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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers

Definitions

• DNA-based array technology is especially relevant for the rapid screening of expression of a large number of genes.
• a genetic predisposition, disease or therapeutic treatment may affect, directly or indirectly, the expression of a large number of genes.
• the interactions may be expected, such as when 0 the genes are part of the same signaling pathway.
• the interactions may be totally unexpected. Therefore, DNA-based arrays can be used to investigate how genetic predisposition, disease, or therapeutic treatment affects the expression of a large number of genes.
• NADJNADH-dependent oxidoreductase activity include those of L-DOPA (precursor of dopamine, a neuronal excitatory compound), glycine (an inhibitory neurotransmitter in the brain and spinal cord), histamine (liberated from mast cells during the inflammatory response), and taurine (an inhibitory neurotransmitter of the brain stem, spinal cord and retina) (Newsholme, supra, pp. 790, 792).
• L-DOPA precursor of dopamine, a neuronal excitatory compound
• glycine an inhibitory neurotransmitter in the brain and spinal cord
• histamine liberated from mast cells during the inflammatory response
• taurine an inhibitory neurotransmitter of the brain stem, spinal cord and retina
• Epigenetic or genetic defects in neurotransmitter o metabolic pathways can result in a spectrum of disease states in different tissues including Parkinson disease and inherited myoclonus (McCance, K.L. and S.E. Huether (1994) Pathophvsi
• 2,4-dienoyl-CoA reductase is located in both mitochondria and peroxisomes. Inherited deficiencies in mitochondrial and peroxisomal beta-oxidation enzymes are 5 associated with severe diseases, some of which manifest themselves soon after birth and lead to death within a few years. Defects in beta-oxidation are associated with Reye's syndrome, Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum's disease, acyl-CoA oxidase deficiency, and bifunctional protein deficiency (Suzuki, Y. et al. (1994) Am. J. Hum.
• Prenyl transferases are heterodimers, consisting of an alpha and a beta subunit, that catalyze the transfer of an isoprenyl group.
• An example of a prenyl transferase is the mammalian protein farnesyl transferase.
• the alpha subunit of farnesyl transferase consists of 5 repeats of 34 amino acids each, with each repeat containing an invariant tryptophan (PROSITE: PDOC00703).
• Hydrolysis is the breaking of a covalent bond in a substrate by introduction of a molecule of water.
• the reaction involves a nucleophilic attack by the water molecule's oxygen atom on a target bond in the substrate.
• the water molecule is split across the target bond, breaking the bond and generating two product molecules.
• Hydrolases participate in reactions essential to such functions as synthesis and degradation of cell components, and for regulation of cell functions including cell signaling, cell proliferation, inflamation, apoptosis, secretion and excretion. Hydrolases are involved in key steps in disease processes involving these functions.
• Glyoxylases are involved in hyperglycemia, non-insulin-dependent diabetes mellitus, the detoxification of bacterial toxins, and in the control of cell proliferation and microtubule assembly. Lyases
• Isomerases are a class of enzymes that catalyze geometric or structural changes within a molecule to form a single product. This class includes racemases and epimerases, cis-trans- isomerases, intramolecular oxidoreductases, intramolecular transferases (mutases) and intramolecular lyases. Isomerases are critical components of cellular biochemistry with roles in metabolic energy production including glycolysis, as well as other diverse enzymatic processes (Stryer, L. (1995) Biochemistry. W.H. Freeman and Co., New York NY, pp.483-507).
• Oxidoreductases can be isomerases as well. Oxidoreductases catalyze the reversible transfer of electrons from a substrate that becomes oxidized to a substrate that becomes reduced. This class of enzymes includes dehydrogenases, hydroxylases, oxidases, oxygenases, peroxidases, and reductases. 5 Proper maintenance of oxidoreductase levels is physiologically important. For example, genetically- linked deficiencies in lipoamide dehydrogenase can result in lactic acidosis (Robinson, B.H. et al. (1977) Pediat. Res. 11:1198-1202).
• Transferases transfera chemical group from one compound (the donor) to another compound (the acceptor).
• the types of groups transferred by these enzymes include acyl groups, amino groups, phosphate groups
• the 20 aminoacyl-tRNA synthetase enzymes can be divided into two structural classes, and each class is characterized by a distinctive topology of the catalytic domain.
• Class I enzymes contain a catalytic domain based on the nucleotide-binding Rossman fold.
• Class II enzymes contain a central catalytic domain, which consists of a seven-stranded antiparallel ⁇ -sheet motif, as well as N- and C- terminal regulatory domains.
• Class II enzymes are separated into two groups based on the heterodimeric or homodimeric structure of the enzyme; the latter group is further subdivided by the structure of the N- and C-terminal regulatory domains (Hartlein, M. and S. Cusack (1995) J. Mol. Evol.
• Examples of such reactions include the tricarboxylic acid cycle, synthesis of fatty acids and long-chain phospholipids, synthesis of alcohols and aldehydes, synthesis of intermediary metabolites, and reactions involved in the amino acid degradation pathways. Some of these reactions require input of energy, usually in the form of conversion of ATP to either ADP or AMP and pyrophosphate.
• acyl-CoA synthetase activity i) acetyl-CoA synthetase, which activates acetate and several other low molecular weight carboxylic acids and is found in muscle mitochondria and the cytosol of other tissues; ii) medium-chain acyl-CoA synthetase, which activates fatty acids containing between four and eleven carbon atoms (predominantly from dietary sources), and is present only in liver mitochondria; and iii) acyl CoA synthetase, which is specific for long chain fatty acids with between six and twenty carbon atoms, and 5 is found in microsomes and the mitochondria.
• Adenylosuccinate synthetase catalyzes a later step in purine biosynthesis that converts inosinic acid to o adenylosuccinate, a key step on the path to ATP synthesis.
• This enzyme is also similar to another carbon-nitrogen ligase, argininosuccinate synthetase, that catalyzes a similar reaction in the urea cycle (Powell, S.M. et al. (1992) FEBS Lett. 303:4-10).
• de novo synthesis of the pyrimidine nucleotides uridylate and cytidylate also arises from a common precursor, in this instance the nucleotide 5 orotidylate derived from orotate and phosphoribosyl pyrophosphate (PPRP).
• PPRP phosphoribosyl pyrophosphate
• ATCase aspartate transcarbamylase
• carbamyl phosphate synthetase II carbamyl phosphate synthetase II
• DHOase dihydroorotase
• Ligases forming phosphoric ester bonds include the DNA ligases involved in both DNA replication and repair.
• DNA ligases seal phosphodiester bonds between two adjacent nucleotides in a DNA chain using the energy from ATP hydrolysis to first activate the free 5 ' -phosphate of one nucleotide and then react it with the 3' -OH group of the adjacent nucleotide. This reseating reaction is used in both DNA replication to join small DNA fragments called Okazaki fragments that are transiently formed in the process of replicating new DNA, and in DNA repair.
• DNA repair is the process by which accidental base changes, such as those produced by oxidative damage, hydrolytic attack, or uncontrolled methylation of DNA, are corrected before replication or transcription of the DNA can occur.
• Regulated progression of the cell cycle depends on the integration of growth control pathways with the basic cell cycle machinery.
• Cell cycle regulators have been identified by selecting for human and yeast cDNAs that block or activate cell cycle arrest signals in the yeast mating pheromone pathway when they are overexpressed.
• Known regulators include human CPR (cell cycle progression restoration) genes, such as CPR8 and CPR2, and yeast CDC (cell division control) genes, including CDC91, that block the arrest signals.
• the CPR genes express a variety of proteins including cyclins, tumor suppressor binding proteins, chaperones, transcription factors, translation factors, and RNA-binding proteins (Edwards, M.C. et al.(1997) Genetics 147:1063-1076).
• FSH follicle-stimulating hormone
• LH luteinizing hormone
• Cell Differentiation and Proliferation Tissue growth involves complex and ordered patterns of cell proliferation, cell differentiation, and apoptosis.
• Cell proliferation must be regulated to maintain both the number of cells and their spatial organization. This regulation depends upon the appropriate expression of proteins which control cell cycle progression in response to extracellular signals, such as growth factors and other mitogens, and intracellular cues, such as DNA damage or nutrient starvation. Molecules which directly or indirectly modulate cell cycle progression fall into several categories, including growth factors and their receptors, second messenger and signal transduction proteins, oncogene products, tumor-suppressor proteins, and mitosis-promoting factors.
• ECM molecules such as laminin or fibronectin
• laminin or fibronectin may act as growth factors.
• Tenascin-C and -R expressed in developing and lesioned neural tissue, provide stimulatory/anti-adhesive or inhibitory properties, respectively, for axonal growth (Faissner, A. (1997) Cell Tissue Res. 290:331-341).
• Cancers are associated with the activation of oncogenes which are derived from normal cellular genes. These oncogenes encode oncoproteins which convert normal cells into malignant cells. Some oncoproteins are mutant isoforms of the normal protein, and other oncoproteins are abnormally expressed with respect to location or amount of expression. The latter category of oncoprotein causes cancer by altering transcriptional control of cell proliferation.
• Five classes of oncoproteins are known to affect cell cycle controls. These classes include growth factors, growth factor receptors, intracellular signal transducers, nuclear transcription factors, and cell-cycle control proteins.
• Viral oncogenes are integrated into the human genome after infection of human cells by certain viruses. Examples of viral oncogenes include v-src, v-abl, and v-fps.
• the Philadelphia chromosome characteristic of chronic myeloid leukemia and a subset of acute lymphoblastic leukemias, results from a reciprocal translocation between chromosomes 9 and 22 that moves a truncated portion of the proto-oncogene c-abl to the breakpoint cluster region (bcr) on chromosome 22.
• Tumor-suppressor genes are involved in regulating cell proliferation. Mutations which cause reduced or loss of function in tumor-suppressor genes result in uncontrolled cell proUferation.
• the retinoblastoma gene product (RB) in a non-phosphorylated state, binds several early- response genes and suppresses their transcription, thus blocking cell division. Phosphorylation of RB causes it to dissociate from the genes, releasing the suppression, and allowing cell division to proceed. Apoptosis
• Apoptosis is the genetically controlled process by which unneeded or defective cells undergo programmed cell death. Selective elimination of cells is as important for morphogenesis and tissue remodeling as is cell proliferation and differentiation. Lack of apoptosis may result in hyperplasia and other disorders associated with increased cell proliferation. Apoptosis is also a critical component of the immune response. Immune cells such as cytotoxic T-cells and natural killer cells prevent the spread of disease by inducing apoptosis in tumor cells and virus-infected cells. In addition, immune cells that fail to distinguish self molecules from foreign molecules must be eliminated by apoptosis to avoid an autoimmune response.
• DNA Deoxyribonucleic acid
• DNA the genetic material, is found in both the nucleus and mitochondria of human cells.
• the bulk of human DNA is nuclear, in the form of linear chromosomes, while mitochondrial DNA is circular.
• DNA replication begins at specific sites called origins of replication. Bidirectional synthesis occurs from the origin via two growing forks that move in opposite directions. Rephcation is semi-conservative, with each daughter duplex containing one old strand and its newly synthesized complementary partner.
• Proteins involved in DNA replication include DNA polymerases, DNA primase, telomerase, DNA helicase, topoisomerases, DNA Ugases, replication factors, and DNA-binding proteins.
• hnRNPs Heterogeneous nuclear ribonucleoproteins
• RNA helicases alter and regulate RNA conformation and secondary structure by using energy derived from ATP hydrolysis to destabihze and unwind RNA duplexes.
• the most well-characterized and ubiquitous family of RNA heUcases is the DEAD-box family, so named for the conserved B-type ATP-binding motif which is diagnostic of proteins in this family.
• DEAD-box heUcases Over 40 DEAD-box heUcases have been identified in organisms as diverse as bacteria, insects, yeast, amphibians, mammals, and plants. DEAD-box helicases function in diverse processes such as translation initiation, splicing, ribosome assembly, and RNA editing, transport, and stability. Some DEAD-box helicases play tissue- and stage- specific roles in spermatogenesis and embryogenesis. (Reviewed in Linder, P. et al. (1989) Nature 337:121-122.)
• DEAD-box 1 protein may play a role in the progression of neuroblasto a (Nb) and retinoblastoma (Rb) tumors.
• DEAD-box helicases have been implicated either directly or indirectly in ultraviolet light-induced tumors, B cell lymphoma, and myeloid malignancies. (Reviewed in Godbout, R. et al. (1998) J. Biol. Chem. 273:21161-21168.)
• Initiation of ttanslation can be divided into three stages.
• the first stage brings an initiator transfer RNA (Met-tRNA f ) together with the 40S ribosomal subunit to form the 43S preinitiation complex.
• the second stage binds the 43S preinitiation complex to the mRNA, followed by migration of the complex to the correct AUG initiation codon.
• the third stage brings the 60S ribosomal subunit to the 40S subunit to generate an 80S ribosome at the initiation codon.
• Regulation of translation primarily involves the first and second stage in the initiation process (Pain, V.M. (1996) Eur. J. Biochem. 236:747-771).
• Cholesterol composed of four fused hydrocarbon rings with an alcohol at one end, moderates the fluidity of membranes in which it is inco ⁇ orated.
• cholesterol is used in the synthesis of steroid hormones such as cortisol, progesterone, estrogen, and testosterone.
• 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-franslationally 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, supra; Stryer, supra, pp.
• OSBP oxysterol- binding protein
• NH 4 + is assimilated into amino acids by the actions of two enzymes, glutamate dehydrogenase and glutamine synthetase.
• the carbon skeletons of amino acids come from the intermediates of glycolysis, the pentose phosphate pathway, or the citric acid cycle.
