EP1278846A2 - Proteines associees au cytosquelette - Google Patents

Proteines associees au cytosquelette

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Publication number
EP1278846A2
EP1278846A2 EP01932973A EP01932973A EP1278846A2 EP 1278846 A2 EP1278846 A2 EP 1278846A2 EP 01932973 A EP01932973 A EP 01932973A EP 01932973 A EP01932973 A EP 01932973A EP 1278846 A2 EP1278846 A2 EP 1278846A2
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European Patent Office
Prior art keywords
polynucleotide
seq
polypeptide
amino acid
sequence
Prior art date
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EP01932973A
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German (de)
English (en)
Inventor
Henry Yue
Y. Tom Tang
Janice Au-Young
Dyung Aina M. Lu
Mariah R. Baughn
Jennifer L. Hillman
Yalda Azimzai
Preeti Lal
Monique G. Yao
Olga Bandman
Neil Burford
Sajeev Batra
Liam Kearney
Jennifer L. Policky
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Incyte Corp
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Incyte Genomics Inc
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Publication of EP1278846A2 publication Critical patent/EP1278846A2/fr
Withdrawn legal-status Critical Current

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Definitions

  • This invention relates to nucleic acid and amino acid sequences of cytoskeleton-associated proteins and to the use of these sequences in the diagnosis , treatment, and prevention of cell proliferative, autoimmune/iriflammatory, vesicle trafficking, neurological, cell motility, reproductive, and muscle disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of cytoskeleton-associated proteins.
  • the cytoskeleton a cytoplasmic system of protein fibers, mediates cell shape, structure, and movement.
  • the cytoskeleton supports the cell membrane and forms tracks along which organelles and other elements move in the cytosol.
  • the cytoskeleton is a dynamic structure that allows cells to adopt various shapes and to carry out directed movements. Additonally. molecules can be sequestered to a specific cellular location through interaction with cytoskeleton associated proteins.
  • Major cytoskeletal fibers are the microfflaments, the microtubules, and the intermediate filaments.
  • Motor proteins including myosin, dynein, and kinesin, drive movement of, or along, the fibers.
  • Accessory or associated proteins modify the structure or activity of the fibers while cytoskeletal membrane anchors connect the fibers to the cell membrane.
  • Other proteins associated with the cytoskeleton have roles in processes such as secretion and intracellular signaling. (The cytoskeleton is reviewed in Lodish, H. et al. (1995) Molecular Cell Biology Scientific American Books, New York NY.) Microtubules and Associated Proteins Tubulins
  • Microtubules cytoskeletal fibers with a diameter of 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 bi-directional 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, in the form of the spindle, are also critical to chromosomal movement during cell division. Both stable and short-lived populations of microtubules exist in the cell.
  • Microtubules are a polymer of GTP-binding tubulin protein subunits. Each subunit is a heterodimer of ⁇ - and ⁇ - tubulin, multiple iso orms of which exist. Alpha-tubulin undergoes a number of post-translational modifications, including acetylation, polyglutamylation, truncation of two amino acids (forming ⁇ 2 tubulin), and tyrosination. In some cases, these modifications can affect microtubule stability.
  • the hydrolysis of GTP is linked to the addition of tubulin subunits at the end of a microtubule. The subunits interact head to tail to form protofilaments ; the protofilaments interact side to side to form a microtubule.
  • a microtubule is polarized, one end ringed with ⁇ -tubulin and the other with ⁇ -tubulin, and the two ends differ in their rates of assembly.
  • Each microtubule is generally composed of 13 protofilaments although 11 or 15 protofilament-microtubules are sometimes found.
  • Cilia and flagella contain doublet microtubules.
  • Microtubules grow from specialized structures known as centrosomes or microtubule-organizing centers (MTOCs). MTOCs may contain one or two centrioles, which are pinwheel arrays of triplet microtubules.
  • the basal body, the organizing center located at the base of a cilium or flagellum, contains one centriole.
  • ⁇ - tubulin present in the MTOG is important for nucleating the polymerization of ⁇ - and ⁇ - tubulin heterodimers but does not polymerize into microtubules.
  • the protein pericentrin is found in the MTOC and has a role in microtubule assembly.
  • Microtubule-associated proteins have roles in the assembly and stabilization of microtubules.
