Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
  • C12N9/20—Triglyceride splitting, e.g. by means of lipase
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to nucleic acid and amino acid sequences of lipid metabolism enzymes and to the use of these sequences in the diagnosis, treatment, and prevention of cancer, neurological disorders, autoimmune/inflammatory disorders, gastrointestinal disorders, and cardiovascular disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of lipid metabolism enzymes.
• Lipids are water-insoluble, oily or greasy substances that are soluble in nonpolar solvents such as chloroform or ether.
• Neutral fats triacylglycerols
• Polar lipids such as phospholipids, sphingolipids, glycolipids, and cholesterol, are key structural components of cell membranes.
• 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.
• 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.
• CoA acetyl-Coenzyme A
• ACC carboxylase
• FAS fatty acid synthase
• 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.
• 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-Co A' s are produced from fatty acids by fatty acyl-CoA synthetases. Glyercol-3 -phosphate is acylated with two fatty acyl-CoA's 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.
• 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 emanolamine, 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).
• Cholesterol composed of four fused hydrocarbon rings with an alcohol at one end, moderates the fluidity of membranes in which it is incorporated.
• 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. (1991) Textbook of Medical Physiology, W.B. Saunders Company, Philadelphia PA, pp. 760-763; Stryer, supra, pp. 279-280, 691-702, 934). Mammals obtain cholesterol derived from both de novo biosynthesis and the diet.
• Sphingolipids are an important class of membrane lipids that contain sphingosine, a long chain amino alcohol. They are composed of one long-chain fatty acid, one polar head alcohol, and sphingosine or sphingosine derivatives.
• the three classes of sphingolipids are sphingomyelins, cerebrosides, and gangliosides. Sphingomyelins, which contain phosphocholine or phosphoe anolamine as their head group, are abundant in the myelin sheath surrounding nerve cells.
• Galactocerebrosides which contain a glucose or galactose head group, are characteristic of the brain. Other cerebrosides are found in non-neural tissues.
• Eicosanoids including prostaglandins, prostacyclin, thromboxanes, and leukotrienes, 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. Within cells, fatty acids are transported by cytoplasmic fatty acid binding proteins (Online
• 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-regulate the effects of GABA.
• GABA ⁇ -aminobutyric acid
• DBI binds medium- and long-chain acyl-CoA esters with very high affinity and may function as an intracellular carrier of acyl-CoA esters (OMHVI * 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 and cholesterol esters in the blood are transported in lipoprotein particles.
• 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).
• VLDL very-low-density lipoproteins
• DDL intermediate- density lipoproteins
• LDL low-density lipoproteins
• HDL high-density lipoproteins
• 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 mitochondrial 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, xenobiotics, 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.
• Lipases Three classes of lipid metabolism enzymes are discussed in further detail. The three classes are lipases, phospholipases and lipoxygenases. Lipases
• 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 upases also play a major role in the regulation of plasma lipids.
• Pancreatic lipase requires a small protein cofactor, colipase, for efficient dietary lipid hydrolysis. Colipase binds to the C-terminal, non-catalytic domain of lipase, thereby stabilizing an active conformation and considerably increasing the overall hydrophobic binding site. Deficiencies of these enzymes have been identified in man, and all are associated with pathologic levels of circulating lipoprotein particles (Gargouri, Y. et al. (1989) Biochim. Biophys. Acta 1006:255-271; Connelly, P.W. (1999) Clin. Chim. Acta 286:243-255; van Tilbeurgh, H. et al. (1999) Biochim Biophys Acta 1441:173-184).
• 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-II 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.
• 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 inflammatory 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) which are involved in pain, fever, and inflammation (Kaiser, E. et al.
• 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-
• 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 III 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, II, and HI PLA2s found in snake and bee venoms. These venom PLA2s share many features with mammalian PLA2s including a common catalytic mechanism, the same Ca + 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, DA, EC, and N have been described in mammalian and avian cells, and were originally characterized by tissue distribution, although the distinction is no longer absolute. Thus, Group I PLA2s were found in the pancreas, Group HA and DC 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 mflammatory processes and are highly expressed in serum and synovial fluids of patients with iiiflammatory 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 lysolecifl ⁇ nase are widely distributed enzymes that metabolize intracellular lipids, 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; SeUe, 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 inositol 1, 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 Xhas 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-region, one of two conserved regions.
• the hydrolysis of common inositol-containing phospholipids, such as phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4, 5-bisphosphate (P1P2), 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 JH
• choline phosphatase catalyzes the hydrolysis of phosphatidylcholine and other phospholipids to generate phosphatidic acid.
• PLD plays an important role in membrane vesicle frafficking, 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 agonists 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
• Lipoxygenases are non-heme on-containing enzymes that catalyze the dioxygenation of certain polyunsaturated fatty acids such as lipoproteins. Lipoxygenases are found widely in plants, fungi, and animals. Several different lipoxygenase enzymes are known, each having a characteristic oxidation action. In animals, there are specific lipoxygenases that catalyze the dioxygenation of arachidonic acid at the carbon-3, 5, 8, 11, 12, and 15 positions. These enzymes are named after the position of arachidonic acid that they dioxygenate.
• Lipoxygenases have a single polypeptide chain with a molecular mass of -75-80 kDa in animals.
• the proteins have an N-te ⁇ ninal-barrel domain and a larger catalytic domain containing a single atom of non-heme iron. 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.
• 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-Lipoxygenase (5-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-hydroperoxyeicosatetraenoic 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.
• conjugation of LTA4 with glutathione by leukotriene C4 synthase plus downstream metabolism leads to the cysteinyl leukotrienes that influence airway reactivity and mucus secretion, especially in asthmatics.
• Most lipoxygenases require no other cofactors or proteins for activity.
• the mammalian 5-LOX requires calcium and ATP, and is activated in the presence of a 5- LOX activating protein (FLAP).
• FLAP itself binds to arachidonic acid and supplies 5-LOX with substrate (Lewis, R.A. et al. (1990) New Engl. J. Med. 323:645-655).
• the expression levels of 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. Grit. 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).
• Lipid metabolism is involved in human diseases and disorders.
• 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).
• the GM 2 ganglioside a sphingolipid
• the GM 2 ganglioside accumulates in lysosomes of the central nervous system due to a lack of the enzyme N-acetylhexosaminidase.
• Patients suffer nervous system degeneration leading to early death (Fauci, A.S. et al. (1998) Harrison's Principles of Internal Medicine, McGraw-Hill, New York NY, p. 2171).
• the Niemann-Pick diseases are caused by defects in lipid metabolism.
• 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.
• 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.
• the invention features purified polypeptides, lipid metabolism enzymes, referred to collectively as “LMM” and individually as “LMM-1,” “LMM-2,” “LMM-3,” “LMM-4,” “LMM-5,” and “LMM- 6.”
• the invention provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-6, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-6, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-6.
• the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:l-6.
• the invention further provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-6, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l- 6, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-6, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6.
• the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO: 1-6.
• the polynucleotide is selected from the group consisting of SEQ ID NO:7-12.
• the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-6, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-6, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-6.
• the invention also provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-6, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-6, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6.
• the method comprises a) culturing.
• a cell under conditions suitable for expression of the polypeptide wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
• the invention provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-6, 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: 1-6, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6.
• 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:7-12, 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:7-12, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
• the polynucleotide comprises at least 60 contiguous nucleotides.
• the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:7-12, 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:7-12, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
• the method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof.
• the probe comprises at least 60 contiguous nucleotides.
• the invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:7-12, 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:7-12, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
• the method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
• the invention further provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-6, 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: 1-6, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-6, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-6, and a pharmaceutically acceptable excipient.