• humans can synthesize only thirteen (nonessential amino acids). The remaining nine must come from the diet (essential amino acids).
• Acetyl CoA is oxidized to C0 2 with concomitant formation of NADH, FADH 2 , and GTP.
• oxidative phosphorylation the transport of electrons from NADH and FADH 2 to oxygen by dehydrogenases is coupled to the synthesis of ATP from ADP and P. by the F Q F, ATPase complex in 5 the mitochondrial inner membrane.
• Po ⁇ hyrin groups contain four substituted pyrroles covalentiy joined in a ring, often with a bound metal atom.
• Enzymes involved in po ⁇ hyrin synthesis include ⁇ - aminolevulinate synthase, ⁇ -aminolevulinate dehydrase, po ⁇ hobilinogen deaminase, and cosynthase. Deficiencies in heme formation cause po ⁇ hyrias. Heme is broken down as a part of erythrocyte turnover.
• Enzymes involved in heme degradation include heme oxygenase and biliverdin reductase. o Iron is a required cofactor for many enzymes.
• iron is found in iron-sulfur clusters in proteins including aconitase, succinate dehydrogenase, and NADH-Q reductase. Iron is transported in the blood by the protein transferrin. Binding of transferrin to the fransferrin receptor on cell surfaces allows uptake by receptor mediated endocytosis. Cytosolic iron is bound to ferritin protein. 5 A molybdenum-containing cofactor (molybdopterin) is found in enzymes including sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase.
• molybdenum-containing cofactor molybdenum-containing cofactor
• Molybdopterin biosynthesis is performed by two molybdenum cofactor synthesizing enzymes. Deficiencies in these enzymes cause mental retardation and lens dislocation. Other diseases caused by defects in cofactor metabolism include pernicious anemia and methylmalonic aciduria. 0 Secretion and Trafficking
• the final Golgi compartment is the Trans-Golgi Network (TGN), where both membrane and lumenal proteins are sorted for their final destination.
• TGN Trans-Golgi Network
• secretory vesicle which contains proteins destined for the plasma membrane, such as receptors, adhesion molecules, and ion channels, and secretory proteins, such as hormones, neurofransmitters, and digestive enzymes. Secretory vesicles eventually fuse with the plasma membrane (GUck, B.S. and V. Malhofra (1998) Cell 95:883-889).
• Activation of the Fc receptors initiates a signal cascade involving src-family cytosolic kinases and the monomeric GTP-binding (G) protein Rho.
• G GTP-binding
• the resulting actin reorganization leads to phagocytosis of the particle.
• This process is an important component of the humoral immune response, allowing the processing and presentation of bacterial-derived peptides to antigen-specific T-lymphocytes.
• the second form of endocytosis, pinocytosis is a more generalized uptake of material from the external miUeu. Like phagocytosis, pinocytosis is activated by Ugand binding to cell surface receptors.
• Activation of individual receptors stimulates an internal response that includes coalescence of the receptor-ligand complexes and formation of clathrin-coated pits.
• Imagination of the plasma membrane at clathrin-coated pits produces an endocytic vesicle within the cell cytoplasm.
• These vesicles undergo homotypic fusion to form an early endosomal (EE) compartment.
• the tubulovesicular EE serves as a sorting site for incoming material.
• ATP-driven proton pumps in the EE membrane lowers the pH of the EE lumen (pH 6.3-6.8).
• the acidic environment causes many hgands to dissociate from their receptors.
• RecycUng vesicles may return directly to the plasma membrane. Receptors internalized and returned directly to the plasma membrane have a turnover rate of 2-3 minutes. Some RVs undergo microtubule-directed relocation to a perinuclear site, from which they then return to the plasma membrane. Receptors following this route have a turnover rate of 5-10 minutes. Still other RVs are retained within the cell until an appropriate signal is received (Mellman, supra; and James, D.E. et al. (1994) Trends Cell Biol. 4:120-126). Vesicle Formation
• the initial budding and coating processes are controlled by a cytosolic ras-like GTP-binding protein, ADP- ribosylating factor (Arf), and adapter proteins (AP). Different isoforms of both Arf and AP are involved at different sites of budding.
• Another small G-protein, dynamin forms a ring complex around the neck of the forming vesicle and may provide the mechanochemical force to accomplish the final step of the budding process.
• the coated vesicle complex is then transported through the cytosol. During the transport process, Arf -bound GTP is hydrolyzed to GDP and the coat dissociates from the transport vesicle (West, M.A. et al. (1997) J. Cell Biol.
• coat protein Two different classes have also been identified. Clathrin coats form on the TGN and PM surfaces, whereas coatomer or COP coats form on the ER and Golgi. COP coats can further be distinguished as COPI, involved in retrograde traffic through the Golgi and from the Golgi to the ER, and COPII, involved in anterograde traffic from the ER to the Golgi (Mellman, supra).
• the COP coat consists of two major components, a 5 G-protein (Arf or Sar) and coat protomer (coatomer).
• Coatomer is an equimolar complex of seven proteins, termed alpha-, beta-, beta'-, gamma-, delta-, epsilon- and zeta-COP. (Harter, C. and F.T. Wieland (1998) Proc. Nati. Acad. Sci. USA 95:11649-11654.) Membrane Fusion
• Transport vesicles undergo homotypic or heterotypic fusion in the secretory and endocytotic 0 pathways.
• Molecules required for appropriate targeting and fusion of vesicles with their target membrane include proteins inco ⁇ orated in the vesicle membrane, the target membrane, and proteins recruited from the cytosol.
• VAMP vesicle-associated membrane protein
• a cytosolic prenylated GTP-binding protein, Rab a member of the Ras 5 superfamily
• GTP-bound Rab proteins are directed into nascent fransport vesicles where they interact with VAMP. Following vesicle fransport, GTPase activating proteins (GAPs) in the target membrane convert Rab proteins to the GDP-bound form. A cytosolic protein, guanine-nucleotide dissociation inhibitor (GDI) helps return GDP-bound Rab proteins to their membrane of origin.
• GAPs GTPase activating proteins
• GDI guanine-nucleotide dissociation inhibitor
• Rab proteins appear to play a role in mediating the function of a viral gene, Rev, which is essential for replication of HIV-1, the virus responsible for AIDS (Flavell, R.A. et al. (1996) Proc. Nati. Acad. Sci. USA 93:4421-4424).
• N-ethylmaleimide sensitive factor (NSF) and soluble NSF-attachment protein ( ⁇ -SNAP and ⁇ -SNAP) are two such proteins that are conserved from yeast to man and function in most intracellular membrane fusion o reactions.
• Seel represents a family of yeast proteins that function at many different stages in the secretory pathway including membrane fusion. Recently, mammalian homologs of Seel , called Munc-18 proteins, have been identified (Katagiri, H. et al. (1995) J. Biol. Chem. 270:4963-4966; Hata et al. supra).
• the SNARE complex involves three SNARE molecules, one in the vesicular membrane and 5 two in the target membrane.
• Synaptotagmin is an integral membrane protein in the synaptic vesicle which associates with the t-SNARE syntaxin in the docking complex.
• Synaptotagmin binds calcium in a complex with negatively charged phospholipids, which allows the cytosolic SNAP protein to displace synaptotagmin from syntaxin and fusion to occur.
• synaptotagmin is a negative regulator of fusion in the neuron (Littleton, J.T. et al. (1993) Cell 74:1125-1134).
• the most abundant membrane 5 protein of synaptic vesicles appears to be the glycoprotein synaptophysin, a 38 kDa protein with four transmembrane domains.
• NPCs nuclear 5 pore complexes
• All nuclear proteins are imported from the cytoplasm, their site of synthesis.
• tRNA and mRNA are exported from the nucleus, their site of synthesis, to the cytoplasm, their site of function.
• Processing of small nuclear RNAs involves export into the cytoplasm, assembly with proteins and modifications such as hypermethylation to produce small nuclear ribonuclear proteins (snRNPs), and o subsequent import of the snRNPs back into the nucleus.
• snRNPs small nuclear ribonuclear proteins
• ribosomes require the initial import of ribosomal proteins from the cytoplasm, their incorporation with RNA into ribosomal subunits, and export back to the cytoplasm. (Gorlich, D. and l.W. Mattaj (1996) Science 271 :1513- 1518.)
• abnormal hormonal secretion is Unked to disorders such as diabetes insipidus (vasopressin), hyper- and hypoglycemia (insutin, glucagon), Grave's disease 5 and goiter (thyroid hormone), and Cushing's and Addison's diseases (adrenocorticotropic hormone, ACTH).
• cancer cells secrete excessive amounts of hormones or other biologically active peptides.
• Biologically active peptides that are ectopically synthesized in and secreted from tumor cells include ACTH and vasopressin (lung and pancreatic cancers); parathyroid hormone (lung 5 and bladder cancers); calcitonin (lung and breast cancers); and thyroid-stimulating hormone (medullary thyroid carcinoma).
• ACTH and vasopressin lung and pancreatic cancers
• parathyroid hormone lung 5 and bladder cancers
• calcitonin lung and breast cancers
• thyroid-stimulating hormone medullary thyroid carcinoma.
• Such peptides may be useful as diagnostic markers for tumorigenesis (Schwartz, M.Z. (1997) Semin. Pediatr. Surg. 3:141-146; and Said, S.I. and GR. Faloona (1975) N. Engl. J. Med. 293:155-160).
• Aquaporins are channels that fransport water and, in some cases, nonionic small solutes such as urea and glycerol. Water movement is important for a number of physiological processes 5 including renal fluid filtration, aqueous humor generation in the eye, cerebrospinal fluid production in the brain, and appropriate hydration of the lung. Aquaporins are members of the major intrinsic protein (MIP) family of membrane transporters (King, L.S. and P. Agre (1996) Annu. Rev. Physiol. 58:619- 648; Ishibasbi, K. et al. (1997) J. Biol. Chem. 272:20782-20786).
• MIP major intrinsic protein
• MTs The metallothioneins
• cysteine-rich proteins that bind heavy metals such as cadmium, zinc, mercury, lead, and copper and are thought to play a role in metal detoxification or the metabolism and homeostasis of metals.
• Arsenite-resistance proteins have been identified in hamsters that are resistant to toxic levels of arsenite (Rossman, T.G. et al. (1997) Mutat. Res. 386:307-314). 5 Humans respond to light and odors by specific protein pathways. Proteins involved in light perception include rhodopsin, fransducin, and cGMP phosphodiesterase.
• the cellular components of the humoral immune system include six different types of 5 leukocytes: monocytes, lymphocytes, polymo ⁇ honuclear granulocytes (consisting of monrophils, eosinophils, and basopbils) and plasma cells. Additionally, fragments of megakaryocytes, a seventh type of white blood cell in the bone marrow, occur in large numbers in the blood as platelets.
• Leukocytes are formed from two stem cell lineages in bone marrow.
• the myeloid stem cell line produces granulocytes and monocytes and, the lymphoid stem cell produces lymphocytes.
• o Lymphoid cells fravel to the thymus, spleen and lymph nodes, where they mature and differentiate into lymphocytes.
• Leukocytes are responsible for defending the body against invading pathogens. Neufrophils and monocytes attack invading bacteria, viruses, and other pathogens and destroy them by phagocytosis.
• Monocytes enter tissues and differentiate into macrophages which are extremely phagocytic. Lymphocytes and plasma cells are a part of the immune system which recognizes 5 specific foreign molecules and organisms and inactivates them, as well as signals other cells to attack the invaders.
• Tissue inflammation in response to pathogen invasion results in production of chemo-attractants for leukocytes, such as endotoxins or other bacterial products, prostaglandins, and products of leukocytes 0 or platelets.
• Helper T cells constitute up to 75% of the total T cell population. They regulate the immune functions by producing a variety of lymphokines that act on other cells in the immune system and on bone marrow. Among these lymphokines are: interleukins-2,3,4,5,6; granulocyte-monocyte colony stimulating factor, and ⁇ -interferon.
• B-lymphocytes produce antibodies which react with specific antigenic proteins presented by pathogens. Once activated, B cells become filled with extensive rough endoplasmic reticulum and are known as plasma cells. As with T cells, interaction of B cells with antigen stimulates proliferation of only those B cells which produce antibody specific to that antigen.
• AIDS l o Abnormal Immunodeficiency Syndrome
• helper T cells are depleted, leaving the patient susceptible to infection by microorganisms and parasites.
• Another widespread medical condition attributable to the immune system is that of allergic reactions to certain antigens. Allergic reactions include: hay fever, asthma, anaphylaxis, and urticaria (hives).
• Leukemias are an excess production of white blood cells, to the point where a major portion of the body' s metabolic resources
• Intercellular communication is essential for the growth and survival of multicellular organisms, and in particular, for the function of the endocrine, nervous, and immune systems.
• intercellular communication is critical for developmental processes such as tissue construction and organogenesis, in which cell proliferation, cell differentiation, and mo ⁇ hogenesis must be spatially and temporally regulated in a precise and coordinated manner.
• Cells communicate with one another through the secretion and uptake of diverse types of signaling molecules such as hormones, growth factors, neuropeptides, and cytokines.
• Hormones include hormones, growth factors, neuropeptides, and cytokines.
• disorders of the hypothalamus and pituitary often result from lesions such as primary brain tumors, adenomas, infarction associated with pregnancy, hypophysectomy, aneurysms, vascular o malformations, thrombosis, infections, immunological disorders, and complications due to head frauma. Such disorders have profound effects on the function of other endocrine glands.
• Disorders associated with hypopituitarism include hypogonadism, Sheehan syndrome, diabetes insipidus, Kallman's disease, Hand-SchuUer-Christian disease, Letterer-Siwe disease, sarcoidosis, empty sella syndrome, and dwarfism.