  • assembly MAPs can be identified in neurons as well as non-neuronal cells. Assembly MAPs are responsible for cross-linking microtubules in the cytosol. These MAPs are organized into two domains: a basic microtubule-binding domain and an acidic projection domain. The projection domain is the binding site for membranes, intermediate filaments, or other microtubules. Based on sequence analysis, assembly MAPs can be further grouped into two types: Type I and Type II.
  • Type I MAPs which include MAPI A and MAPIB, are large, filamentous molecules that co- purify with microtubules and are abundantly expressed in brain and testis. They contain several repeats of a positively-charged amino acid sequence motif that binds and neutralizes negatively charged tubulin, leading to stabilization of microtubules.
  • MAPI A and MAPIB are each derived from a single precursor polypeptide that is subsequently proteolytically processed to generate one heavy chain and one light chain. Another light chain, LC3, is a 16.4 kDa molecule that binds MAPI A, MAPIB, and microtubules.
  • LC3 is synthesized from a source other than the MAP1A or MAPIB transcripts, and the expression of LC3 may be important in regulating the microtubule binding activity of MAPI A and MAPIB during cell proliferation (Mann, S. S. et al. (1994) J. Biol. Chem. 269:11492- 11497).
  • Type II MAPs which include MAP2a, MAP2b, MAP2c, MAP4, and Tau, are characterized by three to four copies of an 18-residue sequence in the microtubule-binding domain.
  • MAP2a, MAP2b, and MAP2c are found only in dendrites, MAP4 is found in non-neuronal cells, and Tau is found in axons and dendrites of nerve cells.
  • Alternative splicing of the Tau mRNA leads to the existence of multiple forms of Tau protein.
  • Tau phosphorylation is altered in neurodegenerative disorders such as Alzheimer's disease, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia and Parkinsonism linked to chromosome 17.
  • the altered Tau phosphorylation leads to a collapse of the microtubule network and the formation of intraneuronal Tau aggregates (Spillantini, M.G. and Goedert, M.
  • Microtubule stability may also be regulated by severing the microtubule along its length.
  • the protein katanin a member of the AAA adenosine triphosphatase (ATPase) superfamily, uses ATP hydrolysis energy to sever and disassemble stable microtubules.
  • Katanin may play a role in releasing microtubules from centrosomes, regulating assembly of the mitotic spindle, and accelerating microtubule turnover during cell cycle transitions (Hartman, J.J. and Vale, R.D. (1999) Science 286:782-785).
  • Microtubular aggregates are associated with several disorders.
  • An extraskeletal myxoid chondrosarcoma tumor from human contained parallel arrays of microtubules within the rough endoplasmic reticulum (Suzuki, T. et al. (1988) J. Pathol. 156:51-57).
  • Microtubular aggregates were also found in hepatocytes from chimpanzees infected with hepatitis C virus. Monoclonal antibodies prepared to these aggregates detect a protein called p44 (or microtubular aggregates protein) (Maeda, T. et al. (1989) J. Gen. Virol. 70:1401-1407).
  • a human homolog of p44 is inducible by interferon- ⁇ and interferon- ⁇ , but not by interferon- ⁇ .
  • p44 protein may be a mediator in the antiviral action of interferon (Kita ura, A. et al. (1994) Eur. J. Biochem. 224:877-883).
  • Dvnein-related Motor Proteins Dyneins are (-) end-directed motor proteins which act on microtubules. Two classes of dyneins exist, cytosolic and axonemal.
  • Cytosolic dyneins are responsible for translocation of materials along cytoplasmic microtubules; for example, transport from the nerve terminal to the cell body and transport of endocytic vesicles to lysosomes. Cytoplasmic dyneins are also reported to play a role in mitosis. Axonemal dyneins are responsible for the beating of flagella and cilia. Dynein on one microtubule doublet walks along the adjacent microtubule doublet. This shding force produces bending forces that cause the flagellum or cilium to beat. Dyneins have a native mass between 1000 and 2000 kDa and contain either two or three force-producing heads driven by the hydrolysis of ATP. The heads are linked via stalks to a basal domain which is composed of a highly variable number of accessory intermediate and light chains. Kinesin-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 transport in neurons.