• the composition comprises an amino acid sequence selected from the group consistmg of SEQ ID NO: 1-6.
• the invention additionally provides a method of treating a disease or condition associated with decreased expression of functional LMM, comprising administering to a patient in need of such treatment the composition.
• the invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-6, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-6.
• 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 LMM, 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:l-6, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-6, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-6, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6.
• 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 LMM, comprising administering to a patient in need of such treatment the composition.
• the invention further provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6, 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: 1-6, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-6.
• 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 ID NO:l-6, 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: 1-6, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6.
• the method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
• the invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:7-12, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide.
• the invention further provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:7-12, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:7-12, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
• Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:7-12, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:7-12, iii) a poly ⁇ ucleotide 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 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the present invention.
• Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog for polypeptides of the invention. The probability score for the match between each polypeptide and its GenBank homolog is 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.
• a reference to “a host cell” includes a plurality of such host cells
• a reference to “an antibody” is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
• all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.
• any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the prefened machines, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. None herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. DEFINITIONS
• LMM refers to the amino acid sequences of substantially purified LMM 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 LMM.
• Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of LMM either by directly interacting with LMM or by acting on components of the biological pathway in which LMM participates.
• allelic variant is an alternative form of the gene encoding LMM. 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 LMM include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as LMM or a polypeptide with at least one functional characteristic of LMM. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding LMM, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding LMM.
• the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent LMM.
• Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of LMM is retained.
• negatively charged amino acids may include aspartic acid and glutamic acid
• positively charged amino acids may include lysine and arginine.
• Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine.
• Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
• amino acid and amino acid sequence refer to an oligopepti.de, 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 earned out using polymerase chain reaction (PCR) technologies well known in the art.
• PCR polymerase chain reaction
• Antagonist refers to a molecule which inhibits or attenuates the biological activity of LMM.
• Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of LMM either by directly interacting with LMM or by acting on components of the biological pathway in which LMM 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 LMM 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.
• a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (particular regions or three-dimensional structures on the protein).
• An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
• antisense refers to any composition capable of base-pairing with the "sense”
• 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.
• 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 LMM, 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 LMM or fragments of LMM 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 In hybridizations, the probe maybe deployed in an aqueous solution containing salts (e.g., NaCI), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardfs solution, dry milk, salmon sperm DNA, etc.).
• salts e.g., NaCI
• detergents e.g., sodium dodecyl sulfate; SDS
• other components e.g., Denhardfs solution, dry milk, salmon sperm DNA, etc.
• Consensus sequence refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (Applied Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison WI) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
• 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 substitation, 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 LMM or the polynucleotide encoding LMM 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 may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments maybe 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 ID NO:7-12 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ID NO:7-12, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
• a fragment of SEQ ID NO:7-12 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO:7-12 from related polynucleotide sequences.
• the precise length of a fragment of SEQ ID NO:7-12 and the region of SEQ ID NO:7-12 to which the fragment co ⁇ esponds 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: 1-6 is encoded by a fragment of SEQ ID NO:7-12.
• a fragment of SEQ ID NO:l-6 comprises a region of unique amino acid sequence that specifically identifies SEQ ID NO: 1-6.
• a fragment of SEQ ID NO: 1-6 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:l-6.
• the precise length of a fragment of SEQ ID NO:l-6 and the region of SEQ ID NO:l-6 to which the fragment co ⁇ esponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
• a “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon.
• a “full length” polynucleotide sequence encodes a "full length” polypeptide sequence.
• Homology refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
• percent identity and % identity 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.
• BLAST Basic Local Alignment Search Tool
• NCBI National Center for Biotechnology Information
• BLAST Basic Local Alignment Search Tool
• the BLAST software suite includes various sequence analysis programs including "blastn ,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases.
• blastn 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.nih.gov/gorf/bl2.html.
• the "BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed below). BLAST programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) set at default parameters.
• Such default parameters may be, for example: Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch: -2 Open Gap: 5 and Extension Gap: 2 penalties Gap x drop-off: 50 Expect: 10 Word Size: 11 Filter: on
• Percent identity maybe measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
• Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
• nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
• 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 and_hydrophobicity at the site of substitation, thus preserving the structure (and therefore function) of the polypeptide.
• Percent identity maybe measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
• Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, maybe used to describe a length over which percentage identity may be measured.
• HACs Human artificial chromosomes
• HACs are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain all of the elements required for chromosome replication, segregation and maintenance.
• humanized antibody refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
• Hybridization refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s).
• the washing step(s) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
• Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 ⁇ g/ml sheared, denatured salmon sperm DNA.
• wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (T-.) for the specific sequence at a defined ionic strength and pH.
• T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
• High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1% SDS, for 1 hour. Alternatively, temperatures of about 65 °C, 60°C, 55 °C, or 42°C maybe used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1 %.
• blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ⁇ g/ml.
• Organic solvent such as formamide at a concentration of about 35-50% v/v
• RNA:DNA hybridizations Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art.
• Hybridization particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
• 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 Rot analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
• immunoreactive response can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
• An "immunogenic fragment” is a polypeptide or oligopeptide fragment of LMM which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal.
• immunogenic fragment also includes any polypeptide or oligopeptide fragment of LMM 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 "a ⁇ ay 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 LMM. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of LMM.
• 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.
• PNA peptide nucleic acid
• operably linked refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence.
• a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
• Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
• PNA protein nucleic acid
• PNA refers to an antisense molecule or anti-gene agent which comprises an 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 LMM 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 LMM.
• Probe refers to nucleic acid sequences encoding LMM, 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.
• 'Trimers are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme.
• Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, 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.
• the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope.
• the Primer3 primer selection program (available to the public from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) allows the user to input a "n ⁇ spriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microa ⁇ ays.
• the source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.
• the PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments.
• 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 maybe part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
• a "regulatory element” refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
• Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.
• 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 occu ⁇ ences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
• sample is used in its broadest sense.
• a sample suspected of containing LMM, nucleic acids encoding LMM, 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.
• binding and “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
• substantially purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.
• substitution refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
• Substrate refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
• the substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
• a "transcript image” refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.
• Transformation describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on 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 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%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length.
• a variant may be described as, for example, an "allelic” (as defined above), "splice,” “species,” or “polymorphic” variant.
• a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
• the conesponding. polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
• Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides will generally have significant amino acid identity relative to each other.
• a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
• SNPs single nucleotide polymorphisms
• 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 metabolism enzymes (LMM), the polynucleotides encoding LMM, and the use of these compositions for the diagnosis, treatment, or prevention of cancer, neurological disorders, automimune/inflammatory disorders, gastrointestinal disorders, and cardiovascular disorders.
• LMM human lipid metabolism enzymes
• Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the invention.
• Each polynucleotide and its conesponding polypeptide are conelated to a single Incyte project identification number (Incyte Project ID).
• Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide ID) as shown.
• polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as shown.
• Table 2 shows sequences with homology to the polypeptides of the invention as identified by
• FIG. 1 shows the polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the conesponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention.
• Column 3 shows the GenBank identification number (Genbank ID NO:) of the nearest GenBank homolog.
• Column 4 shows the probability score for the match between each polypeptide and its GenBank homolog.
• Column 5 shows the annotation of the GenBank homolog 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
• FIG. 3 shows the number of amino acid residues in each polypeptide.
• Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS program of the GCG sequence analysis software package (Genetics Computer Group, Madison WI).
• Column 6 shows amino acid residues comprising signature sequences, domains, and motifs.
• Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.
• SEQ ID NO:l is 53% identical to human lysosomal acid lipase (GenBank ID g505053) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 5.2e- 116, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:l also contains an alpha/beta hydrolase fold as dete ⁇ nined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein domains.
