antipeptide and anti-LJPAM activity by, for example, binding the peptide or LIPAM to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
  • Naturally occurring or recombinant LIPAM is substantially purified by immunoaffinity chromatography using antibodies specific for LIPAM.
  • An immunoaffinity column is constructed by covalently coupling anti-LIPAM antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Biosciences). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
  • LIPAM Media containing LJPAM are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of LIPAM (e.g., high ionic strength buffers in the presence of detergent).
  • the column is eluted under conditions that disrupt antibody/LIPAM 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 LIPAM is collected.
  • LJPAM or biologically active fragments thereof, are labeled with 1 5 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 arrayed in the wells of a multi-well plate are incubated with the labeled LJPAM, washed, and any wells with labeled LJPAM complex are assayed. Data obtained using different concentrations of LIPAM are used to calculate values for the number, affinity, and association of LJPAM with the candidate molecules.
  • LJPAM molecules interacting with LJPAM are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
  • LIPAM 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 all interactions between the proteins encoded by two large libraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101). XVIII. Demonstration of LIPAM Activity
  • LJPAM activity is determined in a continuous fluorescent transfer assay using as substrate l-palmitoyl-2-pyrenyldecanoyl-phosphatidyHnositol (Phy(lO)PI).
  • the assay measures the increase of pyrene monomer fluorescence intensity as a result of the transfer of pyrenylacyl (Pyr(x ))-labeled phospholipid from quenched donor vesicles to unquenched acceptor vesicles (Van Paridon et al. (1988) Biochemistry 27:6208-6214).
  • Donor vesicles consist of Pyr(x ) phosphatidylinositol (Pyr( )PI), 2,4,6-trinitiophenylphosphatidyle anolamine (TNP-PE) and egg phosphatidylcholine (PC) in a mol % ratio of 10:10:80 (2 nmol of total phospholipid).
  • Acceptor vesicles consist of phosphatidic acid (PA) and egg PC in a mol % ratio of 5:95 (25-fold excess of total phospholipid).
  • the reaction is carried out in 2 ml of 20 mM Tris-HCl, 5 mM EDTA, 200 mM NaCI (pH 7.4) containing 0.1 mg of BSA at 37 °C.
  • the reaction is initiated by the addition of 10-50 ⁇ l of LIPAM. Measurements are performed using a fluorimeter equipped with a thermostated cuvette holder and a stirring device. The initial slope of the progress curve is taken as an arbitrary unit of transfer activity (van Tiel, CM. et al. (2000) J. Biol. Chem. 275:21532-21538; Westerman, J. et al. (1995) J. Biol. Chem. 270:14263-14266).
  • LJPAM activity is determined by measuring the rate of incorporation of a radioactive fatty acid precursor into fatty acyl-CoA.
  • the final reaction contains 200 mM Tris-HCl, pH 7.5, 2.5 mM ATP, 8 mM MgC ⁇ , 2mM EDTA, 20 mM NaF, 0.1% Triton X-100, 10 mM [ 3 H]oleate, pHjmyristate or [ 14 C]decanoate, 0.5 mM coenzyme A, and LIPAM in a total volume of 0.5 ml.
  • the reaction is initiated with the addition of coenzyme A, incubated at 35 °C for 10 min, and teiminated by the addition of 2.5 ml of isopropyl alcohol, n-heptane, l M H j SO 4 (40:10:1). Radioactive fatty acid is removed by organic extraction using n-heptane. Fatty acyl-CoA formed during the reaction remains in the aqueous fraction and is quantified by scintillation counting (Black, P.N. et al. (1997) J. Biol. Chem. 272: 4896-4904).
  • LJPAM activity is determined by measuring the degradation of the sphingohpid glucosylceramide. 25-50 microunits glucocerebrosidase are incubated with varying concentrations of LIPAM in a 40 ⁇ l reaction at 37 °C for 20 min. The final reaction contains 50mM sodium citrate pH 4.5, 20 ng human serum albumin, and 3.125 mM lipids in the form of liposomes, which contain Hpids in the following proportions: [ 14 C]glucosylceramide (3 mol %, 2.4 Ci/mol), cholesterol (23 mol %), phosphatidic acid (20 mol %), phosphatidylcholine (54 mol %).
  • LJPAM activity can be demonstrated by an in vitro hydrolysis assay with vesicles containing l-palmitoyl-2-[l- 14 C]oleoyl phosphatidylcholine (Sigma- Aldrich).