  • Kinesin is also important in all cell types for the transport of vesicles from the Golgi complex to the endoplasmic reticulum. This role is critical for maintaining the identity and functionality of these secretory organelles.
  • Kinesins define a ubiquitous, conserved family of over 50 proteins that can be classified into at least 8 subfamilies based on primary amino acid sequence, domain structure, velocity of movement, and cellular function. (Reviewed in Moore, J.D. and S.A. Endow (1996) Bioessays 18:207-219; and Hoyt, A.M. (1994) Curr. Opin. Cell Biol. 6:63-68.)
  • the prototypical kinesin molecule is a heterotetramer comprised of two heavy polypeptide chains (KHCs) and two light polypeptide chains (KLCs).
  • KHC subunits are typically referred to as "kinesin.” KHC is about 1000 amino acids in length, and KLC is about 550 amino acids in length.
  • Two KHCs dimerize to form a rod-shaped molecule with three distinct regions of secondary structure.
  • a globular motor domain that functions in ATP hydrolysis and microtubule binding.
  • Kinesin motor domains are highly conserved and share over 70% identity.
  • an ⁇ -helical coiled-coil region which mediates dimerization.
  • a fan-shaped tail that associates with molecular cargo. The tail is formed by the interaction of the KHC C-termini with the two KLCs.
  • KRPs kinesin-related proteins
  • centromere protein E localizes to the kinetochore of human mitotic chromosomes and may play a role in their segregation to opposite spindle poles.
  • Microfilaments are vital to cell locomotion, cell shape, cell adhesion, cell division, and muscle contraction. Assembly and disassembly of the microfilaments allow cells to change their morphology. Microfilaments are the polymerized form of actin, the most abundant intracellular protein in the eukaryotic cell. Human cells contain six isoforms of actin. The three ⁇ -actins are found in different kinds of muscle, nonmuscle ⁇ -actin and nonmuscle ⁇ - actin are found in nonmuscle cells, and another ⁇ -actin is found in intestinal smooth muscle cells.
  • 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 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.
  • Other actin-related filaments are not part of the actin cytoskeleton, but rather associate with microtubules and dyenin. Actin- Associated Proteins
  • Actin-associated proteins have roles in cross-Unking, severing, and stabilization of actin filaments and in sequestering actin monomers.
  • actin-associated proteins have multiple functions. Bundles and networks of actin filaments are held together by actin cross-linking proteins. These proteins have two actin-binding sites, one for each filament. Short cross-linking proteins promote bundle formation while longer, more flexible cross-linking proteins promote network formation. Calmodulin-like calcium-binding domains in actin cross-linking proteins allow calcium regulation of cross-hnking.
  • Group I cross-linking proteins have unique actin-binding domains and include the 30 Kd protein, EF-la, fascin, and scruin.
  • Group II cross-hnking proteins have a 7,000-MW actin-binding domain and include villin and dematin.
  • Group III cross-linking proteins have pairs of a 26,000-MW actin-binding domain and include alpha-actinin, fimbrin, spectrin, dystrophin, ABP 120, andfilamin.
  • 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 include CapZ, tropomodulin, and tensin.
  • Tensin which is found in focal adhesions, also crosslinks actin filaments. Integrin activation by the extracellular matrix leads to the phosphorylation of tensin on tyrosine, serine, and threonine residues; this phosphorylation also occurs in cells transformed with oncogenes. Tensin has an SH2 domain and may bind to other tyrosine-phosphorylated proteins. (Lo, S.H. et al. (1997) J. Cell Biol. 136:1349-1361.) The N-terminus of tensin contains a region homologous to the catalytic domain of a putative tyrosine phosphatase (PTP) from Saccharomvces cerevisiae.
  • PTP putative tyrosine phosphatase
  • This PTP domain in tensin may mediate binding interactions with phosphorylated polypeptides (Haynie, D.T. andPonting, CP. (1996) Protein Sci. 5 :2643-2646). Mice which lack the tensin gene have kidney abnormalities, indicating that the loss of tensin leads to weakening of focal adhesions in the kidney (Lo, supra).
  • Profilin may also stimulate F-actin formation by effectively lowering the critical concentration required for actin monomer addition (Gertler, F.B. et al. (1996) Cell 87:227-239).
  • the actin-associated proteins tropomyosin, troponin, and caldesmon regulate muscle contraction in response to calcium.