• HMM hidden Markov model
• SEQ ID NO:4 is 63% identical to human pregastric lipase (GenBank ID g579561) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 1.4e- 142, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:4 also contains an alpha/beta hydrolase fold 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:4 is a lipase.
• SEQ ID NO:5 is 35% identical to human arylacetamide deacetylase (GenBank ID g537514) as dete ⁇ nined by the Basic Local Alignment Search Tool (BLAST).
• the BLAST probability score is 1.6e-47, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
• SEQ ID NO:6 is 51 % identical to murine phospholipase C-L2 (GenBank ID g6705987) as determined by the Basic Local Alignment Search Tool (BLAST).
• SEQ ID NO:6 also contains phosphatidylinositol-specific phospholipase X and Y domains, a C2 domain, and a pleckstrin homology 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
• PROFTLESCAN analyses provide further conoborative evidence that SEQ ID NO:6 is a phospholipase.
• SEQ ID NO:2 and SEQ ID NO:3 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ ID NO:l-6 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.
• Columns 1 and 2 list the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and the conesponding Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) for each polynucleotide of the invention.
• Column 3 shows the length of each polynucleotide sequence in basepairs.
• Column 4 lists fragments of the polynucleotide sequences which are useful, for example, in hybridization or amplification technologies that identify SEQ ID NO:7-12 or that distinguish between SEQ ID NO:7-12 and related polynucleotide sequences.
• Column 5 shows identification numbers corresponding to cDNA sequences, coding sequences (exons) predicted from genomic DNA, and/or sequence assemblages comprised of both cDNA and genomic DNA. These sequences were used to assemble the full length polynucleotide sequences of the invention.
• Columns 6 and 7 of Table 4 show the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences in column 5 relative to their respective full length sequences.
• the identification numbers in Column 5 of Table 4 may refer specifically, for example, to Incyte cDNAs along with their conesponding cDNA libraries.
• 6875328H1 is the identification number of an Incyte cDNA sequence
• EP1MUNN04 is the cDNA library from which it is derived.
• Incyte cDNAs for which cDNA libraries are not indicated were derived from pooled cDNA libraries (e.g., 71075936V1).
• the identification numbers in column 5 may refer to GenBank cDNAs or ESTs (e.g., g5369512) which contributed to the assembly of the full length polynucleotide sequences.
• the identification numbers in column 5 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences including the designation "ENST”).
• the identification numbers in column 5 maybe derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences including the designation "NM” or “NT”) or the NCBI RefSeq Protein Sequence Records (ie. , those sequences including the designation "NP”).
• the identification numbers in column 5 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm.
• FLJXXXXXXX_N 1 _N 2 _YYYY_N 3 _N 4 represents a "stitched" sequence in which XXXXX is the identification number of the cluster of sequences to which the algorithm was applied, and ITOTis the number of the prediction generated by the algorithm, and N 1A3, stamp , if present, represent specific exons that may have been manually edited during analysis (See Example V).
• the identification numbers in column 5 may refer to assemblages of exons brought together by an "exon-stretching" algorithm.
• a RefSeq identifier (denoted by "NM,” “NP,” or “NT”) may be used in place of the 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 conesponding sequence analysis methods associated with the prefixes (see Example IV and Example V).
• Incyte cDNA coverage redundant with the sequence coverage shown in column 5 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
• Table 5 shows the representative cDNA libraries for those full length polynucleotide 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 LMM variants.
• a prefened LMM 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 LMM amino acid sequence, and which contains at least one functional or structural characteristic of LMM.
• the invention also encompasses polynucleotides which encode LMM.
• the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:7-12, which encodes LMM.
• the invention also encompasses a variant of a polynucleotide sequence encoding LMM.
• 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 LMM.
• a particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:7- 12 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:7-12. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of LMM.
• nucleotide sequences which encode LMM and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occu ⁇ ing LMM under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding LMM 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 LMM and LMM derivatives, or fragments thereof, entirely by synthetic chemistry.
• the synthetic sequence may be 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 LMM or any fragment thereof.
• polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NO:7-12 and fragments thereof under various conditions of stringency.
• polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NO:7-12 and fragments thereof under various conditions of stringency.
• 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 Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD).
• sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Applied Biosystems).
• Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art.
• the resulting sequences are analyzed using a variety of algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997) Short Protocols in Molecular 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 LMM may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
• PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
• one method which may be employed restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic. 2:318-322.)
• Another method, inverse PCR uses primers that extend in divergent directions to amplify unknown sequence from a circularized template.
• the template is derived from restriction fragments comprising a known genomic locus and su ⁇ ounding sequences.
• a third method, capture PCR involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
• capture PCR involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
• multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
• Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res.
• primers may be designed using commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
• 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 may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled.
• Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
• polynucleotide sequences or fragments thereof which encode LMM maybe cloned in recombinant DNA molecules that direct expression of LMM, 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 LMM.
• nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter LMM-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product.
• DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences.
• 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 maybe subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent Number 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 LMM, 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 Number 5,837,458; Chang, C.-C. et al. (1999) Nat. BiotechnoL 17:793-797; Christians, F.C. et al. (1999) Nat
• 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 prefened 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 may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
• sequences encoding LMM 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.)
• LMM 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 431A peptide synthesizer (Applied Biosystems).
• the amino acid sequence of LMM, 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 maybe confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, supra, pp. 28-53.)
• 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 LMM. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of sequences encoding LMM. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding LMM and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals maybe needed.
• 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.)
• a variety of expression vector/host systems may be utilized to contain and express sequences encoding LMM. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
• 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
• Expression vectors derived from rettoviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids maybe used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population.
• the invention is not limited by the host cell employed.
• a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding LMM.
• routine cloning, subcloning, and propagation of polynucleotide sequences encoding LMM can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding LMM into the vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules.
• these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
• vectors which direct high level expression of LMM may be used.
• vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
• Yeast expression systems may be used for production of LMM.
• a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
• 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 LMM. Transcription of sequences encoding LMM maybe driven by viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:17-311). Alternatively, plant promoters such as the 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. Cell Differ.
• 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 LMM 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 LMM in host cells.
• transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
• SV40 or EB V- 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 LMM can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
• the purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which 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 sad pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
• Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites.
• Visible markers e.g., anlhocyanins, 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 LMM is inserted within a marker gene sequence
• transformed cells containing sequences encoding LMM can be identified by the absence of marker gene function.
• a marker gene can be placed in tandem with a sequence encoding LMM 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.
• LMM 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 LMM 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
• a two-site, monoclonal-based imimraoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on LMM is prefened, but a competitive binding assay may be employed.
• a competitive binding assay may be employed.
• Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding LMM include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
• the sequences encoding LMM, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
• Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
• reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
• Host cells transformed with nucleotide sequences encoding LMM maybe 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 LMM may be designed to contain signal sequences which direct secretion of LMM 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.
• Such modifications of the polypeptide include, but are not hmited to, acetylation, carboxylation, glycosylation, phosphorylation. lipidation, and acylation.
• Post-translational processing which cleaves a "prepro” or “pro” form of the protein may also be used to specify protein targeting, folding, and/or activity.
• Different host cells which have specific cellular macliinery 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 conect modification and processing of the foreign protein.
• natural, modified, or recombinant nucleic acid sequences, encoding LMM may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
• a chimeric LMM protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of LMM 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 LMM encoding sequence and the heterologous protein sequence, so that LMM may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10). A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
• synthesis of radiolabeled LMM maybe achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35 S-me1hionine.
• LMM of the present invention or fragments thereof may be used to screen for compounds that specifically bind to LMM. At least one and up to a plurality of test compounds may be screened for specific binding to LMM. Examples of test compounds include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
• the compound thus identified is closely related to the natural ligand of LMM, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner.