• Disorders associated with hype ⁇ ituitarism include acromegaly, giantism, and syndrome of 5 inappropriate ADH secretion (SIADH), often caused by benign adenomas.
• Thyroid hormones secreted by the thyroid and parathyroid primarily control metabolic rates and the regulation of serum calcium levels, respectively.
• Thyroid hormones include calcitonin, somatostatin, and thyroid hormone.
• the parathyroid secretes parathyroid hormone.
• Disorders associated with hypothyroidism include goiter, myxedema, acute thyroiditis associated with bacterial infection, o subacute thyroiditis associated with viral infection, autoimmune thyroiditis (Hashimoto's disease), and cretinism.
• Disorders associated with hyperthyroidism include thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and Plummer's disease.
• Diabetes melUtus is generally classified as either Type I (insulin-dependent, juvenile diabetes) or Type II (non- 5 insuUn-dependent, adult diabetes).
• Cytokine structure and function have been extensively characterized in vitro. Most cytokines are small polypeptides of about 30 kilodaltons or less. Over 50 cytokines have been identified from human and rodent sources. Examples of cytokine subfamiUes include the interferons (IFN- ⁇ , - ⁇ , and - ⁇ ), the interleukins (IL1-IL13), the tumor necrosis factors (TNF- ⁇ and - ⁇ ), and the chemokines. Many cytokines have been produced using recombinant DNA techniques, and the activities of individual cytokines have been determined in vitro. These activities include regulation of leukocyte proliferation, differentiation, and motiUty.
• cytokine activity in vitro may not reflect the full scope of that cytokine' s activity in vivo.
• Cytokines are not expressed individually in vivo but are instead expressed in combination with a multitude of other cytokines when the organism is challenged with a stimulus. Together, these cytokines collectively modulate the immune response in a manner appropriate for that particular stimulus. Therefore, the physiological activity of a cytokine is determined by the stimulus itself and by complex interactive networks among co-expressed cytokines which may demonstrate both synergistic and antagonistic relationships.
• the CC chemokines for example, each contain a conserved motif consisting of two consecutive cysteines followed by two additional cysteines which occur downstream at 24- and 16- residue intervals, respectively (ExPASy PROSITE database, documents PS00472 and PDOC00434).
• the presence and spacing of these four cysteine residues are highly conserved, whereas the intervening residues diverge significantly.
• a conserved tyrosine located about 15 residues downstream of the cysteine doublet seems to be important for chemotactic activity.
• Most of the human genes encoding CC chemokines are clustered on chromosome 17, although there are a few examples of CC chemokine genes that map elsewhere.
• Receptor Molecules 0 SEQ ID NO:6 and SEQ ID NO:7 encode, for example, receptor molecules.
• receptor describes proteins that specifically recognize other molecules.
• the category is broad and includes proteins with a variety of functions.
• the bulk of receptors are cell surface proteins which bind exfracellular ligands and produce cellular responses in the areas of growth, differentiation, endocytosis, and immune response.
• Other receptors facilitate the selective fransport of 5 proteins out of the endoplasmic reticulum and localize enzymes to particular locations in the cell.
• the term may also be applied to proteins which act as receptors for ligands with known or unknown chemical composition and which interact with other cellular components. For example, the steroid hormone receptors bind to and regulate transcription of DNA.
• Exfracellular loops of the GPCR alternate with intracellular loops and link the transmembrane domains.
• the most conserved domains of GPCRs are the transmembrane domains and the first two cytoplasmic loops.
• the transmembrane domains account for structural and functional features of the receptor. In most cases, the bundle of ⁇ helices forms a binding pocket.
• the extracellular N-terminal segment or one or more of the three exfracellular loops may also participate in ligand binding. Ligand binding activates the receptor by inducing a conformational change in intracellular portions of the receptor.
• GPCRs include those for acetylchoUne, adenosine, epinephrine and norepinephrine, bombesin, bradykinin, chemokines, dopamine, endothelin, ⁇ -aminobutyric acid (GABA), follicle-stimulating hormone (FSH), glutamate, gonadotropin-releasing hormone (GnRH), hepatocyte growth factor, histamine, leukotrienes, melanocortins, neuropeptide Y, opioid peptides, opsins, prostanoids, serotonin, 5 somatostatin, tachykinins, thrombin, thyrottopin-releasing hormone (TRH), vasoactive intestinal polypeptide family, vasopressin and oxytocin, and o ⁇ han receptors.
• GABA ⁇ -aminobutyric acid
• FSH follicle-stimulating hormone
• GPCR mutations which may cause loss of function or constitutive activation, have been associated with numerous human diseases (Coughlin, supra). For instance, retinitis pigmentosa may arise from mutations in the rhodopsin gene. Rhodopsin is the retinal photoreceptor which is located 0 within the discs of the eye rod cell. Parma, J. et al. (1993, Nature 365:649-651) report that somatic activating mutations in the thyrofropin receptor cause hyperfunctioning thyroid adenomas and suggest that certain GPCRs susceptible to constitutive activation may behave as protooncogenes. Nuclear Receptors
• Macrophage scavenger receptors with broad ligand specificity may participate in the binding of low density lipoproteins (LDL) and foreign antigens.
• Scavenger receptors types I and II are trimeric 5 membrane proteins with each subunit containing a small N-terminal intracellular domain, a transmembrane domain, a large extracellular domain, and a C-terminal cysteine-rich domain.
• the extracellular domain contains a short spacer domain, an ⁇ -helical coiled-coil domain, and a friple helical collagenous domain.
• Enterokinase the initiator of intestinal digestion, is a serine protease found in the intestinal brush border, where it cleaves the acidic propeptide from trypsinogen to yield active trypsin (Kitamoto, Y. et al. (1994) Proc. Nati. Acad. Sci. USA 91:7588-7592).
• o Prolylcarboxypeptidase a lysosomal serine peptidase that cleaves peptides such as angiotensin II and III and [des-Arg9] bradykinin, shares sequence homology with members of both the serine carboxypeptidase and prolylendopeptidase families (Tan, F. et al. (1993) J. Biol. Chem. 268:16631- 16638).
• Cysteine proteases have a cysteine as the major catalytic residue at an active site where 5 catalysis proceeds via an intermediate thiol ester and is facilitated by adjacent histidine and aspartic acid residues.
• CPs are involved in diverse cellular processes ranging from the processing of precursor proteins to intracellular degradation. Mammalian CPs include lysosomal cathepsins and cytosolic calcium activated proteases, calpains. CPs are produced by monocytes, macrophages and other cells of the immune system which migrate to sites of inflammation and secrete molecules involved in o tissue repair. Overabundance of these repair molecules plays a role in certain disorders.
• RNases also serve a variety of functions.
• RNase P is a ribonucleoprotein enzyme which cleaves the 5' end of pre-tRNAs as part of their maturation process.
• RNase H digests the RNA strand of an RNA/DNA hybrid. Such hybrids occur in cells invaded by refroviruses, and RNase H is an important enzyme in the retroviral replication cycle.
• Pancreatic RNase secreted by the pancreas into 5 the intestine hydrolyzes RNA present in ingested foods.
• RNase activity in serum and cell extracts is elevated in a variety of cancers and infectious diseases (Schein, CH. (1997) Nat. Biotechnol. 15:529- 536).
• RNA helicases utilize energy derived from ATP hydrolysis to destabilize and unwind RNA duplexes.
• the most well-characterized and ubiquitous family of RNA helicases is the DEAD-box family, so named for the conserved B-type ATP-binding motif which is diagnostic of proteins in this family.
• DEAD-box helicases Over 40 DEAD-box helicases have been identified in organisms as diverse as bacteria, insects, o yeast, amphibians, mammals, and plants. DEAD-box helicases function in diverse processes such as translation initiation, splicing, ribosome assembly, and RNA editing, fransport, and stability. Some DEAD-box helicases play tissue- and stage-specific roles in spermatogenesis and embryogenesis.
• murine p68 is mutated in ultraviolet Ught-induced tumors
• human DDX6 is located at a chromosomal breakpoint associated with B-cell lymphoma.
• a chimeric protein comprised of DDX10 and NUP98, a nucleoporin protein, may be involved in the pathogenesis of certain myeloid maUgnancies. Topoisomerases
• the spUceosomal complex is composed of five small nuclear ribonucleoprotein particles 0 (snRNPs) designated Ul, U2, U4, U5, and U6, and a number of additional proteins.
• snRNP small nuclear ribonucleoprotein particles 0
• Ul small nuclear ribonucleoprotein particles 0
• U2, U4, U5, and U6 small nuclear ribonucleoprotein particles 0
• snRNP contains a single species of snRNA and about ten proteins.
• the RNA components of some snRNPs recognize and base pair with intron consensus sequences.
• the protein components mediate spliceosome assembly and the splicing reaction.
• Autoantibodies to snRNP proteins are found in the blood of patients with systemic lupus erythematosus (Stryer, L. (1995) Biochemistry, W.H. Freeman and 5 Company, New York NY, p. 863).
• N-cadherin is present on nerve, muscle, and lens cells and is also critical for embryonic development.
• P-cadherin is present on cells of the placenta and epidermis. Recent studies report that protocadherins are involved in 5 a variety of cell-cell interactions (Suzuki, S.T. (1996) J. Cell Sci. 109:2609-2611).
• the infracellular anchorage of cadherins is regulated by their dynamic association with catenins, a family of cytoplasmic signal transduction proteins associated with the actin cytoskeleton.
• o Galectins contain a characteristic carbohydrate recognition domain (CRD). The CRD is about
• Galectins play a number of roles in diseases and conditions associated with cell-cell and cell- matrix interactions. For example, certain galectins associate with sites of inflammation and bind to cell surface immunoglobulin E molecules. In addition, galectins may play an important role in cancer metastasis.
• Selectins comprise a specialized lectin subfamily involved primarily in o inflammation and leukocyte adhesion (Reviewed in Lasky, supra). Selectins mediate the recruitment of leukocytes from the circulation to sites of acute inflammation and are expressed on the surface of vascular endothelial cells in response to cytokine signaling. Selectins bind to specific ligands on the leukocyte cell membrane and enable the leukocyte to adhere to and migrate along the endothehal surface. Binding of selectin to its ligand leads to polarized rearrangement of the actin cytoskeleton and 5 stimulates signal transduction within the leukocyte (Brenner, B. et al. (1997) Biochem. Biophys. Res.
• MHC proteins are cell surface markers that bind to and present foreign antigens to T cells. MHC molecules are classified as either class I or class II. Class I MHC molecules (MHC I) are expressed on the surface of almost all cells and are involved in the presentation of antigen to cytotoxic T cells. For example, a cell infected with virus will degrade intracellular viral proteins and express the protein fragments bound to MHC I molecules on the cell surface. The MHC I/antigen 5 complex is recognized by cytotoxic T-cells which destroy the infected cell and the virus within.
• MHC I major histocompatibility
• the five antibody classes, IgA, IgD, o IgE, IgG and IgM, are defined by the ⁇ , ⁇ , e, ⁇ , and ⁇ H-chain types.
• IgG the most common class of antibody found in the circulation, is tettameric, while the other classes of antibodies are generally variants or multimers of this basic structure.
• Both H-chains and L-chains contain repeated Ig domains.
• a typical H-chain contains four Ig domains, three of which occur within the constant region and one of which occurs 5 within the variable region and contributes to the formation of the antigen recognition site.
• a typical L-chain contains two Ig domains, one of which occurs within the constant region and one of which occurs within the variable region.
• the immune system is capable of recognizing and responding to any foreign molecule that enters the body. Therefore, the immune system must be armed with a full repertoire of antibodies 5 against all potential antigens.
• antibody diversity is generated by somatic rearrangement of gene segments encoding variable and constant regions. These gene segments are joined together by site- specific recombination which occurs between highly conserved DNA sequences that flank each gene segment. Because there are hundreds of different gene segments, millions of unique genes can be generated combinatorially. In addition, imprecise joining of these segments and an unusually high 0 rate of somatic mutation within these segments further contribute to the generation of a diverse antibody population.
• T-cell receptors are both structurally and functionally related to antibodies. (Reviewed in Alberts, supra, pp. 1228-1229.) T-cell receptors are cell surface proteins that bind foreign antigens and mediate diverse aspects of the immune response.
• a typical T-cell receptor is a heterodimer comprised 5 of two disulfide-linked polypeptide chains called ⁇ and ⁇ . Each chain is about 280 amino acids in length and contains one variable region and one constant region. Each variable or constant region folds into an Ig domain. The variable regions from the ⁇ and ⁇ chains come together in the heterodimer to form the antigen recognition site.
• T-cell receptor diversity is generated by somatic rearrangement of gene segments encoding the ⁇ and ⁇ chains.
• Protein secretion is essential for cellular function. Protein secretion is mediated by a signal peptide located at the amino terminus of the protein to be secreted.
• the signal peptide is comprised of about ten to twenty hydrophobic amino acids which target the nascent protein from the ribosome to the endoplasmic reticulum (ER). Proteins targeted to the ER may either proceed through the secretory o pathway or remain in any of the secretory organelles such as the ER, Golgi apparatus, or lysosomes.
• Proteins that transit through the secretory pathway are either secreted into the exfracellular space or retained in the plasma membrane.
• Secreted proteins are often synthesized as inactive precursors that are activated by post-franslational processing events during transit through the secretory pathway. Such events include glycosylation, proteolysis, and removal of the signal peptide by a signal peptidase. 5
• Other events that may occur during protein fransport include chaperone-dependent unfolding and folding of the nascent protein and interaction of the protein with a receptor or pore complex.