  • the tropomyosin proteins, found in muscle and nonmuscle cells, are ⁇ -helical and form coiled-coil dimers.
  • Striated muscle tropomyosin mediates the interactions between the troponin complex and actin, regulating muscle contraction (PROSITE PDOC00290 Tropomyosins signature).
  • the troponin complex is composed of troponin-T, troponin-I, and troponin-C.
  • Troponin-T binds tropomyosin, linking troponin-I and troponin-C to tropomyosin.
  • the actin-associated proteins tropomyosin, troponin, and caldesmon regulate muscle contraction in response to calcium.
  • the tropomyosin proteins found in muscle and nonmuscle cells, are ⁇ -helical and form coiled-coil dimers.
  • Striated muscle tropomyosin mediates the interactions between the troponin complex and actin, regulating muscle contraction.
  • the troponin complex is composed of troponin-T, troponin-I, and troponin- C.
  • Troponin-T binds tropomyosin, linking troponin-I and troponin-C to tropomyosin.
  • calponin homology domains found in actin cross- linking proteins including alpha- actinin, spectrin, and fimbrin.
  • CH calponin homology
  • LIM domains protein-protein interaction motifs known as LIM domains.
  • zyxin is a protein that plays a role in the spatial control of actin assembly and contains three tandem LIM domains.
  • Zyxin also interacts with alpha-actinin through its prolinerichN-terminus (Beckerle, M. C.
  • Alpha-actinin and several MAPs are present in Hirano bodies, which are observed more frequently in the elderly and in patients with neurodegenerative diseases such as Alzheimer's disease (Maciver, S.K. and Harrington, C.R. (1995) Neuroreport. 6:1985-1988).
  • Actinin-4 a novel actin- bundling protein, appears to be associated with the cell motility of metastatic cancer cells.
  • Other disease associations include premature chromosome condensation which is frequently observed in dividing cells from tumor tissue (Murnane, J.P. (1995) Cancer Metastasis Rev. 14:17-29) and the significant roles of axonemal and assembly MAPs in viral pathogenesis (Sodeik, B. et al. (1997) J. Cell Biol. 136:1007-1021).
  • Intermediate filaments are cytoskeletal fibers with a diameter of 10 nm, intermediate between that of microfilaments and microtubules. They 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.
  • IF proteins include acidic keratins, basic keratins, desmin, glial fibrillary acidic protein, vimentin, peripherin, neurofilaments, nestin, andlamins.
  • IFs have a central ⁇ -helical rod region interrupted by short nonhelical linker segments.
  • the rod region is bracketed, in most cases, by non-helical 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.
  • IF-associated proteins mediate the interactions of IFs with one another and with other cell structures.
  • IFAPs cross-link IFs into a bundle, into a network, or to the plasma membrane, and may cross-link IFs to the microfilament and microtubule cytoskeleton. Microtubules and IFs in particular are closely associated.
  • IFAPs include BPAG1, plakoglobin, desmoplakin I, desmoplakin II, plectin, ankyrin, filaggrin, and lamin B receptor.
  • ankyrin consists of a repeated 33 -amino acid motif, the ankyrin repeat, which is involved in specific protein-protein interactions. Variable regions within the motif are responsible for specific protein binding, such that different ankyrin repeats are involved in binding to tubulin, anion exchange protein, voltage-gated sodium channel, Na7K + -ATPase, and neurofascin.
  • the ankyrin motif is also found in transcription factors, such as NF-K-B, and in the yeast cell cycle proteins CDC10, SW14, and SW16. Proteins involved in tissue differentiation, such as Drosophila Notch and C, elegans LIN-12 and GLP-1, also contain ankyrin-like repeats.
  • Lux et al. (1990; Nature 344:36-42) suggest that ankyrin-hke repeats function as 'built-in' ankyrins and form binding sites for integral membrane proteins, tubulin, and other proteins.
  • Cytoskeletal fibers are attached to the plasma membrane by specific proteins. These attachments are important for maintaining cell shape and for muscle contraction.
  • the spectrin-actin cytoskeleton is attached to cell membrane by three proteins, band 4.1 , 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.
  • muscle cells the protein dystrophin Unks actin filaments to the plasma membrane; mutations in the dystrophin gene lead to Duchenne muscular dystrophy.