• the compound can be closely related to the natural receptor to which LMM binds, or to at least a fragment of the receptor, e.g., the ligand binding site.
• the compound can be rationally designed using known techniques.
• screening for these compounds involves producing appropriate cells which express LMM, either as a secreted protein or on the cell membrane.
• Prefened cells include cells from mammals, yeast, Drosophila, or R coli. Cells expressing LMM or cell membrane fractions which contain LMM are then contacted with a test compound and binding, stimulation, or inhibition of activity of either LMM or the compound is analyzed.
• An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label.
• the assay may comprise the steps of combining at least one test compound with LMM, either in solution or affixed to a solid support, and detecting the binding of LMM to the compound.
• the assay may detect or measure binding of a test compound in the presence of a labeled competitor.
• the assay may be carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compound(s) maybe free in solution or affixed to a solid support.
• LMM of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of LMM.
• Such compounds may include agonists, antagonists, or partial or inverse agonists.
• an assay is performed under conditions permissive for LMM activity, wherein LMM is combined with at least one test compound, and the activity of LMM in the presence of a test compound is compared with the activity of LMM in the absence of the test compound. A change in the activity of LMM in the presence of the test compound is indicative of a compound that modulates the activity of LMM.
• a test compound is combined with an in vitro or cell-free system comprising LMM under conditions suitable for LMM activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of LMM may do so indirectly and need not come in direct contact with the test compound. At least one and up to a plurality of test compounds maybe screened.
• polynucleotides encoding LMM or their mammalian homologs maybe "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 conesponding region of the host genome by homologous recombination.
• homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
• Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain.
• the blastocysts are surgically transfened to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.
• Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
• Polynucleotides encoding LMM 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 cardiomyocyt.es (Thomson, J.A. et al. (1998) Science 282:1145-1147).
• Polynucleotides encoding LMM can also be used to create 'lcnockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease.
• pigs e.g., pigs
• transgenic animals e.g., mice or rats
• knockin technology a region of a polynucleotide encoding LMM is injected into animal ES cells, and the injected sequence integrates into the animal cell genome.
• Transformed cells are injected into blastulae, and the blastalae are implanted as described above.
• Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
• a mammal inbred to overexpress LMM e.g., by secreting LMM 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
• LMM lipid metabolism enzymes.
• the expression of LMM is closely associated with leukocytes, pituitary glands, and reproductive and bronchial tissue.
• LMM lipid metabolism enzymes
• LMM 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 LMM.
• 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, tfiymus, thyroid, and uterus; a neurological disorder such as epilepsy, ischemic cerebro vascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other
• a vector capable of expressing LMM 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 LMM including, but not limited to, those described above.
• a composition comprising a substantially purified LMM in conjunction with a suitable pharmaceutical carrier ma be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of LMM including, but not limited to, those provided above.
• an agonist which modulates the activity of LMM may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of LMM including, but not limited to, those listed above.
• an antagonist of LMM may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of LMM.
• disorders include, but are not limited to, those cancer, neurological disorders, autoimmune/inflammatory disorders, gastrointestinal disorders, and cardiovascular disorders described above.
• an antibody which specifically binds LMM maybe used directly as an antagonist or indirectly as a targeting or dehvery mechanism for bringing a pharmaceutical agent to cells or tissues which express LMM.
• a vector expressing the complement of the polynucleotide encoding LMM maybe administered to a subject to treat or prevent a disorder associated with increased expression or activity of LMM 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 maybe able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
• LMM An antagonist of LMM may be produced using methods which are generally known in the art.
• purified LMM may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind LMM.
• Antibodies to LMM 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
• various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with LMM 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 lysolecilhin, 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 LMM 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 LMM amino acids maybe fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
• Monoclonal antibodies to LMM maybe 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.
• the hybridoma technique the human B-cell hybridoma technique
• 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, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol.
• 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 LMM 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 maybe 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 LMM and its specific antibody.
• a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering LMM epitopes is generally used, but a competitive binding assay may also be employed (Pound, supra).
• K a is defined as the molar concentration of LMM-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions.
• K a is defined as the molar concentration of LMM-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions.
• the K a dete ⁇ rtined for a preparation of monoclonal antibodies, which are monospecific for a particular LMM epitope, represents a true measure of affinity.
• High-affinity antibody preparations with K a ranging from about IO 9 to IO 12 L/mole are prefened for use in immunoassays in which the LMM-antibody complex must withstand rigorous manipulations.
• Low-affinity antibody preparations with K a ranging from about IO 6 to IO 7 L/mole are prefened for use in immunopurification and similar procedures, which ultimately require dissociation of LMM, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL Press, Washington DC; Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
• 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 LMM-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.)
• the polynucleotides encoding LMM, or any fragment or complement thereof may be used for therapeutic purposes.
• modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory regions of the gene encoding LMM.
• complementary sequences or antisense molecules DNA, RNA, PNA, or modified oligonucleotides
• antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding LMM.
• 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.
• viral vectors such as retrovirus and adeno-associated virus vectors.
• retrovirus and adeno-associated virus vectors See, e.g., Miller, A.D. (1990) Blood 76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63(3):323-347.
• Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art.
• polynucleotides encoding LMM may be used for somatic or germline gene therapy.
• Gene therapy may be performed to (i) conect a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-Xl disease characterized by X- linked inheritance (Cavazzana-Calvo, M. et al.
• diseases or disorders caused by deficiencies in LMM are treated by constructing mammalian expression vectors encoding LMM and introducing these vectors by mechanical means into LMM-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).
• Expression vectors that may be effective for the expression of LMM include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors
• LMM maybe expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H.
• a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes
• an inducible promoter e.g., the tetracycline-regulated promoter (Gossen, M. and H.
• liposome transformation kits e.g., the PERFECT LIPID TRANSFECTION KIT, available from Invitrogen
• PERFECT LIPID TRANSFECTION 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 LMM expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding LMM under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE) along with additional retrovirus cis-acting RNA sequences and coding sequences required for efficient vector propagation.
• Rettovirus 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 A.D. Miller (1988) J. Virol. 62:3802-3806; DuU, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey, R. et al. (1998) J.
• VPCL vector producing cell line
• U.S. Patent Number 5,910,434 to Rigg discloses a method for obtaining retrovirus packaging cell lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4 + T-cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. ViroL 71:7020-7029; Bauer, G. et al.
• an adeno virus-based gene therapy delivery system is used to deliver polynucleotides encoding LMM to cells which have one or more genetic abnormalities with respect to the expression of LMM.
• the construction and packaging of adeno virus-based vectors are well known to those with ordinary skill in the art.
• Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268).
• Potentially useful adenoviral vectors are described in U.S. Patent Number 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby incorporated by reference.
• a herpes-based, gene therapy dehvery system is used to deliver polynucleotides encoding LMM to target cells which have one or more genetic abnormalities with respect to the expression of LMM.
• the use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing LMM to cells of the central nervous system, for which HS V has a tropism.
• the construction and packaging of herpes-based vectors are well known to those with ordinary skill in the art.
• a replication-competent herpes simplex virus (HSV) type 1-based vector has been used to deliver a reporter gene to the eyes of primates (Liu, X. et al. (1999) Exp. Eye Res. 169:385-395).
• HSV-1 virus vector has also been disclosed in detail in U.S. Patent Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference.
• U.S. Patent Number 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transfened to a cell under the control of the appropriate promoter for purposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22.
• HSV vectors see also Goins, W.F. et al. (1999) J. Virol.