• the collagens comprise a family of ECM proteins that provide structure to bone, teeth, skin, Ugaments, tendons, cartilage, blood vessels, and basement membranes. Multiple collagen proteins have been identified. Three collagen molecules fold together in a friple helix stabilized by interchain disulfide bonds. Bundles of these friple helices then associate to form fibrils. Collagen primary structure o consists of hundreds of (Gly-X-Y) repeats where about a third of the X and Y residues are Pro.
• Glycines are crucial to helix formation as the bulkier amino acid sidechains cannot fold into the friple helical conformation. Because of these strict sequence requirements, mutations in collagen genes have severe consequences. Osteogenesis imperfecta patients have brittle bones that fracture easily; in severe cases patients die in utero or at birth. Ehlers-Danlos syndrome patients have hyperelastic skin, 5 hypermobile joints, and susceptibility to aortic and intestinal rupture. Chondrodysplasia patients have short stature and ocular disorders. Alport syndrome patients have hematuria, sensorineural deafness, and eye lens deformation. (Isselbacher, K.J. et al.
• Elastin is a highly hydrophobic protein of about 750 amino acids that is rich in proline and glycine residues. Elastin molecules are highly cross-linked, forming an extensive extracellular network of fibers and sheets. Elastin fibers are surrounded by a sheath of microfibrils which are composed of a number of glycoproteins, including fibrillin. Mutations in the gene encoding fibrillin are responsible for 5 Marfan's syndrome, a genetic disorder characterized by defects in connective tissue. In severe cases, the aortas of afflicted individuals are prone to rupture. (Reviewed in Alberts, supra, pp. 984-986.)
• proteoglycans are composed of unbranched polysaccharide chains (glycosaminoglycans) attached to protein cores. Common proteoglycans include aggrecan, betaglycan, decorin, perlecan, serglycin, and syndecan-1. Some of these molecules not only provide mechanical support, but also bind to extracellular signaling molecules, such as fibroblast growth factor and transforming growth factor ⁇ , suggesting a role for proteoglycans in cell-cell communication and cell growth. (Reviewed in Alberts, supra, pp.
• glycoproteins tenascin-C and tenascin-R are expressed in developing and lesioned neural tissue and provide stimulatory and anti- adhesive (inhibitory) properties, respectively, for axonal growth. (Faissner, A. (1997) Cell Tissue Res. 290:331-341.)
• Microtubules cytoskeletal fibers with a diameter of about 24 nm, have multiple roles in the cell. Bundles of microtubules form cilia and flagella, which are whip-like extensions of the cell membrane that are necessary for sweeping materials across an epithelium and for swimming of sperm, respectively. Marginal bands of microtubules in red blood cells and platelets are important for these cells' pliability. Organelles, membrane vesicles, and proteins are transported in the cell along tracks of microtubules. For example, microtubules run through nerve cell axons, allowing bidirectional transport of materials and membrane vesicles between the cell body and the nerve terminal. Failure to supply the nerve terminal with these vesicles blocks the transmission of neural signals. Microtubules are also critical to chromosomal movement during cell division. Both stable and short-lived populations of microtubules exist in the cell.
• G-actin the monomeric form of actin, polymerizes into polarized, helical F- actin filaments, accompanied by the hydrolysis of ATP to ADP.
• Actin filaments associate to form o bundles and networks, providing a framework to support the plasma membrane and determine cell shape. These bundles and networks are connected to the cell membrane.
• thin filaments containing actin slide past thick filaments containing the motor protein myosin during contraction.
• a family of actin-related proteins exist that are not part of the actin cytoskeleton, but rather associate with microtubules and dynein.
• Severing proteins regulate the length of actin filaments by breaking them into short pieces or by blocking their ends.
• Severing proteins include gCAP39, severin (fragmin), gelsolin, and villin.
• Capping proteins can cap the ends of actin filaments, but cannot break filaments.
• Capping proteins 5 include CapZ and fropomodulin.
• the proteins thymosin and profilin sequester actin monomers in the cytosol, allowing a pool of unpolymerized actin to exist.
• the actin-associated proteins tropomyosin, froponin, and caldesmon regulate muscle confraction in response to calcium.
• Intermediate filaments are cytoskeletal fibers with a diameter of about 10 nm, o intermediate between that of microfilaments and microtubules. IFs serve structural roles in the cell, reinforcing cells and organizing cells into tissues. IFs are particularly abundant in epidermal cells and in neurons. IFs are extremely stable, and, in contrast to microfilaments and microtubules, do not function in cell motility.
• Type III IF proteins include desmin, glial fibrillary acidic protein, vimentin, and peripherin.
• Desmin filaments in muscle cells link myofibrils into bundles and stabilize sarcomeres in contracting 5 muscle.
• GUal fibrillary acidic protein filaments are found in the glial cells that surround neurons and astrocytes.
• Vimentin filaments are found in blood vessel endothelial cells, some epitheUal cells, and mesenchymal cells such as fibroblasts, and are commonly associated with microtubules. Vimentin filaments may have roles in keeping the nucleus and other organelles in place in the cell.
• Type IV IFs include the neurofilaments and nestin.
• Neurofilaments composed of three polypeptides NF-L, NF-M, and NF-H, are frequently associated with microtubules in axons. Neurofilaments are responsible for the radial growth and diameter of an axon, and ultimately for the speed of nerve impulse transmission. Changes in phosphorylation and metabolism of neurofilaments are observed in neurodegenerative diseases including amyofrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease (Julien, J.P. and W.E. Mushynski (1998) Prog. Nucleic Acid Res. Mol. Biol. 61:1-23). Type V IFs, the lamins, are found in the nucleus where they support the nuclear membrane.
• IFs have a central ⁇ -helical rod region interrupted by short nonhelical linker segments.
• the rod region is bracketed, in most cases, by non-hehcal head and tail domains.
• the rod regions of intermediate filament proteins associate to form a coiled-coil dimer.
• a highly ordered assembly process leads from the dimers to the IFs. Neither ATP nor GTP is needed for IF assembly, unlike that of microfilaments and microtubules.
• Cytoskeletal fibers are attached to the plasma membrane by specific proteins. These attachments are important for maintaining cell shape and for muscle confraction.
• the spectrin- actin cytoskeleton is attached to cell membrane by three proteins, band 4J, ankyrin, and adducin. Defects in this attachment result in abnormally shaped cells which are more rapidly degraded by the spleen, leading to anemia.
• the spectrin-actin cytoskeleton is also linked to the membrane by ankyrin; a second actin network is anchored to the membrane by filamin.
• the protein dysfrophin links actin filaments to the plasma membrane; mutations in the dysfrophin gene lead to Duchenne muscular dystrophy.
• adherens junctions and adhesion plaques the peripheral membrane proteins ⁇ -actinin and vinculin attach actin filaments to the cell membrane.
• IFs are also attached to membranes by cytoskeletal-membrane anchors.
• the nuclear lamina is attached to the inner surface of the nuclear membrane by the lamin B receptor.
• Vimentin IFs are attached to the plasma membrane by ankyrin and plectin.
• Desmosome and hemidesmosome membrane junctions hold together epithelial cells of organs and skin. These membrane junctions allow shear forces to be distributed across the entire epithelial cell layer, thus providing strength and rigidity to the epithelium.
• IFs in epithelial cells are attached to the desmosome by plakoglobin and desmoplakins. The proteins that link IFs to hemidesmosomes are not known.
• Myosin provides the motor function for muscle confraction and intracellular movements such as phagocytosis and rearrangement of cell contents during mitotic cell division (cytokinesis).
• the contractile unit of skeletal muscle termed the sarcomere, consists of highly ordered arrays of thin actin-containing filaments and thick myosin-containing filaments. Crossbridges form o between the thick and thin filaments, and the ATP-dependent movement of myosin heads within the thick filaments pulls the thin filaments, shortening the sarcomere and thus the muscle fiber.
• Myosins are composed of one or two heavy chains and associated light chains.
• Myosin heavy chains contain an amino-terminal motor or head domain, a neck that is the site of light-chain binding, and a carboxy-terminal tail domain.
• the tail domains may associate to form an ⁇ -helical 5 coiled coil.
• Conventional myosins such as those found in muscle tissue, are composed of two myosin heavy-chain subunits, each associated with two light-chain subunits that bind at the neck region and play a regulatory role.
• Unconventional myosins believed to function in infracellular motion, may contain either one or two heavy chains and associated light chains. There is evidence for about 25 myosin heavy chain genes in vertebrates, more than half of them unconventional. 0 Dynein-related Motor Proteins
• Kinesins are (+) end-directed motor proteins which act on microtubules.
• the prototypical kinesin molecule is involved in the transport of membrane-bound vesicles and organelles. This function is particularly important for axonal fransport in neurons.
• Kinesin is also important in all cell types for 5 the fransport of vesicles from the Golgi complex to the endoplasmic reticulum. This role is critical for maintaining the identity and functionality of these secretory organelles.
• dynamin disassembly. Following disassembly the dynamin may either dissociate from the membrane or remain associated to the vesicle and be fransported to another region of the cell.
• Three homologous dynamin genes have been discovered, in addition to several dynamin-related proteins. conserveed dynamin regions are the N-terminal GTP-binding domain, a central pleckstrin homology domain that binds membranes, a central coiled-coil region that may activate dynamin' s GTPase activity, and a C-terminal proline-rich domain that contains several motifs that bind SH3 domains on other proteins.
• the cytoskeleton is reviewed in Lodish, H. et al. (1995) Molecular Cell Biology, Scientific 5 American Books, New York NY.
• Chromatin is associated with disorders of protein expression such as thalassemia, a genetic anemia resulting from the removal of the locus confrol region (LCR) required for decondensation of the 15 globin gene locus .
• LCR locus confrol region
• Glucose is initially converted 0 to pyruvate in the cytoplasm.
• Fatty acids and pyruvate are fransported to the mitochondria for complete oxidation to C0 2 coupled by enzymes to the fransport of electrons from NADH and FADH 2 to oxygen and to the synthesis of ATP (oxidative phosphorylation) from ADP and P..
• Pyruvate is transported into the mitochondria and converted to acetyl-CoA for oxidation via the citric acid cycle, involving pyruvate dehydrogenase components, dihydrolipoyl fransacetylase, and 5 dihydrolipoyl dehydrogenase.
• Enzymes involved in the cifric acid cycle include: citrate synthetase, aconitases, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase complex including transsuccinylases, succinyl CoA synthetase, succinate dehydrogenase, fumarases, and malate dehydrogenase.
• Mitochondria are oval-shaped organelles comprising an outer membrane, a tightly folded 5 inner membrane, an intermembrane space between the outer and inner membranes, and a matrix inside the inner membrane.
• the outer membrane contains many porin molecules that allow ions and charged molecules to enter the intermembrane space, while the inner membrane contains a variety of fransport proteins that transfer only selected molecules.
• Mitochondria are the primary sites of energy production in cells. o Mitochondria contain a small amount of DNA.
• Human mitochondrial DNA encodes 13 proteins, 22 tRNAs, and 2 rRNAs.
• Mitochondrial-DNA encoded proteins include NADH-Q reductase, a cytochrome reductase subunit, cytochrome oxidase subunits, and ATP synthase subunits.
• mitochondrial proteins The majority of mitochondrial proteins are encoded by nuclear genes, are synthesized on cytosolic ribosomes, and are imported into the mitochondria. Nuclear-encoded proteins which are destined for the mitochondrial matrix typically contain positively-charged amino terminal signal sequences. Import of these preproteins from the cytoplasm requires a multisubunit protein complex in the outer membrane known as the translocase of outer mitochondrial membrane (TOM; previously designated MOM; Pfanner, N. et al. (1996) Trends Biochem. Sci. 21 :51-52) and at least three inner membrane proteins which comprise the translocase of inner mitochondrial membrane (TIM; previously designated MIM; Pfanner, supra). An inside-negative membrane potential across the inner mitochondrial membrane is also required for preprotein import.
• TOM translocase of outer mitochondrial membrane
• TIM inner membrane proteins which comprise the translocase of inner mitochondrial membrane
• Mitochondria are implicated in disorders of cell proliferation, since they play an important role in a cell's decision to proliferate or self-destruct through apoptosis.
• the oncoprotein Bcl-2 for example, promotes cell proliferation by stabilizing mitochondrial membranes so that apoptosis signals are not released (Susin, S.A. (1998) Biochim.
• SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43 encode, for example, franscription factor molecules.
• the double helix structure and repeated sequences of DNA create topological and chemical features which can be recognized by franscription factors. These features are hydrogen bond donor 15 and acceptor groups, hydrophobic patches, major and minor grooves, and regular, repeated stretches of sequence which induce distinct bends in the helix.
• franscription factors recognize specific DNA sequence motifs of about 20 nucleotides in length. Multiple, adjacent franscription factor-binding motifs may be required for gene regulation.
• the helix-turn-helix motif consists of two ⁇ helices connected at a fixed angle by a short chain of amino acids. One of the helices binds to the major groove. Helix-turn-helix motifs are 5 exemplified by the homeobox motif which is present in homeodomain proteins. These proteins are critical for specifying the anterior-posterior body axis during development and are conserved throughout the animal kingdom.
• the Antennapedia and Ultrabithorax proteins of Drosophila melanogaster are prototypical homeodomain proteins (Pabo, CO. and R.T. Sauer (1992) Annu. Rev. Biochem. 61 :1053-1095).