  • LIPAM triglyceride lipase activity and phospholipase A 2 activity are demonstrated by analysis of the cleavage products isolated from the hydrolysis reaction mixture.
  • the supernatant maybe stored at 4 °C
  • the final reaction mixtures contain 0.25 ml of Hanks buffered salt solution supplemented with 2.0 mM taurochenodeoxycholate, 1.0% bovine serum albumin, 1.0 mM CaCl j , pH 7.4, 150 ⁇ g of l-palmitoyl-2-[l- 14 C]oleoyl phosphatidylcholine vesicles, and various amounts of LIPAM diluted in PBS. After incubation for 30 min at 37 °C, 20 ⁇ g each of lyso-phosphatidylcholine and oleic acid are added as carriers and each sample is extracted for total lipids.
  • the lipids are separated by thin layer chromatography using a two solvent system of chloroform:methanol:acetic acid:water (65:35:8:4) until the solvent front is halfway up the plate. The process is then continued with hexane:ether:acetic acid (86:16:1) until the solvent front is at the top of the plate.
  • the Epid-containing areas are visualized with I 2 vapor; the spots are scraped, and their radioactivity is determined by scintillation counting. The amount of radioactivity released as fatty acids will increase as a greater amount of LIPAM is added to the assay mixture while the amount of radioactivity released as lysophosphatidylcholine will remain low.
  • LJPAM cleaves at the sn-2 and not the sn-1 position, as is characteristic of phospholipase A 2 activity.
  • phospholipase activity of LJPAM is measured by the hydrolysis of a fatty acyl residue at the sn-1 position of phosphatidylserine.
  • LJPAM is combined with the tritium [ 3 H] labeled substrate phosphatidylserine at stoichiometric quantities in a suitable buffer. Following an appropriate incubation time, the hydrolyzed reaction products are separated from the substrates by chromatographic methods.
  • Lipoxygenase activity of LIPAM can be measured by chromatographic methods. Extracted LJPAM lipoxygenase protein is incubated with 100 ⁇ M [1- 14 C] arachidonic acid or other unlabeled fatty acids at 37°C for 30 min. After the incubation, stop solution (acetonitrile:methanol:water, 350:150:1) is added. The samples are extracted and analyzed by reverse-phase HPLC using a solvent system of methanol/water/acetic acid, 85:15:0.01 (vol/vol) at a flow rate of 1 ml/min.
  • the effluent is monitored at 235 nm and analyzed for the presence of the major arachidonic metabolite such as 12- HPET ⁇ (catalyzed by 12-LOX).
  • the fractions are also subjected to liquid scintillation counting.
  • the final counts represent the products, which is directly proportional to the activity of LJPAM in biological samples.
  • the metabolites of arachidonic acid are analyzed further by chiral phase-HPLC and by mass spectrometry (Sun, D. et al. (1998) J. Biol. Chem. 273:33540-33547).
  • the reaction mixture contains 30 nmol substrate, 0.2 mg bovine serum albumin, 10 ⁇ mol sodium acetate (pH 4.6), 0.2 mg Triton X-100, and purified LJPAM (or a sample containing LJPAM).
  • the released sialic acid is quantified using the thiobarbituric acid method (Aminoff, D. (1961) Biochem. J. 81:384-392 ).
  • One unit of sialidase activity is defined as the amount of LJPAM that catalyzes the release of 1 nmol of sialic acid from substrate per hour (Hasegawa, T. et al. (2000) J. Biol. Chem.

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Abstract

L'invention concerne des molécules associées à des lipides de l'homme et des polynucléotides qui identifient et codent ces molécules. L'invention a aussi pour objet des vecteurs d'expression, des cellules hôtes, des anticorps, des agonistes et des antagonistes. L'invention traite également de procédés permettant de diagnostiquer, traiter ou prévenir des troubles associés à l'expression aberrante de ces molécules.
EP02736944A 2001-05-18 2002-05-17 Molecules associees a des lipides Withdrawn EP1481081A2 (fr)

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US29224201P 2001-05-18 2001-05-18
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US29534601P 2001-06-01 2001-06-01
US295346P 2001-06-01
US30340401P 2001-07-06 2001-07-06
US303404P 2001-07-06
US31475401P 2001-08-24 2001-08-24
US314754P 2001-08-24
US35126202P 2002-01-22 2002-01-22
US351262P 2002-01-22
US36879902P 2002-03-29 2002-03-29
US368799P 2002-03-29
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