• herpesvirus sequences The manipulation of cloned herpesvirus sequences, the generation of recombinant virus following the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of cells with herpesvirus are techniques well known to those of ordinary skill in the art.
• an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding LMM 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 LMM into the alphavirus genome in place of the capsid-coding region results in the production of a large number of LMM- coding RNAs and the synthesis of high levels of LMM 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 LMM 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 LMM.
• ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC
• ribozyme cleavage sites including the following sequences: GUA, GUU, and GUC
• short RNA sequences of between 15 and 20 ribonucleotides, conesponding to the region of the target gene containing the cleavage site maybe evaluated for secondary structural features which may render the oligonucleotide inoperable.
• the suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
• Complementary ribonucleic acid molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding LMM. Such DNA sequences maybe incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues.
• RNA molecules maybe 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 LMM.
• 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 LMM may be therapeutically useful, and in the treatment of disorders associated with decreased LMM expression or activity, a compound which specifically promotes expression of the polynucleotide encoding LMM maybe therapeutically useful.
• At least one, and up to a plurality, of test compounds maybe screened for effectiveness in altering expression of a specific polynucleotide.
• a test compound maybe 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 nataraUy-occuning 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 LMM 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 LMM 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 LMM.
• the amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds.
• a screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomvces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem. Biophys. Res. Commun.
• a particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified ohgonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No. 6,022,691).
• oligonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified ohgonucleotides
• 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.
• any of the therapeutic methods described above maybe applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
• An additional embodiment of the invention relates to the administration of a composition which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient.
• Excipients may include, for example, sugars, starches, celluloses, gums, and proteins.
• Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's
• compositions may consist of LMM, antibodies to LMM, and mimetics, agonists, antagonists, or inhibitors of LMM.
• 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 are generally aerosolized immediately prior to inhalation by the patient.
• small molecules e.g. traditional low molecular weight organic drugs
• aerosol delivery of fast- acting formulations is well-known in the art.
• macromolecules e.g. larger peptides and proteins
• Pulmonary dehvery has the advantage of administration without needle injection, and obviates the need for potentially toxic penetration enhancers.
• Compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.
• compositions maybe prepared for direct intracellular dehvery of macromolecules comprising LMM or fragments thereof.
• liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular dehvery of the macromolecule.
• LMM or a fragment thereof may be joined to a short cationic N- terminal portion from the HTV 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 LMM or fragments thereof, antibodies of LMM, and agonists, antagonists or inhibitors of LMM, which ameliorates the symptoms or condition.
• Therapeutic efficacy and toxicity maybe determined 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 prefened.
• 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 S0 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.
• Dosage and adnunistration 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, dehvery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
• antibodies which specifically bind LMM may be used for the diagnosis of disorders characterized by expression of LMM, or in assays to monitor patients being treated with LMM or agonists, antagonists, or inhibitors of LMM.
• Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for LMM include methods which utilize the antibody and a label to detect LMM in human body fluids or in extracts of cells or tissues.
• the antibodies may be used with or without modification, and maybe 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.
• LMM A variety of protocols for measuring LMM, including ELISAs, RlAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of LMM expression.
• Normal or standard values for LMM expression are estabhshed by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibodies to LMM under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of LMM 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 LMM 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 LMM maybe conelated with disease.
• the diagnostic assay may be used to determine absence, presence, and excess expression of LMM, and to monitor regulation of LMM levels during therapeutic intervention.
• hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding LMM or closely related molecules may be used to identify nucleic acid sequences which encode LMM.
• the specificity of the probe whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amphfication will determine whether the probe identifies only naturally occurring sequences encoding LMM, allelic variants, or related sequences.
• Probes may also be used for the detection of related sequences, and may have at least 50% sequence identity to any of the LMM encoding sequences.
• the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO:7-12 or from genomic sequences including promoters, enhancers, and introns of the LMM gene.
• Means for producing specific hybridization probes for DNAs encoding LMM include the cloning ofpolynucleoti.de sequences encoding LMM or LMM derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
• Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
• Polynucleotide sequences encoding LMM may be used for the diagnosis of disorders associated with expression of LMM.
• 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 neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic
• the polynucleotide sequences encoding LMM maybe 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 microanays utilizing fluids or tissues from patients to detect altered LMM expression. Such qualitative or quantitative methods are well known in the art.
• the nucleotide sequences encoding LMM may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
• the nucleotide sequences encoding LMM ma be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding LMM in the sample indicates the presence of the associated disorder.
• Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
• a normal or standard profile for expression is estabhshed. 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 LMM, 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 maybe compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to estabhsh the presence of a disorder.
• hybridization assays maybe repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
• the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
• 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.
• ohgonucleotides designed from the sequences encoding LMM may involve the use of PCR. These ohgomers may be chemically synthesized, generated enzymaticahy, or produced in vitro. Ohgomers will preferably contain a fragment of a polynucleotide encoding LMM, or a fragment of a polynucleotide complementary to the polynucleotide encoding
• oligonucleotide primers derived from the polynucleotide sequences encoding LMM 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 LMM 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 amphmers 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 MASSARRAY system (Sequenom, Inc., San Diego CA), Methods which may also be used to quantify the expression of LMM include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves.
• the speed of quantitation of multiple samples may be accelerated by mnning the assay in a Mgh-throughput format where the ohgomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
• ohgonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as elements on a microa ⁇ ay.
• the microanay can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below.
• the microanay may also be used to identify genetic variants, mutations, and polymorphisms. This information may be used to dete ⁇ nine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease.
• this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient.
• therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
• LMM, fragments of LMM, or antibodies specific for LMM may be used as elements on a microanay. The microanay maybe 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 Number 5,840,484, expressly incorporated by reference herein.)
• a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or cell type.
• the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurahty of elements on a microanay.
• the resultant transcript image would provide a profile of gene activity.
• Transcript images may be generated using transcripts isolated from tissues, cell lines, biopsies, or other biological samples. The transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
• Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals, as well as toxicological testing of industrial and nataraUy-occurring environmental compounds. All compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L. Anderson (2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein).
• a test compound has a signatare similar to that of a compound with known toxicity, it is likely to share those toxic properties.
• These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families. Ideally, a genome-wide measurement of expression provides the highest quality signature. Even genes whose expression is not altered by any tested compounds are important as well, as the levels of expression of these genes are used to normalize the rest of the expression data. The normahzation procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant signatare aids in interpretation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity.
• the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound. Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels conesponding to the polynucleotides of the present invention maybe quantified. The transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
• proteome refers to the global pattern of protein expression in a particular tissue or cell type.
• proteome expression patterns, or profiles are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time.
• a profile of a cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type.
• the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra).
• the proteins are visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains.
• the optical density of each protein spot is generally proportional to the level of the protein in the sample.
• the optical densities of equivalently positioned protein spots from different samples are compared to identify any changes in protein spot density related to the treatment:
• the proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry.
• the identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of the present invention. In some cases, further sequence data may be obtained for definitive protein identification.
• a proteomic profile may also be generated using antibodies specific for LMM to quantify the levels of LMM expression.
• the antibodies are used as elements on a microanay, and protein expression levels are quantified by exposing the microanay to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem. 270:103- lll; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be performed by a variety of methods known in the art, for example, by reactmg the proteins in the sample with a thiol- or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each anay element.
• Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in parallel with toxicant signatures at the transcript level.
• There is a poor conelation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures maybe useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile.
• the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling maybe more reliable and informative in such cases.
• the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound.
• Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified.
• the amount of each protein is compared to the amount of the conesponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
• Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
• the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
• Microa ⁇ ays may be prepared, used, and analyzed using methods known in the art. (See, e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad.