• the zinc finger motif which binds zinc ions, generally contains tandem repeats of about 30 amino acids consisting of periodically spaced cysteine and histidine residues. Examples of this sequence pattern, designated C2H2 and C3HC4 ("RING" finger), have been described (Lewin, supra). Zinc finger proteins each contain an ⁇ helix and an antiparallel ⁇ sheet whose proximity and conformation are maintained by the zinc ion. Contact with DNA is made by the arginine prece ding 5 the ⁇ helix and by the second, third, and sixth residues of the ⁇ hehx. Variants of the zinc finger motif include poorly defined cysteine-rich motifs which bind zinc or other metal ions. These motifs may not contain histidine residues and are generally nonrepetitive.
• the helix-loop-helix motif (HLH) consists of a short ⁇ hehx connected by a loop to a longer ⁇ hetix.
• the loop is flexible and allows the two helices to fold back against each other and to bind to DNA.
• the transcription factor Myc contains a prototypical HLH motif.
• neoplastic disorders in humans can be attributed to inappropriate gene expression. Malignant cell growth may result from either excessive expression of tumor promoting genes or insufficient expression of tumor suppressor genes (Cleary, M.L. (1992) Cancer Surv. 15:89-104).
• Chromosomal franslocations may also produce chimeric loci which fuse the coding sequence of one gene with the regulatory regions of a second unrelated gene. Such an arrangement likely results in inappropriate gene franscription, potentially contributing to malignancy.
• the immune system responds to infection or frauma by activating a cascade of events that coordinate the progressive selection, amplification, and mobilization of cellular defense mechanisms. A complex and balanced program of gene activation and repression is involved in this process.
• hyperactivity of the immune system as a result of improper or insufficient regulation of gene expression may result in considerable tissue or organ damage. This damage is well documented in immunological responses associated with arthritis, allergens, heart attack, sfroke, and infections (Isselbacher, K.J. et al. (1996) Harrison's Principles of Internal Medicine, 13/e, McGraw
• CeU Membrane Molecules SEQ ID NO:28 and SEQ ID NO:29 encode, for example, cell membrane molecules.
• Eukaryotic cells are surrounded by plasma membranes which enclose the cell and maintain an environment inside the cell that is distinct from its surroundings.
• eukaryotic organisms are distinct from prokaryotes in possessing many intracellular organelle and vesicle structures. Many of the metabolic reactions which distinguish eukaryotic biochemistry from prokaryotic biochemistry take place within these structures.
• the plasma membrane and the membranes surrounding organelles and vesicles are composed of phosphoglycerides, fatty acids, cholesterol, phosphoUpids, glycolipids, proteoglycans, and proteins. These components confer identity and functionality to the membranes with which they associate.
• Integral Membrane Proteins The majority of known integral membrane proteins are fransmembrane proteins (TM) which are characterized by an exfracellular, a transmembrane, and an infracellular domain. TM domains are typically comprised of 15 to 25 hydrophobic amino acids which are predicted to adopt an ⁇ -helical conformation. TM proteins are classified as bitopic (Types I and II) and polytopic (Types III and IV) (Singer, S.J. (1990) Annu. Rev. Cell Biol. 6:247-296). Bitopic proteins span the membrane once while polytopic proteins contain multiple membrane-spanning segments. TM proteins function as cell-surface receptors, receptor-interacting proteins, fransporters of ions or metabolites, ion channels, cell anchoring proteins, and cell type- specific surface antigens.
• TM proteins function as cell-surface receptors, receptor-interacting proteins, fransporters of ions or metabolites, ion channels, cell anchoring proteins, and cell type- specific surface antigens.
• NGR NGR
• GSL motif-containing peptides have been used as drug delivery agents in targeted cancer treatment of tumor vasculature (Arap, W. et al. (1998) Science 279:377-380).
• MPs may also contain amino acid sequence motifs, such as the carbohydrate recognition domain (CRD), that mediate interactions with exfracellular or infracellular molecules.
• CCD carbohydrate recognition domain
• GPCR G-protein coupled receptors
• GPCRs include receptors for biogenic amines, lipid mediators of inflammation, peptide hormones, and sensory signal mediators.
• the structure of these highly-conserved receptors consists of seven hydrophobic fransmembrane regions, an extracellular N-terminus, and a cytoplasmic C-terminus. Three extracellular loops alternate with three infracellular loops to link the seven transmembrane regions. Cysteine disulfide bridges connect the second and third extracellular loops.
• the most conserved regions of GPCRs are the transmembrane regions and the first two cytoplasmic loops.
• a conserved, acidic-Arg-aromatic residue triplet present in the second cytoplasmic loop may interact with G proteins.
• a GPCR consensus pattern is characteristic of 5 most proteins belonging to this superfamily (ExPASy PROSITE document PS00237; and Watson, S. and S. Arkinstall (1994) The G-protein Linked Receptor Facts Book, Academic Press, San Diego CA, pp. 2-6). Mutations and changes in franscriptional activation of GPCR-encoding genes have been associated with neurological disorders such as schizophrenia, Parkinson's disease, Alzheimer's disease, drug addiction, and feeding disorders. 0 Scavenger Receptors
• Macrophage scavenger receptors with broad ligand specificity may participate in the binding of low density lipoproteins (LDL) and foreign antigens.
• Scavenger receptors types I and II are trimeric membrane proteins with each subunit containing a small N-terminal infracellular domain, a fransmembrane domain, a large exfracellular domain, and a C-terminal cysteine-rich domain.
• the 5 exfracellular domain contains a short spacer region, an ⁇ -helical coiled-coil region, and a triple helical collagen-like region.
• the fransmembrane 4 superfamily (TM4SF) or tetraspan family is a multigene family encoding type III integral membrane proteins (Wright, M.D. and M.G. Tomlinson (1994) Immunol. 5 Today 15 :588-594).
• the TM4SF is comprised of membrane proteins which traverse the cell membrane four times.
• Members of the TM4SF include platelet and endothelial cell membrane proteins, melanoma-associated antigens, leukocyte surface glycoproteins, colonal carcinoma antigens, tumor-associated antigens, and surface proteins of the schistosome parasites (Jankowski, S.A. (1994) Oncogene 9:1205-1211).
• Members of the TM4SF share about 25-30% amino acid sequence identity o with one another.
• Tumor antigens are cell surface molecules that are differentially expressed in tumor cells relative to normal cells. Tumor antigens distinguish tumor cells immunologically from normal cells and provide diagnostic and therapeutic targets for human cancers (Takagi, S. et al. (1995) Int. J. Cancer 61:706-715; Liu, E. et al. (1992) Oncogene 7:1027-1032). 5 Leukocyte Antigens
• cell surface antigens include those identified on leukocytic cells of the immune system. These antigens have been identified using systematic, monoclonal antibody (mAb)-based "shot gun” techniques. These techniques have resulted in the production of hundreds of mAbs directed against unknown cell surface leukocytic antigens. These antigens have been grouped into o "clusters of differentiation” based on common immunocytochemical localization patterns in various differentiated and undifferentiated leukocytic cell types. Antigens in a given cluster are presumed to identify a single cell surface protein and are assigned a "cluster of differentiation" or "CD" designation.
• mAb monoclonal antibody
• CD antigens Some of the genes encoding proteins identified by CD antigens have been cloned and verified by standard molecular biology techniques. CD antigens have been characterized as both 5 fransmembrane proteins and cell surface proteins anchored to the plasma membrane via covalent attachment to fatty acid-containing glycolipids such as glycosylphosphatidylinositol (GPI). (Reviewed in Barclay, A.N. et al. (1995) The Leucocyte Antigen Facts Book, Academic Press, San Diego CA, pp. 17-20.) Ion Channels o Ion channels are found in the plasma membranes of virtually every cell in the body.
• GPI glycosylphosphatidylinositol
• chloride channels mediate a variety of cellular functions including regulation of membrane potentials and abso ⁇ tion and secretion of ions across epithelial membranes.
• Chloride channels also regulate the pH of organelles such as the Golgi apparatus and endosomes (see, e.g., Greger, R. (1988) Annu. Rev. Physiol. 50:111-122).
• Elecfrophysiological and pharmacological properties of chloride 5 channels including ion conductance, current-voltage relationships, and sensitivity to modulators, suggest that different chloride channels exist in muscles, neurons, fibroblasts, epithelial cells, and lymphocytes.
• the resultant gradient may be used to transport other ions across the membrane (Na + , K + , or Cl " ) or to maintain organelle pH.
• Proton ATPases are further subdivided into the mitochondrial F- ATPases, the plasma membrane ATPases, and the vacuolar ATPases.
• the vacuolar ATPases establish and maintain an acidic pH 5 within various organelles involved in the processes of endocytosis and exocytosis (Mellman, I. et al. (1986) Annu. Rev. Biochem. 55:663-700).
• ABC ATP-binding cassette
• fransporters also called the "traffic ATPases”
• ATP-binding cassettes comprise a o superfamily of membrane proteins that mediate fransport and channel functions in prokaryotes and eukaryotes (Higgins, C.F. (1992) Annu. Rev. Cell Biol. 8:67-113).
• ABC proteins share a similar overall structure and significant sequence homology. All ABC proteins contain a conserved domain of approximately two hundred amino acid residues which includes one or more nucleotide binding domains.
• Membrane o anchors are covalentiy joined to a protein post-franslationally and include such moieties as prenyl, myristyl, and glycosylphosphatidyl inositol groups.
• Membrane localization of peripheral and anchored proteins is important for their function in processes such as receptor-mediated signal transduction. For example, prenylation of Ras is required for its localization to the plasma membrane and for its normal and oncogenic functions in signal transduction. 5 Vesicle Coat Proteins Intercellular communication is essential for the development and survival of multicellular organisms. Cells communicate with one another through the secretion and uptake of protein signaling molecules.
• the uptake of proteins into the cell is achieved by the endocytic pathway, in which the interaction of exfracellular signaling molecules with plasma membrane receptors results in 5 the formation of plasma membrane-derived vesicles that enclose and transport the molecules into the cytosol. These transport vesicles fuse with and mature into endosomal and lysosomal (digestive) compartments.
• the secretion of proteins from the cell is achieved by exocytosis, in which molecules inside of the cell proceed through the secretory pathway. In this pathway, molecules transit from the ER to the Golgi apparatus and finally to the plasma membrane, where they are secreted from the cell.
• Clathrin coats form on vesicles derived from the TGN and PM, whereas coatomer (COP) coats form on vesicles derived from the ER and Golgi.
• COP coats can be further classified as COPI, involved in retrograde traffic through the Golgi and from the Golgi to the ER, and COPII, involved in anterograde traffic from the 0 ER to the Golgi (Mellman, supra).
• adapter proteins bring vesicle cargo and coat proteins together at the surface of the budding membrane.
• Adapter protein- 1 and -2 select cargo from the TGN and plasma membrane, respectively, based on molecular information encoded on the cytoplasmic tail of integral membrane cargo proteins.
• Adapter proteins also recruit clathrin to the bud 5 site.
• Clathrin is a protein complex consisting of three large and three small polypeptide chains arranged in a three-legged structure called a friskelion. Multiple triskelions and other coat proteins appear to self-assemble on the membrane to form a coated pit. This assembly process may serve to deform the membrane into a budding vesicle.
• GTP-bound ADP-ribosylation factor (Arf) is also inco ⁇ orated into the coated assembly.
• Another small G-protein, dynamin forms a ring complex o around the neck of the forming vesicle and may provide the mechanochemical force to seal the bud, thereby releasing the vesicle.
• the coated vesicle complex is then transported through the cytosol.
• Arf-bound GTP is hydrolyzed to GDP, and the coat dissociates from the fransport vesicle (West, M.A. et al. (1997) J. Cell Biol. 138:1239-1254).
• the coat protein is assembled from cytosolic precursor molecules at specific budding regions on the organelle.
• the COP coat consists of two major components, a G-protein (Arf or Sar) and coat protomer (coatomer).
• Coatomer is an equimolar complex of seven proteins, termed alpha-, beta-, beta'-, gamma-, delta-, epsilon- and zeta-COP.
• the coatomer complex binds to dilysine motifs contained on the cytoplasmic tails of integral membrane 5 proteins.
• the p24 family of type I membrane proteins represent the major membrane proteins of COPI vesicles (Harter, C and F.T. Wieland (1998) Proc. Nati. Acad. Sci. USA 95:11649-11654).
• SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46 encode, for example, organelle associated molecules.
• DNA-binding structural motifs which comprise either ⁇ helices or ⁇ sheets that bind to the major groove of DNA.
• Four well-characterized structural motifs are helix-turn-heUx, zinc finger, leucine zipper, and helix-loop-helix. Proteins containing these motifs may act alone as monomers, or they may form homo- or heterodimers that interact with DNA. 5 Many neoplastic disorders in humans can be attributed to inappropriate gene expression.
• Chromosomal franslocations may also produce chimeric loci which fuse the coding sequence of one gene with the regulatory regions of a second unrelated gene. Such an arrangement likely results in o inappropriate gene franscription, potentially contributing to malignancy.
• the immune system responds to infection or frauma by activating a cascade of events that coordinate the progressive selection, amplification, and mobilization of cellular defense mechanisms.
• a complex and balanced program of gene activation and repression is involved in this process.
• hyperactivity of the immune system as a result of improper or insufficient 5 regulation of gene expression may result in considerable tissue or organ damage. This damage is well documented in immunological responses associated with arthritis, allergens, heart attack, sfroke, and infections (Isselbacher, KJ. et al. (1996) Harrison's Principles of Internal Medicine, 13/e, McGraw Hill, Inc. and Teton Data Systems Software).
• RNA polymerase I makes large ribosomal RNA
• the nucleolus is a highly organized subcompartment in the nucleus that contains high o concenfrations of RNA and proteins and functions mainly in ribosomal RNA synthesis and assembly
• Ribosomal RNA is a structural RNA that is complexed with proteins to form ribonucleoprotein structures called ribosomes. Ribosomes provide the platform on which protein synthesis takes place.