  • adherens junctions and adhesion plaques the peripheral membrane proteins ⁇ -actinin and vincuUn 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 epifheUal cells of organs and skin. These membrane junctions allow shear forces to be distributed across the entire epifheUal cell layer, thus providing strength and rigidity to the epitheUum.
  • IFs in epifheUal cells are attached to the desmosome by plakoglobin and desmoplakins. The proteins that Unk TJFs to hemidesmosomes are not known.
  • Desmin IFs surround the sarcomere in muscle and are Unked to the plasma membrane by paranemin, synemin, and ankyrin. Proteins of the Erythrocyte Membrane Skeleton
  • Oxygen diffuses from surrounding water or from the atmosphere through either gill epitheUum or pulmonary epifheUal type I cells. Oxygen then diffuses through the blood capillary endothelium directly to the blood circulatory system and through the erythrocyte membrane and is stored as soluble oxyhemoglobin in the cytoplasm. Oxygen is released from hemoglobin at sites throughout the organism and diffuses out from the erythrocyte to other target cells. The structure of the erythrocyte membrane as well as that of other non-erythrocyte cells must be maintained to enable efficient diffusion of oxygen to intracellular compartments.
  • the erythrocyte membrane is comprised of i) a cholesterol-rich phosphoUpid bilayer in which many trans-bilayer proteins are embedded, U) external glycosylphosphatidyUnositol-anchored proteins (GPI-proteins), and ui) the erythrocyte or membrane skeleton that laminates the inner surface of the bilayer.
  • the trans-bilayer proteins include anion exchangers, glycophorins, glucose transporters, and a variety of cation transporters and pumps.
  • the erythrocyte GPI-proteins include acetylchoUnesterase and decay-accelerating factor (CD 55).
  • the skeletal proteins are organized on the cortical, or cytoplasmic, face of the plasma membrane.
  • proteins include protein 4.1 , protein p55 , ⁇ - and ⁇ - spectrin, actin, and actin-binding proteins such as dematin, tropomyosin, and tropomoduUn.
  • ⁇ - and ⁇ - spectrin combine to form a heterotetramer in vivo.
  • the spectrin heterotetramer organizes into a cortical bidimensional network with a hexagonal mesh.
  • the network is Unked to trans-bilayer proteins through a protein complex comprising ⁇ -spectrin, ankyrin, anion exchanger, and protein 4.2 and through the "triangular" interaction between protein 4.1 , glycophorin C, and protein p55.
  • erythrocyte membrane proteins have been found in a variety of tissues. Variants may be transcribed from multigene famiUes, e.g., anion exchanger, ankyrin, or spectrin, or from single gene famiUes, e.g., protein 4.1 or protein 4.2. mRNA transcripts undergo tissue-specific alternative spUcing. Many congenital hemolytic anemias result from mutations in the above-mentioned genes encoding erythrocyte membrane proteins.
  • multigene famiUes e.g., anion exchanger, ankyrin, or spectrin
  • single gene famiUes e.g., protein 4.1 or protein 4.2.
  • mRNA transcripts undergo tissue-specific alternative spUcing.
  • Many congenital hemolytic anemias result from mutations in the above-mentioned genes encoding erythrocyte membrane proteins.
  • hereditary elUptocytosis stems from an array of mutations in the spectrin genes at or near the head-to-head self-association region of the spectrin tetramer , or from mutations in the protein 4.1 gene which reduce levels of protein 4.1.
  • hereditary spherocytosis is associated with mutations in the ankyrin gene, the anion exchanger gene, the protein 4.2 gene, or the ⁇ - and ⁇ -spectrin genes.
  • Protein 4.1 is an 80 kDa erythrocyte membrane protein with four functional domains. These domains include: i) a 30 kDa basic N-terminal domain, homologous to the ERM
  • Protein 4.1 is a member of a structurally and functionally related protein 4.1 family.
  • the protein 4.1 family is part of an evolutionarily related protein superfamily that includes many tyrosine phosphatases. (Baklouti, F. et al. (1997) Genomics 39:289-302.)
  • protein 4.1 epitopes have been observed throughout the cytoplasmic compartment and the nucleoskeleton in nucleated cells.