• nucleic acid sequences encoding LMM maybe used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping.
• sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA libraries.
• HACs human artificial chromosomes
• YACs yeast artificial chromosomes
• BACs bacterial artificial chromosomes
• PI constructions or single chromosome cDNA libraries.
• nucleic acid sequences of the invention may be used to develop genetic linkage maps, for example, which conelate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP).
• RFLP restriction fragment length polymorphism
• Fluorescent in situ hybridization may be conelated with other physical and genetic map data.
• FISH Fluorescent in situ hybridization
• Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMhM) World Wide Web site. Conelation between the location of the gene encoding LMM on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional cloning efforts.
• LMM LMM, 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 LMM and the agent being tested may be measured.
• Another technique for drug screening provides for high throughput screening of compounds hav g suitable bmding 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 LMM, or fragments thereof, and washed. Bound LMM is then detected by methods well known in the art. Purified LMM can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutraUzing antibodies can be used to capture the peptide and immobihze it on a solid support.
• nucleotide sequences which encode LMM maybe used in any* molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are cunently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
• Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denatarants, such as TRLZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
• poly(A)+ RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
• RNA was provided with RNA and constructed the conesponding cDNA libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were hgated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes.
• the cDNA was size-selected (300- 1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis.
• cDNAs were hgated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA (Invitrogen), or pINCY (Incyte Genomics, Palo Alto CA), or derivatives thereof.
• Recombinant plasmids were transformed into competent E. coli ceUs including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Life Technologies. II. Isolation of cDNA Clones
• Plasmids obtained as described in Example I were recovered from host ceUs by in vivo excision using the UNIZAP vector system (Stratagene) or by ceU lysis. Plasmids were purified using at least one of the foUowing: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN. FoUowing precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4°C
• plasmid DNA was amphfied from host ceU lysates using direct link PCR in a Mgh-fhroughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host ceU lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-weU plates, and the concentration of amphfied plasmid DNA was quantified fluorometricaUy using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland). III. Sequencing and Analysis
• Incyte cDNA recovered in plasmids as described in Example II were sequenced as foUows. Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supphed in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
• Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (Apphed Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example NDX
• 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 dinucleoti.de 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, and hidden Markov model (HMM)-based protein family databases such as PFAM.
• HMM hidden Markov model
• Incyte cDNA sequences were assembled to produce fuU length polynucleotide sequences.
• GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences were used to extend Incyte cDNA assemblages to fuU length.
• FuU length polynucleotide sequences were translated to derive the corresponding fuU length polypeptide sequences.
• a polypeptide of the invention may begin at any of the methionine residues of the fuU length translated polypeptide.
• FuU length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and hidden Markov model ( ⁇ MM)-based protein family databases such as PFAM.
• FuU length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering,
• Incyte cDNA and fuU 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, aU of which are incorporated by reference herein in their entirety, and the fourth column presents, where apphcable, 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 probabihty 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) Cun. Opin. Struct. Biol. 8:346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon.
• Genscan is a FASTA database of polynucleotide and polypeptide sequences.
• the maximum range of sequence for Genscan to analyze at once was set to 30 kb.
• the encoded polypeptides were analyzed by querying against PFAM models for hpid metabolism enzymes. Potential hpid metabolism enzymes were also identified by homology to Incyte cDNA sequences that had been annotated as hpid metabolism enzymes. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri pubhc databases.
• Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to conect enors in the sequence predicted by Genscan, such as extra or omitted exons.
• BLAST analysis was also used to find any Incyte cDNA or pubhc cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription.
• Incyte cDNA coverage was available, this information was used to conect or confirm the Genscan predicted sequence.
• FuU length polynucleotide sequences were obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or pubhc cDNA sequences using the assembly process described in Example HI.
• fuU length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences.
• Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example HI were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible splice variants that were subsequently confirmed, edited, or extended to create a 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.
• Jhco ⁇ ect exons predicted by Genscan were conected by comparison to the top BLAST hit from genpept. Sequences were further extended with additional cDNA sequences, or by inspection of genomic DNA, when necessary. "Stretched" Sequences
• Partial DNA sequences were extended to fuU length with an algorithm based on BLAST analysis.
• the nearest GenBank protein homolog was then compared by BLAST analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV.
• a chimeric protein was generated by using the resultant high-scoring segment pairs (HSPs) to map the translated sequences onto the GenBank protein homolog. Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog.
• HSPs high-scoring segment pairs
• GenBank protein homolog, me chimeric protein, or both were used as probes to search for homologous genomic sequences from the pubhc human genome databases. Partial DNA sequences were therefore "stretched” or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene. VI. Chromosomal Mapping of LMM Encoding Polynucleotides
• sequences which were used to assemble SEQ DD NO:7-12 were compared with sequences from the Incyte LIFESEQ database and pubhc domain databases using BLAST and other implementations of the Smith-Waterman algorithm. Sequences from these databases that matched SEQ DD NO:7-12 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data available from pubhc resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of aU sequences of that cluster, including its particular SEQ ID NO:, to that map location.
• pubhc resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion
• Map locations are represented by ranges, or intervals, of human chromosomes.
• the map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p- arm.
• centiMorgan cM
• centiMorgan is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.
• the cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters.
• Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular ceU type or tissue have been bound.
• a membrane on which RNAs from a particular ceU type or tissue have been bound See, e.g., Sambrook, supra, ch. 7; Ausubel (1995) supra, ch. 4 and 16.
• Analogous computer techniques applying BLAST were used to search for identical or related molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This analysis is much faster than multiple membrane-based hybridizations.
• the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar.
• the basis of the search is the product score, which is defined as:
• the product score takes into account both the degree of similarity between two sequences and the length of the sequence match.
• the product score is a normalized value between 0 and 100, and is calculated as foUows: the BLAST score is 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 ahgnment. For example, a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap.
• polynucleotide sequences encoding LMM are analyzed with respect to the tissue sources from which they were derived. For example, some fuU 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 foUowing organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structares; endocrine system; exocrine glands; genitaha, female; genitaha, male; germ ceUs; hemic and immune system; hver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract.
• the number of hbraries in each category is counted and divided by the total number of hbraries across aU categories.
• each human tissue is classified into one of the foUowing disease/condition categories: cancer, ceU line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of hbraries in each category is counted and divided by the total number of hbraries across aU categories. The resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding LMM.
• cDNA sequences and cDNA library/tissue information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA). VIII.
• FuU length polynucleotide sequences were also produced by extension of an appropriate fragment of the fuU length molecule using ohgonucleotide primers designed from this fragment. One primer was synthesized to initiate 5' extension of the known fragment, and the other primer was synthesized to initiate 3 ' extension of the known fragment.
• the initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C Any stretch of nucleotides which would result in hairpin structares and primer-primer dimerizations was avoided.s
• the reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg 2+ , (NEL ⁇ SO ⁇ and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the foUowing parameters for primer pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 68 °C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 °C, 5 min; Step 7: storage at 4°C
• the parameters for primer pair T7 and SK+ were as foUows: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 68
• the concentration of DNA in each weU was determined by dispensing 100 ⁇ l PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in IX TE and 0.5 ⁇ l of undiluted PCR product into each weU of an opaque fluorimeter plate (Corning Costar, Acton MA), aUowing the DNA to bind to the reagent.
• the plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
• Step 1 94 °C, 3 min
• Step 2 94 °C, 15 sec
• Step 3 60 °C, 1 min
• Step 4 72 °C, 2 min
• Step 5 steps 2, 3, and 4 repeated 29 times
• Step 6 72°C, 5 min
• Step 7 storage at 4°C DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamphfied using the same conditions as described above.