• proteins are synthesized within the endoplasmic reticulum (ER), delivered from 5 the ER to the Golgi apparatus for post-franslational processing and sorting, and transported from the Golgi to specific infracellular and exfracellular destinations. Synthesis of integral membrane proteins, secreted proteins, and proteins destined for the lumen of a particular organelle occurs on the rough endoplasmic reticulum (ER).
• the rough ER is so named because of the rough appearance in electron micrographs imparted by the attached ribosomes on which protein synthesis proceeds.
• Synthesis of l o proteins destined for the ER actually begins in the cytosol with the synthesis of a specific signal peptide which directs the growing polypeptide and its attached ribosome to the ER membrane where the signal peptide is removed and protein synthesis is completed.
• Soluble proteins destined for the ER lumen, for secretion, or for transport to the lumen of other organelles pass completely into the ER lumen.
• Transmembrane proteins destined for the ER or for other cell membranes are translocated
• Translocated polypeptide chains destined for other organelles or for secretion also fold and assemble in the ER lumen with the aid of certain "resident" ER proteins. Protein folding in the ER is aided by two principal types of protein isomerases, protein disulfide isomerase (PDI), and peptidyl-
• prolyl isomerase catalyzes the oxidation of free sulfhydryl groups in cysteine residues to form inframolecular disulfide bonds in proteins.
• PPI an enzyme that catalyzes the isomerization of certain proline imide bonds in oligopeptides and proteins, is considered to govern one of the rate limiting steps in the folding of many proteins to their final functional conformation.
• the cyclophilins represent a major class of PPI that was originally identified as the major receptor for the 5 immunosuppressive drug cyclosporin A (Handschumacher, R.E. et al. (1984) Science 226:544-547).
• Molecular "chaperones" such as BiP (binding protein) in the ER recognize incorrectly folded proteins as well as proteins not yet folded into their final form and bind to them, both to prevent improper aggregation between them, and to promote proper folding.
• the Golgi apparatus is a complex structure that lies adjacent to the ER in eukaryotic cells and serves primarily as a sorting and dispatching station for products of the ER (Alberts, et al. supra, pp. 5 600-610). Additional postfranslational processing, principally additional glycosylation, also occurs in the Golgi. Indeed, the Golgi is a major site of carbohydrate synthesis, including most of the glycosaminoglycans of the extracellular matrix. N-linked oligosaccharides, added to proteins in the ER, are also further modified in the Golgi by the addition of more sugar residues to form complex N- linked oligosaccharides.
• the terminal compartment of the Golgi is the Trans-Golgi Network (TGN), where both membrane and lumenal proteins are sorted for their final destination.
• Other fransport vesicles bud off containing proteins destined for the plasma membrane, such as receptors, adhesion 5 molecules, and ion channels, and secretory proteins, such as hormones, neurofransmitters, and digestive enzymes.
• Lysosomes 5 Lysosomes are membranous vesicles containing various hydrolytic enzymes used for the controlled intracellular digestion of macromolecules.
• Lysosomes contain some 40 types of enzymes including proteases, nucleases, glycosidases, Upases, phospholipases, phosphatases, and sulfatases, all of which are acid hydrolases that function at a pH of about 5. Lysosomes are surrounded by a unique membrane containing fransport proteins that allow the final products of macromolecule degradation, 5 such as sugars, amino acids, and nucleotides, to be fransported to the cytosol where they may be either excreted or reutilized by the cell. A vp-ATPase, such as that described above, maintains the acidic environment necessary for hydrolytic activity (Alberts, supra, pp. 610-611). Endosomes
• Late endosomes 5 appear later in the endocytic process close to the Golgi apparatus and the nucleus, and appear to be associated with delivery of endocytosed material to lysosomes or to the TGN where they may be recycled.
• Specific proteins are associated with particular transport vesicles and their target compartments that may provide selectivity in targeting vesicles to their proper compartments.
• a cytosolic prenylated GTP-binding protein, Rab is one such protein.
• Rabs 4, 5, and 11 are associated o with the early endosome, whereas Rabs 7 and 9 associate with the late endosome.
• Glucose is initially converted to pyruvate in the cytoplasm.
• Fatty acids and pyruvate are fransported to the mitochondria for o complete oxidation to C0 2 coupled by enzymes to the transport of elecfrons from NADH and FADH 2 to oxygen and to the synthesis of ATP (oxidative phosphorylation) from ADP and P..
• Pyruvate is fransported into the mitochondria and converted to acetyl-CoA for oxidation via the citric acid cycle, involving pyruvate dehydrogenase components, dihydrolipoyl fransacetylase, and dihydrolipoyl dehydrogenase.
• Enzymes involved in the cifric acid cycle include: citrate synthetase, 5 aconitases, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase complex including ttanssuccinylases, succinyl CoA synthetase, succinate dehydrogenase, fumarases, and malate dehydrogenase.
• ⁇ oxidation results in shortening of the alkyl chain of fatty acids by blocks of two carbon atoms that are converted to acetyl CoA and exported to the cytosol for reuse in biosynthetic reactions.
• peroxisomes import their proteins from the cytosol using a specific signal sequence located near the C-terminus of the protein.
• the importance of this import process is o evident in the inherited human disease Zellweger syndrome, in which a defect in importing proteins into perixosomes leads to a perixosomal deficiency resulting in severe abnormalities in the brain, liver, and kidneys, and death soon after birth.
• One form of this disease has been shown to be due to a mutation in the gene encoding a perixosomal integral membrane protein called peroxisome assembly factor- 1. 5
• the discovery of new human molecules for diagnostics and therapeutics satisfies a need in the art by providing new compositions which are useful in the diagnosis, study, prevention, and treatment of diseases associated with human molecules.
• the present invention relates to nucleic acid sequences comprising human diagnostic and therapeutic polynucleotides (dithp) as presented in the Sequence Listing. Some of the dithp uniquely identify genes encoding human structural, functional, and regulatory molecules.
• the invention provides an isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of 5 SEQ ID NOJ-52; b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOJ-52; c) a polynucleotide sequence complementary to a); d) a polynucleotide sequence complementary to b); and e) an RNA equivalent of a) through d).
• the polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOJ-52.
• the polynucleotide comprises at least 60 contiguous nucleotides of a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NOJ-52; b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOJ-52; c) a polynucleotide sequence complementary to a); d) a polynucleotide sequence complementary to b); and e) an RNA equivalent of a) through 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, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof.
• the probe comprises at least 30 contiguous nucleotides.
• the probe comprises at least 60 contiguous nucleotides.
• the invention provides a cell transformed with the recombinant polynucleotide.
• the invention provides a transgenic organism comprising the recombinant polynucleotide.
• the invention provides a method 5 for producing a human diagnostic and therapeutic polypeptide, the method comprising a) culturing a cell under conditions suitable for expression of the human diagnostic and therapeutic polypeptide, wherein said cell is transformed with the recombinant polynucleotide, and b) recovering the human diagnostic and therapeutic polypeptide so expressed.
• the invention also provides a purified human diagnostic and therapeutic polypeptide (DITHP) o encoded by at least one polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NOJ-52. Additionally, the invention provides an isolated antibody which specifically binds to the human diagnostic and therapeutic polypeptide.
• DITHP human diagnostic and therapeutic polypeptide
• the invention further provides a method of identifying a test compound which specifically binds to the human diagnostic and therapeutic polypeptide, the method comprising the steps of a) providing a test compound; b) combining 5 the human diagnostic and therapeutic polypeptide with the test compound for a sufficient time and under suitable conditions for binding; and c) detecting binding of the human diagnostic and therapeutic polypeptide to the test compound, thereby identifying the test compound which specifically binds the human diagnostic and therapeutic polypeptide.
• the invention further provides a microarray wherein at least one element of the microarray is 0 an isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NOJ-52; b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOJ-52; c) a polynucleotide sequence complementary to a); d) a polynucleotide sequence 5 complementary to b); and e) an RNA equivalent of a) through d).
• the invention also provides a method for generating a transcript image of a sample which contains polynucleotides.
• the method comprises a) labeling the polynucleotides of the sample, b) contacting the elements of the microarray with the labeled polynucleotides of the sample under conditions suitable for the formation of a hybridization complex, and c) quantifying the expression of the polynucleotides in the sample.
• the invention 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 a) a polynucleotide sequence selected from the group consisting of SEQ ID NOJ-52; b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ 5 ID NOJ-52; c) a polynucleotide sequence complementary to a); d) a polynucleotide sequence complementary to b); and e) an RNA equivalent of a) through d).
• the method comprises 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 detecting a target polynucleotide in a sample for toxicity testing of a compound, said target polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NOJ-52; b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOJ-52; c) a polynucleotide sequence complementary to a); d) a polynucleotide sequence complementary to b); and e) an RNA equivalent of a) through d).
• Table 2 shows the sequence identification numbers (SEQ ID NO:s) and template identification numbers (template IDs) corresponding to the polynucleotides of the present invention, along with polynucleotide segments of each template sequence as defined by the indicated “start” and “stop” nucleotide positions.
• SEQ ID NO:s sequence identification numbers
• template IDs template identification numbers
• Table 3 shows the sequence identification numbers (SEQ ID NO:s) and template identification numbers (template IDs) corresponding to the polynucleotides of the present invention, along with polynucleotide segments of each template sequence as defined by the indicated “start” and “stop” nucleotide positions.
• the reading frames of the polynucleotide segments are shown, and the polypeptides encoded by the polynucleotide segments constitute either signal peptide (SP) or fransmembrane (TM) domains, as indicated.
• SP signal peptide
• TM fransmembrane
• Table 5 summarizes the bioinformatics tools which are useful for analysis of the polynucleotides of the present invention.
• the first column of Table 5 lists analytical tools, programs, o and algorithms, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are inco ⁇ orated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score, the greater the homology between two sequences). 5
• dithp refers to a nucleic acid sequence
• DITHP amino acid sequence encoded by dithp
• a “full-length” dithp refers to a nucleic acid sequence containing the entire coding region of a gene endogenously expressed in human tissue.
• Adjuvants are materials such as Freund's adjuvant, mineral gels (aluminum hydroxide), and 5 surface active substances (lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole Umpet hemocyanin, and dinifrophenol) which may be administered to increase a host's immunological response.
• Alleles refers to an alternative form of a nucleic acid sequence. Alleles result from a “mutation,” a change or an alternative reading of the genetic code. Any given gene may have none, one, or many allelic forms. Mutations which give rise to alleles include deletions, additions, or substitutions of nucleotides. Each of these changes may occur alone, or in combination with the others, one or more times in a given nucleic acid sequence.
• the present invention encompasses allelic dithp.
• Amplification refers to the production of additional copies of a sequence and is carried out using polymerase chain reaction (PCR) technologies well known in the art.
• PCR polymerase chain reaction
• Antibody refers to intact molecules as well as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding the epitopic determinant.
• Antibodies that bind DITHP polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen.
• the polypeptide or peptide 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
• RNA e.g., a mouse, a rat, or a rabbit
• Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobuUn, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
• Antisense sequence refers to a sequence capable of specifically hybridizing to a target sequence.
• the antisense sequence may include DNA, RNA, or any nucleic acid mimic or analog such as 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.
• PNA peptide nucleic acid
• Antisense sequence refers to a sequence capable of specifically hybridizing to a target sequence.
• the antisense sequence can be DNA, RNA, or any nucleic acid mimic or analog.
• Antisense technology refers to any technology which relies on the specific hybridization of an antisense sequence to a target sequence.
• a "bin” is a portion of computer memory space used by a computer program for storage of data, and bounded in such a manner that data stored in a bin may be retrieved by the program.
• Bioly active refers to an amino acid sequence having a structural, regulatory, or biochemical function of a naturally occurring amino acid sequence.
• “Clone joining” is a process for combining gene bins based upon the bins' containing sequence information from the same clone. The sequences may assemble into a primary gene transcript as well as one or more splice variants.
• a "consensus sequence” or “template sequence” is a nucleic acid sequence which has been assembled from overlapping sequences, using a computer program for fragment assembly such as the GEL VIEW fragment assembly system (Genetics Computer Group (GCG), Madison WI) or using a relational database management system (RDMS).
• GCG Genetics Computer Group
• RDMS relational database management system
• Constant amino acid substitutions are those substitutions that, when made, 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 substitutions.
• Lys Arg Gin, Glu Met Leu, He Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val T ⁇ Phe, Tyr Tyr His, Phe, T ⁇ Val He, Leu, Thr
• Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
• “Deletion” refers to a change in either a nucleic or amino acid sequence in which at least one nucleotide or amino acid residue, respectively, is absent.
• “Derivative” refers to the chemical modification of a nucleic acid sequence, such as by replacement of hydrogen by an alkyl, acyl, amino, hydroxyl, or other group.
• E-value refers to the statistical probability that a match between two sequences occurred by chance.
• a “fragment” is a unique portion of dithp or DITHP which is identical in sequence to but o 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 10 to 1000 contiguous amino acid residues or nucleotides.
• a fragment used as a probe, primer, antigen, therapeutic molecule, or for other pu ⁇ oses may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous amino acid residues or nucleotides in length. 5 Fragments may be preferentially selected from certain regions of a molecule.
• a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence.
• a fragment of dithp comprises a region of unique polynucleotide sequence that specifically identifies dithp, for example, as distinct from any other sequence in the same genome.
• a fragment of dithp is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish dithp from related polynucleotide sequences.
• the precise length of a fragment of dithp and the region of dithp to which the fragment corresponds are routinely determinable by one of ordinary 5 skill in the art based on the intended pu ⁇ ose for the fragment.