  • protein 4.1 is present in the nucleoskeleton during interphase, in the mitotic spindle during mitosis, in perichromatin during telophase, and in the midbody during cytokinesis. (Krauss, S.W. et al. (1997) J. Cell Biol. 137:275-289.)
  • the cortical actin cytoskeleton participates in various membrane-based processes which necessitate a large amount of functional plasticity in the molecular components involved.
  • a family of proteins homologous to band 4.1 is involved in the reorganization of the actin cytoskeleton in response to various stimuli and probably plays a role in transmembrane signaUng.
  • This family includes tyrosine phosphatases, substrates of tyrosine kinases and a candidate for a tumor-suppressor gene. (Arpin M, et al. (1994) Curr. Opin. Cell Biol. 6:136-141.)
  • Neurofibromatosis type 2 is an autosomal dominant disease of the nervous system.
  • Schwann cells isolated from patients with neurofibromatosis type 2 have characteristic morphology and growth parameters which differ from control Schwann cells.
  • a gene associated with neurofibromatosis type 2 has been identified and is termed NF2.
  • the NF2 gene product known as schwannomin or erUn, is a member of the protein 4.1 superf amily, and mutations in the NF2 gene have been shown to be associated with the disease. (Rosenbaum, C. et al. (1998) Neurobiol. Dis.
  • a form of psoriasis may be due to altered expression or distribution in epidermal epitheUum of analogs of erythrocyte protein 4.1.
  • Erythrocytes carrying mutations in spectrin and protein 4.1 showed differing sensitivities to invasion by Plasmodium falciparum.
  • antibodies raised against erythrocyte protein 4.1 stained the majority of neurofibrillary tangles in the prefrontal cortex and hippocampus of brain tissue from patients with Alzheimer's disease.
  • a 68 kDa protein was identified as the most Ukely brain analog of erythrocyte protein 4.1.
  • cytoskeleton-associated proteins and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of cell proliferative, autoimmune/inflammatory, vesicle trafficking, neurological, cell motiUty, reproductive, and muscle disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of cytoskeleton-associated proteins.
  • the invention features purified polypeptides, cytoskeleton-associated proteins, referred to collectively as “CYSKP” and individually as “CYSKP-1,” “CYSKP-2,””CYSKP-3,” “CYSKP-4,” “CYSKP-5,” “CYSKP-6,” “CYSKP-7,” “CYSKP-8,” “CYSKP-9,” “CYSKP-10,” “CYSKP-11,” “CYSKP-12,” “CYSKP-13,” “CYSKP-14,” “CYSKP-15,” “CYSKP-16,” “CYSKP-17,” “CYSKP- 18,” “CYSKP-19,” “CYSKP-20,” “CYSKP-21,” “CYSKP-22,” “CYSKP-23,” “CYSKP-24,” “CYSKP-25,” “CYSKP-26,” “CYSKP-27,” “CYSKP-28,” “CYSKP-29,” “CYSKP-30,” “CYSKP- 31,” “CYSKP-32,” “CYSKP-33,” and “CYSKP-34
  • the invention further provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34, b) a naturally occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-34, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34.
  • the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO: 1-34.
  • the polynucleotide is selected from the group consisting of SEQ ID NO:35-68.
  • the invention provides a recombinant polynucleotide comprising a promoter sequence operably Unked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-34, b) a naturally occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-34, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34.
  • the invention provides a cell transformed with the recombinant polynucleotide.
  • the invention provides a transgenic organism comprising the recombinant polynucleotide.
  • the invention also provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34, b) a naturally occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34.
  • the method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably Unked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
  • the invention provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34, b) a naturally occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-34.
  • the invention further provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:35-68, b) a naturally occurring polynucleotide comprising a polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:35-68, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • the polynucleotide comprises at least 60 contiguous nucleotides.
  • the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:35-68, b) a naturally occurring polynucleotide comprising a polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:35-68, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • the method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof.
  • the probe comprises at least 60 contiguous nucleotides.
  • the invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 35 -68, b) a naturally occurring polynucleotide comprising a polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:35-68, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • the method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said ampUfied target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
  • the invention further provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 -34, b) a naturally occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34, and a pharmaceutically acceptable excipient
  • the composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34.