• fuU length polynucleotide sequences are verified using the above procedure or are used to obtain 5' regulatory sequences using the above procedure along with ohgonucleotides designed for such extension, and an appropriate genomic library.
• Hybridization probes derived from SEQ DD NO. -12 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of ohgonucleotides, consisting of about 20 base pairs, is specificaUy described, essentiaUy the same procedure is used with larger nucleotide fragments.
• Ohgonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ⁇ Ci of [ ⁇ - 3 P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
• the labeled ohgonucleotides are substantiaUy purified using a SEPHADEX G-25 superfine size exclusion dextranbead column (Amersham Pharmacia Biotech).
• An ahquot containing IO 7 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the foUowing endonucleases: Ase I, Bgi ⁇ , Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
• the DNA from each digest is fractionated on a 0.7% agarose gel and transfe ⁇ ed to nylon membranes (Nytran Plus, Schleicher & SchueU, Durham NH). Hybridization is carried out for 16 hours at 40 °C To remove nonspecific signals, blots are sequentiaUy washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared. X. Microarrays
• the linkage or synthesis of anay elements upon a microanay can be achieved utilizing photohthography, piezoelectric printing (ink-jet printing, See, e.g., Baldeschweiler, supra.), mechanical microspotting technologies, and derivatives thereof.
• the substrate in each of the aforementioned technologies should be uniform and solid with a non-porous surface (Schena (1999), supra). Suggested substrates include sihcon, sihca, glass shdes, glass chips, and sihcon wafers.
• a procedure analogous to a dot or slot blot may also be used to a ⁇ ange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
• a typical anay may be produced using available methods and machines weU known to those of ordinary skiU in the art and may contain any appropriate number of elements. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; MarshaU, A. and J. Hodgson (1998) Nat. BiotechnoL 16:27-31.)
• FuU length cDNAs, Expressed Sequence Tags (ESTs), or fragments or ohgomers thereof may comprise the elements of the microanay. Fragments or ohgomers suitable for hybridization can be selected using software weU known in the art such as LASERGENE software (DNASTAR).
• the anay 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 anay 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 microanay may be assessed.
• microanay preparation and usage is described in detail below.
• Total RNA is isolated from tissue samples using the guanidinium tbiocyanate method and poly(A) + RNA is purified using the ohgo-(dT) ceUulose method.
• Each poly(A) + RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/ ⁇ l ohgo-(dT) primer (21mer), IX first strand buffer, 0.03 units/ ⁇ l RNase inhibitor, 500 ⁇ M dATP, 500 ⁇ M dGTP, 500 ⁇ M dTTP, 40 ⁇ M dCTP, 40 ⁇ M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech).
• the reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A) + RNA with GEMBRIGHT kits (Incyte).
• Specific control poly(A) + RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.
• Sequences of the present invention are used to generate anay elements.
• Each anay element is amphfied from bacterial ceUs containing vectors with cloned cDNA inserts.
• PCR amphfication uses primers complementary to the vector sequences flanking the cDNA insert.
• Anay elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g.
• Amplified anay elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech). Purified anay elements are immobilized on polymer-coated glass shdes.
• Glass microscope shdes are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distiUed water washes between and after treatments. Glass shdes are etched in 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester PA), washed extensively in distiUed water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated shdes are cured in a 110°C oven.
• Anay elements are applied to the coated glass substrate using a procedure described in US Patent No. 5,807,522, incorporated herein by reference.
• 1 ⁇ l of the anay element DNA is loaded into the open capiUary printing element by a high-speed robotic apparatus.
• the apparatus then deposits about 5 nl of anay element sample per shde.
• Microanays are UV-crosslihked using a STRATALINKER UV-crosslinker (Stratagene). Microanays are washed at room temperature once in 0.2% SDS and three times in distiUed water. Non-specific binding sites are blocked by incubation of microanays in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60° C foUowed by washes in 0.2% SDS and distiUed water as before.
• 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 microanay surface and covered with an 1.8 cm 2 covershp.
• the anays are transfened to a waterproof chamber having a cavity just shghtly larger than a microscope shde.
• the chamber is kept at 100% humidity internaUy by the addition of 140 ⁇ l of 5X SSC in a corner of the chamber.
• the chamber containing the anays is incubated for about 6.5 hours at 60°C
• the anays are washed for 10 min at 45°C in a first wash buffer (IX SSC, 0.1% SDS), three times for 10 minutes each at 45° C in a second wash buffer (0.1X SSC), and dried. Detection
• Reporter-labeled hybridization complexes are detected with a microscope equipped with an 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 anay using a 20X microscope objective (Nikon, Inc., MelviUe NY).
• the shde containing the anay is placed on a computer-controUed X-Y stage on the microscope and raster- scanned past the objective.
• the 1.8 cm x 1.8 cm anay used in the present example is scanned with a resolution of 20 micrometers.
• a mixed gas multiline laser excites the two fluorophores sequentiaUy. Emitted light is spht, based on wavelength, into two photomultipher tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) conesponding to the two fluorophores. Appropriate filters positioned between the anay and the photomultipher tubes are used to filter the signals.
• the emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5.
• Each anay is typicaUy scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
• the sensitivity of the scans is typicaUy cahbrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration.
• a specific location on the anay contains a complementary DNA sequence, aUowing the intensity of the signal at that location to be conelated with a weight ratio of hybridizing species of 1:100,000.
• the calibration is done by labeling samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
• the output of the photomultipher tube is digitized using a 12-bit RTI-835H analog-to-digital
• A/D conversion board Analog Devices, Inc., Norwood MA
• instaUed in an IBM-compatible PC computer The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
• the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first conected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.
• Sequences complementary to the LMM-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of nataraUy occurring LMM.
• ohgonucleotides comprising from about 15 to 30 base pairs is described, essentiaUy the same procedure is used with smaUer or with larger sequence fragments.
• Appropriate ohgonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of LMM.
• a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
• a complementary oligonucleotide is designed to prevent ribosomal binding to the LMM-encoding transcript.
• LMM LMM expression and purification of LMM 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 tip-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
• Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
• Antibiotic resistant bacteria express LMM upon induction with isopropylbeta-D-thiogalactopyranoside (JP TG).
• LMM in eukaryotic ceUs is achieved by infecting insect or mammalian ceU lines with recombinant Autographica cahfornica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus.
• AcMNPV Autographica cahfornica nuclear polyhedrosis virus
• the nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding LMM by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription.
• Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect ceUs in most cases, or human hepatocytes, in some cases.
• LMM is synthesized as a fusion protein with, e.g., glutathione S- transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, pe ⁇ mtting rapid, single-step, affinity-based purification of recombinant fusion protein from crude ceU 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 LMM obtained by these methods can be used directly in the assays shown in Examples XVI and XVH where applicable. XIII. Functional Assays
• LMM function is assessed by expressing the sequences encoding LMM at physiologicaUy elevated levels in mammalian ceU culture systems.
• cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression.
• Vectors of choice include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human ceU line, for example, an endothelial or hematopoietic ceU line, using either hposome formulations or electroporation.
• 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected.
• Expression of a marker protein provides a means to distinguish transfected ceUs from nontransfected ceUs and is a rehable predictor of cDNA expression from the recombinant vector.
• Marker proteins of choice include, e.g., Green Fluorescent Protein (GEP; Clontech), CD64, or a CD64-GFP fusion protein.
• Flow cytometry (FCM) an automated, laser optics- based technique, is used to identify transfected ceUs expressing GFP or CD64-GFP and to evaluate the apoptotic state of the ceUs and other ceUular properties.
• FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with ceU death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in ceU size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of ceU surface and intraceUular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the ceU surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York NY.