• a fragment of DITHP is encoded by a fragment of dithp.
• a fragment of DITHP comprises a region of unique amino acid sequence that specifically identifies DITHP.
• a fragment of DITHP is useful as an immunogenic peptide for the development of antibodies that specifically recognize DITHP.
• the precise length of a fragment of DITHP and the region of DITHP to which the o fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended pu ⁇ ose for the fragment.
• a “full length” nucleotide sequence is one containing at least a start site for translation to a protein sequence, followed by an open reading frame and a stop site, and encoding a "full length” polypeptide.
• 5 "Hit” refers to a sequence whose annotation will be used to describe a given template. Criteria for selecting the top hit are as follows: if the template has one or more exact nucleic acid matches, the top hit is the exact match with highest percent identity. If the template has no exact matches but has significant protein hits, the top hit is the protein hit with the lowest E-value. If the template has no significant protein hits, but does have significant non-exact nucleotide hits, the top hit is the nucleotide hit with the lowest E-value.
• Homology refers to sequence similarity either between a reference nucleic acid sequence and at least a fragment of a dithp or between a reference amino acid sequence and a fragment of a DITHP.
• Hybridization refers to the process by which a strand of nucleotides anneals with a complementary strand through base pairing. Specific hybridization is an indication that two nucleic acid sequences share a high degree of identity. Specific hybridization complexes form under defined annealing conditions, and remain hybridized after the "washing" step.
• the defined hybridization conditions include the annealing conditions and the washing step(s), the latter of which 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 probes that are not perfectly matched.
• Permissive conditions for annealing of nucleic acid sequences are routinely determinable and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency.
• stringency of hybridization is expressed with reference to the temperature under which the wash step is carried out.
• wash temperatures are selected to be about 5°C to 20°C lower than the thermal melting point (T Rule) 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 0J % SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, or 55°C may be used. SSC concentration may be varied from about 0.2 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, denatured salmon sperm DNA at about 100-200 ⁇ g/ml. Useful variations on these conditions will be readily apparent to those skilled 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 resultant proteins. Other parameters, such as temperature, salt concentration, and detergent concentration may be varied to achieve the desired stringency. Denaturants, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as RNA:DNA hybridizations. Appropriate hybridization conditions are routinely determinable by one of ordinary skill in the art.
• Immunogenic describes the potential for a natural, recombinant, or synthetic peptide, epitope, 5 polypeptide, or protein to induce antibody production in appropriate animals, cells, or cell Unes.
• “Insertion” or “addition” refers to a change in either a nucleic or amino acid sequence in which at least one nucleotide or residue, respectively, is added to the sequence.
• LabeleUng refers to the covalent or noncovalent joining of a polynucleotide, polypeptide, or antibody with a reporter molecule capable of producing a detectable or measurable signal.
• a reporter molecule capable of producing a detectable or measurable signal.
• 0 "Microarray” is any arrangement of nucleic acids, amino acids, antibodies, etc., on a substrate.
• the substrate may be a solid support such as beads, glass, paper, nitrocellulose, nylon, or an appropriate membrane.
• BLAST 2 Sequences can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf/bl2/.
• 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 o 2.0.9 (May-07-1999) set at default parameters.
• Such default parameters may be, for example:
• Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed. 5 "Transformants" include 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 cells which transiently express inserted DNA or RNA.
• the 5 corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
• Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other.
• SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
• variants of the polynucleotides of the present invention may be generated through recombinant methods.
• One possible method is a DNA shuffling technique such as 5 MOLECULARBREEDING (Maxygen Inc. , Santa Clara C A; 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.
• 5 MOLECULARBREEDING Maxygen Inc. , Santa Clara C A; 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
• sequences of the present invention are used to develop a transcript image for a particular cell or tissue.
• Chain termination reaction products may be elecfrophoresed on urea-poly acrylamide gels and detected either by autoradiography (for radioisotope-labeled nucleotides) or by fluorescence (for fluorophore-labeled nucleotides).
• Automated methods for mechanized reaction preparation, sequencing, and analysis using fluorescence detection methods have been developed.
• Machines used to prepare cDNAs for sequencing can include the MICROLAB 2200 liquid transfer system (Hamilton Company (Hamilton), Reno NV), Peltier thermal cycler (PTC200; MJ Research, Inc.
• Sequencing can be carried out using, for example, the ABI 373 or 377 (PE Biosystems) or MEGABACE 1000 (Molecular Dynamics, Inc. (Molecular Dynamics), Sunnyvale CA) DNA sequencing systems, or other automated and manual sequencing systems well known in the art.
• cDNA sequences are used as "component" sequences that are assembled into “template” or “consensus” sequences as follows. Sequence chromatograms are processed, verified, and quatity scores are obtained using PHRED. Raw sequences are edited using an editing pathway known as Block 1 (See, e.g., the LIFESEQ Assembled User Guide, Incyte Genomics, Palo Alto, CA). A series of BLAST comparisons is performed and low-information segments and repetitive elements (e.g., dinucleotide repeats, Alu repeats, etc.) are replaced by "n's", or masked, to prevent spurious matches. Mitochondrial and ribosomal RNA sequences are also removed.
• Block 1 See, e.g., the LIFESEQ Assembled User Guide, Incyte Genomics, Palo Alto, CA).
• a series of BLAST comparisons is performed and low-information segments and repetitive elements (e.g., dinucleo
• Template sequences may be extended to include additional contiguous sequences derived from the parent RNA transcript using a variety of methods known to those of skill in the art. Extension may thus be used to achieve the full length coding sequence of a gene.
• the cDNA sequences are analyzed using a variety of programs and algorithms which are well known in the art. (See, e.g., Ausubel, 1997, supra. Chapter 7.7; Meyers, R.A. (Ed.) (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853; and Table 5.) These analyses comprise both reading frame determinations, e.g., based on triplet codon periodicity for particular organisms (Fickett, J.W. (1982) Nucleic Acids Res. 10:5303-5318); analyses of potential start and stop codons; and homology searches.
• BLAST Basic Local Ahgnment Search Tool
• BLAST is especially useful in determining exact matches and comparing two sequence fragments of arbitrary but equal lengths, whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score set by the user (Karlin, S. et al. (1988) Proc. Nati. Acad. Sci. USA 85:841-845).
• GenBank e.g., GenBank
• SwissProt e.g., GenBank
• BLOCKS e.g., GenBank
• PFAM e.g., PFAM
• other databases may be searched for sequences containing regions of homology to a query dithp or DITHP of the present invention.
• search tool e.g., BLAST or HMM
• GenBank e.g., GenBank
• SwissProt e.g., BLOCKS
• PFAM e.g., PFAM
• Other approaches to the identification, assembly, storage, and display of nucleotide and polypeptide sequences are provided in "Relational Database for Storing Biomolecule Information," U.S.S.N. 08/947,845, filed October 9, 1997; "Project-Based Full-Length Biomolecular Sequence Database," U.S.S.N.
• SEQ ID NO:30 and SEQ ID NO:31 encode, for example, ribosomal molecules.
• SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43 encode, for example, transcription factor molecules.
• SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46 encode, for example, organelle associated molecules.
• SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50 encode, for example, biochemical pathway molecules.
• SEQ ID NO:51 and SEQ ID NO:52 encode, for example, molecules associated with growth and development.
• This type 5 of analysis is useful, for example, to assess the relative levels of dithp expression in fully or partially differentiated cells or tissues, to determine if changes in dithp expression levels are correlated with the development or progression of specific disease states, and to assess the response of a cell or tissue to a specific therapy, for example, in pharmacological or toxicological studies.
• Methods for the analysis of dithp expression are based on hybridization and amplification technologies and include membrane- 0 based procedures such as northern blot analysis, high-throughput procedures that utilize, for example, microarrays, and PCR-based procedures.
• the dithp, their fragments, or complementary sequences may be used to identify the presence 5 of and/or to determine the degree of similarity between two (or more) nucleic acid sequences.
• the dithp may be hybridized to naturally occurring or recombinant nucleic acid sequences under appropriately selected temperatures and salt concenfrations. Hybridization with a probe based on the nucleic acid sequence of at least one of the dithp allows for the detection of nucleic acid sequences, including genomic sequences, which are identical or related to the dithp of the Sequence Listing. Probes may be o selected from non-conserved or unique regions of at least one of the polynucleotides of SEQ ID NO: 1 -
• Polynucleotide sequences that are capable of hybridizing, in particular, to those shown in SEQ ID NOJ-52 and fragments thereof, can be identified using various conditions of stringency. (See, e.g., 5 Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol. 152:507-511.) Hybridization conditions are discussed in "Definitions.”
• a probe for use in Southern or northern hybridization may be derived from a fragment of a dithp sequence, or its complement, that is up to several hundred nucleotides in length and is either single-stranded or double-stranded. Such probes may be hybridized in solution to biological materials o such as plasmids, bacterial, yeast, or human artificial chromosomes, cleared or sectioned tissues, or to artificial substrates containing dithp. Microarrays are particularly suitable for identifying the presence of and detecting the level of expression for multiple genes of interest by examining gene expression correlated with, e.g., various stages of development, treatment with a drug or compound, or disease progression.
• An array analogous to a dot or slot blot may be used to arrange and link polynucleotides 5 to the surface of a subsfrate using one or more of the following: mechanical (vacuum), chemical, thermal, or UV bonding procedures.
• Such an array may contain any number of dithp and may be produced by hand or by using available devices, materials, and machines.
• dithp may be cloned into commercially available vectors for the production of RNA probes.
• Such probes may be transcribed in the presence of at least one labeled nucleotide (e.g., 32 P-ATP, Amersham Pharmacia Biotech).
• Alzheimer's disease has been linked to a gene on chromosome 21 ; other studies predict a different gene and location. Mapping of disease genes is a complex and reiterative process and generally proceeds from genetic linkage analysis to physical mapping.
• dithp sequences may be used to generate hybridization probes useful in chromosomal mapping of naturally occurring genomic sequences. Either coding or noncoding sequences of dithp may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a dithp 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 cDN A libraries.
• HACs human artificial chromosomes
• YACs yeast artificial chromosomes
• BACs bacterial artificial chromosomes
• PI constructions or single chromosome cDN A libraries.
• Fluorescent in situ hybridization may be correlated with other physical chromosome mapping techniques and genetic map data. (See, e.g., Meyers, supra, pp. 965-968.) Correlation between the location of dithp on a physical chromosomal map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder.
• the dithp sequences may also be used to detect polymo ⁇ hisms that are genetically linked to the inheritance of a particular condition, disease, or disorder.
• any sequences mapping to that area may represent associated or regulatory genes for further investigation.
• the nucleotide sequences of the subject invention may also be used to detect differences in chromosomal architecture due to translocation, inversion, etc., among normal, carrier, or affected individuals.
• a disease-associated gene is mapped to a chromosomal region, the gene must be cloned in order to identify mutations or other alterations (e.g., franslocations or inversions) that may be correlated with disease.
• This process requires a physical map of the chromosomal region containing the disease- gene of interest along with associated markers.
• a physical map is necessary for determining the nucleotide sequence of and order of marker genes on a particular chromosomal region. Physical mapping techniques are well known in the art and require the generation of overlapping sets of cloned DNA fragments from a particular organelle, chromosome, or genome. These clones are analyzed to reconstruct and catalog their order. Once the position of a marker is determined, the DNA from that region is obtained by consulting the catalog and selecting clones from that region. The gene of interest is located through positional cloning techniques using hybridization or similar methods.
• the dithp of the present invention may be used to design probes useful in diagnostic assays. Such assays, well known to those skilled in the art, may be used to detect or confirm conditions, disorders, or diseases associated with abnormal levels of dithp expression. Labeled probes developed from dithp sequences are added to a sample under hybridizing conditions of desired stringency. In some instances, dithp, or fragments or oligonucleotides derived from dithp, may be used as primers in amplification steps prior to hybridization. The amount of hybridization complex formed is quantified and compared with standards for that cell or tissue. If dithp expression varies significantly from the standard, the assay indicates the presence of the condition, disorder, or disease.
• Qualitative or quantitative diagnostic methods may include northern, dot blot, or other membrane or dip-stick based technologies or multiple-sample format technologies such as PCR, enzyme-linked immunosorbent assay (ELISA)-like, pin, or chip-based assays.
• PCR enzyme-linked immunosorbent assay
• the probes described above may also be used to monitor the progress of conditions, disorders, or diseases associated with abnormal levels of dithp expression, or to evaluate the efficacy of a particular therapeutic treatment.
• the candidate probe may be identified from the dithp that are specific to a given human tissue and have not been observed in GenBank or other genome databases. Such a probe may be used in animal studies, preclinical tests, clinical trials, or in monitoring the treatment of an individual patient.
• standard expression is established by methods well known in the art for use as a basis of comparison, samples from patients affected by the disorder or disease are combined with the probe to evaluate any deviation from the standard profile, and a therapeutic agent is administered and effects are monitored to generate a treatment profile. Efficacy is evaluated by determining whether the expression progresses toward or returns to the standard normal pattern. Treatment profiles may be generated over a period of several days or several months. Statistical methods well known to those skilled in the art may be use to determine the significance of such therapeutic agents.
• oligonucleotide primers derived from the dithp of the invention may be used to detect single nucleotide polymo ⁇ hisms (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 polymo ⁇ hism (SSCP) and fluorescent SSCP (fSSCP) methods.
• SSCP single-stranded conformation polymo ⁇ hism
• fSSCP fluorescent SSCP
• oligonucleotide primers derived from the polynucleotide sequences encoding DITHP 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 elecfrophoresis in non-denaturing gels.
• the oUgonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high-throughput equipment such as DNA sequencing machines.
• sequence database analysis methods termed in silico SNP (isSNP) are capable of identifying polymo ⁇ hisms by comparing the sequences of individual overlapping DNA fragments which assemble into a common consensus sequence.
• SNPs may be detected and characterized by mass spectromefry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
• DNA-based identification techniques are critical in forensic technology. DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, semen, etc., can be amplified using, e.g., PCR, to identify individuals. (See, e.g., Erlich, H. (1992) PCR Technology. Freeman and Co., New York, NY). Similarly, polynucleotides of the present invention can be used as polymo ⁇ hic markers.
• reagents capable of identifying the source of a particular tissue.
• Appropriate reagents can comprise, for example, DNA probes or primers prepared from the sequences of the present invention that are specific for particular tissues. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination.
• polynucleotides of the present invention can also be used as molecular weight markers on nucleic acid gels or Southern blots, as diagnostic probes for the presence of a specific mRNA in a 5 particular cell type, in the creation of subfracted cDNA libraries which aid in the discovery of novel polynucleotides, in selection and synthesis of oligomers for attachment to an array or other support, and as an antigen to eUcit an immune response.
• the dithp of the invention or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells.
• ES embryonic stem
• Such techniques are well known in the art and are useful for the generation of animal models of human disease. (See, e.g., U.S. Patent Number 5,175,383 and U.S. Patent Number 5,161,331.)
• mouse ES cells such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture.
• the ES 5 cells are fransformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphofransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292).
• the vector integrates into the corresponding region of the host genome by homologous recombination.
• homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999- 0 2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
• the dithp of the invention can also be used to create "knockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease.
• pigs humanized animals
• mice or rats transgenic animals
• a region of dithp is injected into animal ES cells, and the injected sequence integrates into the animal cell genome.
• Transformed cells are injected into blastulae, and the blastulae are implanted as described above.
• Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to 5 obtain information on treatment of a human disease.
• a mammal inbred to overexpress dithp may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).
• DITHP encoded by polynucleotides of the present invention may be used to screen for molecules that bind to or are bound by the encoded polypeptides.
• the binding of the polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the polypeptide or the bound molecule.
• Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
• the molecule is closely related to the natural ligand of the polypeptide, e.g., a ligand or fragment thereof, a natural subsfrate, or a structural or functional mimetic.
• the molecule can be closely related to the natural receptor to which the polypeptide binds, or to at least a fragment of the receptor, e.g., the active site. In either case, the molecule can be rationally designed using known techniques. 5
• the screening for these molecules involves producing appropriate cells which express the polypeptide, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli.
• the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a soUd support, chemical libraries, or natural product mixtures.
• the assay may also simply 5 comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring polypeptide/molecule activity or binding, and comparing the polypeptide/molecule activity or binding to a standard.
• an ELISA assay using, e.g., a monoclonal or polyclonal antibody can measure polypeptide level in a sample.
• the antibody can measure polypeptide level by either binding, directly or o indirectly, to the polypeptide or by competing with the polypeptide for a subsfrate.
• Transcript images which profile dithp expression may be generated using transcripts isolated from tissues, cell lines, biopsies, or other biological samples.
• the transcript image may thus reflect dithp 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 may be used to profile dithp expression in distinct tissue types. This process can be used to determine the activity of human diagnostic and therapeutic molecules in a particular 5 tissue type relative to this activity in a different tissue type.
• Transcript images may be used to generate a profile of dithp expression characteristic of diseased tissue.
• Transcript images of tissues before and after treatment may be used for diagnostic pu ⁇ oses, to monitor the progression of disease, and to monitor the efficacy of drug treatments for diseases which affect the activity of human diagnostic and therapeutic molecules.
• Transcript images which profile dithp expression may also be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals.
• Transcript images of cell lines can be used to assess the activity of human diagnostic and therapeutic molecules and/or to identify cell lines that lack or misregulate this activity. Such cell lines may then be treated with pharmaceutical agents, and a transcript image following treatment may indicate the efficacy of these agents in restoring desired 5 levels of this activity.
• a similar approach may be used to assess the toxicity of pharmaceutical agents as reflected by undesirable changes in the activity of human diagnostic and therapeutic molecules.
• Candidate pharmaceutical agents may be evaluated by comparing their associated transcript images with those of pharmaceutical agents of known effectiveness.
• the polynucleotides of the present invention are useful in antisense technology.
• Antisense technology or therapy relies on the modulation of expression of a target protein through the specific binding of an antisense sequence to a target sequence encoding the target protein or directing its expression.
• Agrawal, S., ed. 1996 Antisense Therapeutics, Humana Press Inc., Totawa NJ; Alama, A. et al. (1997) Pharmacol. Res. 36(3):171-178; Crooke, S.T. (1997) Adv. Pharmacol. 40:1-49; Sharma, H.W. and R. Narayanan (1995) Bioessays 17(12):1055-1063; and Lavrosky, Y.
• An antisense sequence is a polynucleotide sequence capable of specifically hybridizing to at least a portion of the target sequence. Antisense sequences bind to cellular mRNA and/or genomic DNA, affecting translation and/or franscription. Antisense 5 sequences can be DNA, RNA, or nucleic acid mimics and analogs.
• the polynucleotides of the present invention and fragments thereof can be used as antisense sequences to modify the expression of the polypeptide encoded by dithp.
• the antisense sequences can be produced ex vivo, such as by using any of the ABI nucleic acid synthesizer series (PE Biosystems) or other automated systems known in the art. Antisense sequences can also be produced biologically,
• Antisense sequences can be delivered infracellularly in the form of an expression plasmid which, upon franscription, produces a sequence complementary to at
• Antisense sequences can also be introduced infracellularly through the use of viral vectors, such as refrovirus and adeno-associated virus vectors.
• viral vectors such as refrovirus and adeno-associated virus vectors.
• nucleotide sequences encoding DITHP or fragments thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for ttanscriptional and franslational control of the inserted coding sequence in a suitable host.
• an appropriate expression vector i.e., a vector which contains the necessary elements for ttanscriptional and franslational control of the inserted coding sequence in a suitable host.
• 35 expression vectors containing sequences encoding DITHP and appropriate ttanscriptional and franslational control elements are 35 expression vectors containing sequences encoding DITHP and appropriate ttanscriptional and franslational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook, supra. Chapters 4, 8, 16, and 17; and Ausubel, supra. Chapters 9, 10, 13, and 16.)
• a variety of expression vector/host systems may be utilized to contain and express sequences encoding DITHP. These include, but are not Umited to, microorganisms such as bacteria fransformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast fransformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems fransformed with viral expression vectors (e.g., cauUflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal (mammaUan) cell systems.
• microorganisms such as bacteria fransformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast fransformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculo
• Expression vectors derived from refroviruses, adenoviruses, or he ⁇ es or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population.
• DITHP in cell Unes is preferred.
• sequences encoding DITHP can be transformed into cell Unes 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. Any number of selection systems may be used to recover fransformed cell lines.
• the dithp of the invention may be used for somatic or germhne gene therapy.
• Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-Xl disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an 5 inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C et al.
• SCID severe combined immunodeficiency
• ADA 5 inherited adenosine deaminase
• diseases or disorders caused by deficiencies in dithp 0 are treated by constructing mammalian expression vectors comprising dithp and introducing these vectors by mechanical means into dithp-deficient cells.
• Mechanical fransfer 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 fransfer, and (v) the use of DNA fransposons (Morgan, R.A. and Anderson, W.F. (1993) Annu. Rev. Biochem. 62:191-217; 5 Ivies, Z. (1997) Cell 91:501-510; Boulay, J-L. and Recipon, H. (1998) Curr. Opin. Biotechnol. 9:445- 450).
• Expression vectors that may be effective for the expression of dithp include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF, o PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
• the dithp of the invention may be 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 tetracycUne-regulated promoter (Gossen, M. and Bujard, H. (1992) Proc. Nati. Acad. Sci. U.S.A. 89:5547-5551 ; Gossen, M. et al., (1995) Science 268:1766-1769; Rossi, F.M.V. and Blau, 5 H.M.
• a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -act
• Sequences complementary to the dithp are used to detect, decrease, or inhibit expression of the naturally occurring nucleotide.
• oUgonucleotides comprising from about 15 to 30 base pairs is typical in the art. However, smaller or larger sequence fragments can also be used.
• Appropriate oligonucleotides are designed from the dithp using OLIGO 4.06 software (National Biosciences) or other appropriate programs and are synthesized using methods standard in the art or ordered from a commercial suppUer.
• a complementary oligonucleotide is designed from the most unique 5 ' sequence and used to prevent franscription factor binding to the promoter sequence.
• a complementary oligonucleotide is designed to prevent ribosomal binding and processing of the transcript.
• DITHP is synthesized as a fusion protein with, e.g., glutathione S- fransferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates.
• GST a 26-kilodalton enzyme from Schistosoma iaponicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech).
• Oxidoreductase activity of DITHP is measured by the increase in extinction coefficient of NAD(P)H coenzyme at 340 nmfor the measurement of oxidation activity, or the decrease in extinction coefficient of NAD(P)H coenzyme at 340 nmfor the measurement of reduction activity (Dalziel, K. (1963) J. Biol. Chem. 238:2850-2858).
• One of three substrates may be used: Asn- ⁇ Gal, biocytidine, or ubiquinone- 10.
• the respective subunits of the enzyme reaction for example, cytochtome c_ -b oxidoreductase and cytochrome c, are reconstituted.
• the reaction mixture contains a) 1-2 mg/ml DITHP; and b) 15 mM subsfrate, 2.4 mM NAD(P) + in 0.1 M phosphate buffer, pH 7.1 (oxidation reaction), or 2.0 mM NAD(P)H, in 0.1 M Na 2 HP0 4 buffer, pH 1.4 ( reduction reaction); in a total volume of 0.1 ml.
• Changes in absorbance at 340 nm (A 340 ) are measured at 23.5 ° C using a recording specfrophotometer (Shimadzu Scientific Insfruments, Inc., Pleasanton CA).
• Transferase activity of DITHP is measured through assays such as a methyl transferase assay in which the transfer of radiolabeled methyl groups between a donor substrate and an acceptor substrate is measured (Bokar, J.A. et al. (1994) J. Biol. Chem. 269: 17697-17704).
• Reaction mixtures (50 ⁇ l final volume) contain 15 mM HEPES, pH 7.9, 1.5 mM MgCl 2 , 10 mM dithiothreitol, 3% polyvinylalcohol, 1.5 ⁇ Ci [met ⁇ y/- 3 H]AdoMet (0.375 ⁇ M AdoMet) (DuPont-NEN), 0.6 ⁇ g DITHP, and acceptor substrate (0.4 ⁇ g [ 35 S]RNA or 6-mercaptopurine (6-MP) to 1 mM final concentration). Reaction mixtures are incubated at 30°C for 30 minutes, then 65 °C for 5 minutes. The products are separated by chromatography or elecfrophoresis and the level of methyl fransferase activity is determined by quantification of methyl- 3 recovery.
• Growth factor activity of DITHP is measured by the stimulation of DNA synthesis in Swiss mouse 3T3 cells (McKay, I. and I. Leigh, eds. (1993) Growth Factors: A Practical Approach, Oxford 5 University Press, New York NY). Initiation of DNA synthesis indicates the cells' entry into the mitotic cycle and their commitment to undergo later division. 3T3 cells are competent to respond to most growth factors, not only those that are mitogenic, but also those that are involved in embryonic induction. This competence is possible because the in vivo specificity demonstrated by some growth factors is not necessarily inherent but is determined by the responding tissue.
• An alternative assay for DITHP cytokine activity utilizes a Boyden micro chamber o (Neuroprobe, Cabin John MD) to measure leukocyte chemotaxis (Vicari, supra). In this assay, about
• DITHP activity by immunoblotting Cells are denatured in SDS in the presence of ⁇ -mercaptoethanol, nucleic acids removed by ethanol precipitation, and proteins purified by acetone precipitation. Pellets are resuspended in 20 mM tris buffer at pH 7.5 and incubated with Protein G-Sepharose pre-coated with an antibody specific for DITHP. After washing, the Sepharose beads are boiled in elecfrophoresis i o sample buffer, and the eluted proteins subjected to SDS-PAGE.
• DITHP kinase activity is measured by phosphorylation of a protein subsfrate using ⁇ -labeled
• DITHP activity is measured by the increase in cell proliferation resulting from transformation of a mammalian cell line such as COS7, HeLa or CHO with an eukaryotic expression vector encoding DITHP.
• Eukaryotic expression vectors are commercially available, and the techniques to introduce them into cells are well known to those skilled in the art.
• the cells are 5 incubated for 48-72 hours after transformation under conditions appropriate for the cell line to allow expression of DITHP. Phase microscopy is then used to compare the mitotic index of transformed versus control cells. An increase in the mitotic index indicates DITHP activity.
• an assay for DITHP signaling activity is based upon the ability of GPCR family proteins to modulate G protein-activated second messenger signal transduction 0 pathways (e.g., cAMP; Gaudin, P. et al. (1998) J. Biol. Chem. 273:4990-4996).
• a plasmid encoding full length DITHP is fransfected into a mammalian cell line (e.g., Chinese hamster ovary (CHO) or human embryonic kidney (HEK-293) cell lines) using methods well-known in the art. Transfected cells are grown in 12-well trays in culture medium for 48 hours, then the culture medium is discarded, and the attached cells are gently washed with PBS.
• a mammalian cell line e.g., Chinese hamster ovary (CHO) or human embryonic kidney (HEK-293) cell lines

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