• LMM The influence of LMM on gene expression can be assessed using highly purified populations of ceUs transfected with sequences encoding LMM and either CD64 or CD64-GFP.
• CD64 and CD64-GFP are expressed on the surface of transfected ceUs and bind to conserved regions of human immunoglobulin G (IgG).
• Transfected ceUs are efficiently separated from nontransfected ceUs using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
• mRNA can be purified from the ceUs using methods weU known by those of skiU in the art. Expression of mRNA encoding LMM and other genes of interest can be analyzed by northern analysis or microanay techniques.
• PAGE polyacrylamide gel electrophoresis
• LMM amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a conesponding oligopeptide is synthesized and used to raise antibodies by means known to those of skiU in the art.
• LASERGENE software DNASTAR
• Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophihc regions are weU described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
• oligopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccmimide ester (MBS) to increase immunogenicity.
• ABI 431 A peptide synthesizer Applied Biosystems
• KLH Sigma- Aldrich, St. Louis MO
• MBS N-maleimidobenzoyl-N-hydroxysuccmimide ester
• Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant.
• Resulting antisera are tested for antipeptide and anti-LMM activity by, for example, binding the peptide or LMM to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
• Media containing LMM are passed over the immunoaffinity column, and the column is washed under conditions that aUow the preferential absorbance of LMM (e.g., high ionic strength buffers in the presence of detergent).
• the column is eluted under conditions that disrupt antibody/LMM binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or fhiocyanate ion), and LMM is coUected.
• LMM, or biologically active fragments thereof are labeled with 125 I Bolton-Hunter reagent.
• Bolton-Hunter reagent See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.
• Candidate molecules previously anayed in the weUs of a multi-weU plate are incubated with the labeled LMM, washed, and any weUs with labeled LMM complex are assayed. Data obtained using different concentrations of LMM are used to calculate values for the number, affinity, and association of LMM with the candidate molecules.
• molecules interacting with LMM are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or using commerciaUy available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
• LMM may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine aU interactions between the proteins encoded by two large hbraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
• LME activity can be demonstrated by an in vitro hydrolysis assay with vesicles containing l-palmitoyl-2-[l- 14 C]oleoyl phosphatidylchohne (Sigma-Aldrich). LME triglyceride hpase activity and phospholipase A 2 activity are demonstrated by analysis of the cleavage products isolated from the hydrolysis reaction mixture.
• Vesicles containing l-palmitoyl-2-[l- 14 C]oleoyl phosphatidylchohne are prepared by mixing 2.0 ⁇ Ci of the radiolabeled phospholipid with 12.5 mg of unlabeled l-palmitoyl-2-oleoyl phosphatidylchohne and drying the mixture under N 2 . 2.5 ml of 150 mM Tris-HCl, pH 7.5, is added, and the mixture is sonicated and centrifuged.
• the supernatant maybe stored at 4 °C
• the fihal 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 ⁇ , pH 7.4, 150 ⁇ g of l-palmitoyl-2-[l- 14 C]oleoyl phosphatidylchohne vesicles, and various amounts of LME 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 hpids.
• 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 hpid-containing areas are visuahzed with I 2 vapor; the spots are scraped, and their radioactivity is dete ⁇ nined by scintillation counting.
• the amount of radioactivity released as fatty acids wiU increase as a greater amount of LME is added to the assay mixture while the amount of radioactivity released as lyso-phosphatidylcholine wiU remain low. This demonstrates that LME cleaves at the sn-2 and not the sn-1 position, as is characteristic of phosphohpase A 2 activity.
• LME phosphohpase activity is measured by the hydrolysis of a fatty acyl residue at the sn-1 position of phosphatidylserine.
• LME is combined with the Tritium [ 3 HJ labeled substrate phosphatidylserine at stoichiometric quantities in a suitable buffer. FoUowing an appropriate incubation time, the hydrolyzed reaction products are separated from the substrates by chromatographic methods. The amount of acylglycerophosphoseiine produced is measured by counting tritiated product with the help of a scintiUation counter.
• Various control groups are set up to account for background noise and unincorporated substrate.
• the final counts represent the tritiated enzyme product [ 3 HJ- acylglycerophosphoserine, which is directly proportional to the activity of LME in biological samples.
• LME hpoxygenase activity can be measured by chromatographic methods. Extracted LME hpoxygenase 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 by 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 metabohte such as 12-HPETE (catalyzed by 12-LOX).
• the fractions are also subjected to hquid scintiUation counting.
• the final counts represent the products, which is directly proportional to the activity of LME in biological samples.
• the metabohtes of arachidonic acid are analyzed further by chiral phase-HPLC and by mass spectrometry (Sun, D. et al.
• UTRCDIC01 PSP0RT1 This large size fractionated library was constructed using RNA isolated from uterine cervix tissue removed from a 29-year-old Caucasian female during a vaginal hysterectomy and cystocele repair. Pathology indicated the cervix showed mild chronic cervicitis with focal squamous metaplasia. Pathology for the matched tumor tissue indicated intramural uterine leiomyoma. Patient history included hypothyroidism, pelvic floor relaxation, paraplegia, and self catheterization. Previous surgeries included a normal delivery, a laminectomy, and a rhinoplasty. Patient medications included Synthroid. Family history included benign hypertension in the father; and type II diabetes and hyperlipidemia in the mother.
• ABI FACTURA A program that removes vector sequences and Applied Biosystems, Foster City, CA. masks ambiguous bases in nucleic acid sequences.
• ABJTPARACEL FDF A Fast Data Finder useful in comparing and Applied Biosystems, Foster City, CA; Mismatch ⁇ 50% annotating amino acid or nucleic acid sequences. Paracel Inc., Pasadena, CA. ABI AutoAssembler A program that assembles nucleic acid sequences. Applied Biosystems, Foster City, CA.
• fastx score 100 or greater
• HMM hidden Markov model
• Phred A base-calling algorithm that examines automated Ewing, B. et al. (1998) Genome Res. sequencer traces with high sensitivity and probability. 8:175-185; Ewing, B. and P. Green (1998) Genome Res. 8:186-194.
• TMHMMER A program that uses a hidden Markov model (HMM) to Sonnhammer, E.L. et al. (1998) Proc. Sixth Intl. delineate transmembrane segments on protein sequences Conf. on Intelligent Systems for Mol. Biol., and determine orientation. Glasgow et al., eds., The Am. Assoc. for Artificial Intelligence Press, Menlo Park, CA, pp. 175-182.
• HMM hidden Markov model

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Medicinal Chemistry (AREA)
• Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Veterinary Medicine (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Biomedical Technology (AREA)
• Genetics & Genomics (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Molecular Biology (AREA)
• General Engineering & Computer Science (AREA)
• Cardiology (AREA)
• Biotechnology (AREA)
• Neurology (AREA)
• Microbiology (AREA)
• Immunology (AREA)
• Heart & Thoracic Surgery (AREA)
• Biochemistry (AREA)
• Neurosurgery (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Peptides Or Proteins (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Enzymes And Modification Thereof (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Applications Claiming Priority (9)

Publications (1)

Family

ID=27499475

Family Applications (1)

Country Status (5)

Families Citing this family (2)

Family Cites Families (3)

• 2001
  • 2001-08-22 WO PCT/US2001/026365 patent/WO2002016597A2/en not_active Application Discontinuation
  • 2001-08-22 EP EP01966141A patent/EP1339854A2/de not_active Withdrawn
  • 2001-08-22 AU AU2001286678A patent/AU2001286678A1/en not_active Abandoned
  • 2001-08-22 JP JP2002522270A patent/JP2004527216A/ja active Pending
  • 2001-08-22 CA CA002417787A patent/CA2417787A1/en not_active Abandoned

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events