  • the invention additionally provides a method of treating a disease or condition associated with decreased expression of functional CYSKP, comprising administering to a patient in need of such treatment the composition.
  • the invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34, b) a naturally occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1 -34, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-34, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-34.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample.
  • the invention provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient.
  • the invention provides a method of treating a disease or condition associated with decreased expression of functional CYSKP, comprising administering to a patient in need of such treatment the composition.
  • the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34, b) a naturally occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1 -34, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.
  • the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient
  • the invention provides a method of treating a disease or condition associated with overexpression of functional CYSKP, comprising administering to a patient in need of such treatment the composition.
  • the invention further provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-34, b) a naturally occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-34, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34.
  • the method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specificaUy binds to the polypeptide.
  • the invention further provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34, b) a naturally occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1 -34, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-34, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-34.
  • the method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
  • the invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO:35-68, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide.
  • the invention further provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:35-68, ii) a naturally occurring polynucleotide comprising a polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:35-68, in) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of u), and v) an RNA equivalent of i)-iv).
  • Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:35-68, ii) a naturally occurring polynucleotide comprising a polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:35-68, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of U), and v) an RNA equivalent of i)-iv).
  • a target polynucleotide selected from the group consisting of i) a polynucleotide comprising
  • the target polynucleotide comprises a fragment of a polynucleotide sequence selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
  • Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the present invention.
  • Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog for polypeptides of the invention. The probabiUty score for the match between each polypeptide and its GenBank homolog is also shown.
  • Table 3 shows structural features of polypeptide sequences of the invention, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
  • Table 4 Usts the cDNA and genomic DNA fragments which were used to assemble polynucleotide sequences of the invention, along with selected fragments of the polynucleotide sequences.
  • Table 5 shows the representative cDNAUbrary for polynucleotides of the invention.
  • Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA Ubraries shown in Table 5.
  • Table 7 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with appUcable descriptions, references, and threshold parameters.
  • CYSKP refers to the amino acid sequences of substantially purified CYSKP obtained from any species, particularly a mammaUan species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
  • agonist refers to a molecule which intensifies or mimics the biological activity of CYSKP.
  • Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of CYSKP either by directly interacting with CYSKP or by acting on components of the biological pathway in which CYSKP participates.
  • AlleUc variant is an alternative form of the gene encoding CYSKP. AlleUc variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many alleUc variants of its naturaUy occurring form. Common mutational changes which give rise to alleUc variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • altered nucleic acid sequences encoding CYSKP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as CYSKP or a polypeptide with at least one functional characteristic of CYSKP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding CYSKP, and improper or unexpected hybridization to alleUc variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding CYSKP.
  • the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent CYSKP.
  • DeUberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubiUty, hydrophobicity, hydrophilicity, and/or the ampl ⁇ pathic nature of the residues, as long as the biological or immunological activity of CYSKP is retained.
  • negatively charged amino acids may include aspartic acid and glutamic acid
  • positively charged amino acids may include lysine and arginine.
  • Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine.
  • Amino acids with uncharged side chains having similar hydrophiUcity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
  • amino acid and amino acid sequence refer to an oUgopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where “amino acid sequence” is recited to refer to a sequence of a naturally occurring protein molecule, “amino acid sequence” and Uke terms are not meant to Umit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
  • Amplification relates to the production of additional copies of a nucleic acid sequence. Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.
  • PCR polymerase chain reaction
  • Antagonist refers to a molecule which inhibits or attenuates the biological activity of CYSKP.
  • Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of CYSKP either by directly interacting with CYSKP or by acting on components of the biological pathway in which CYSKP participates.
  • antibody refers to intact immunoglobuUn molecules as well as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding an epitopic determinant.
  • Antibodies that bind CYSKP polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen.
  • the polypeptide or oUgopeptide 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 Ribonucleic acid
  • 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.
  • KLH keyhole limpet hemocyanin
  • antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
  • antisense refers to any composition capable of base-pairing with the "sense" (coding) strand of a specific nucleic acid sequence.
  • Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oUgonucleotides having modified backbone Unkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oUgonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oUgonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine.
  • Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation.
  • the designation "negative” or “minus” can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.
  • the term “biologically active” refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
  • immunologically active or “immunogenic” refers to the capabiUty of the natural, recombinant, or synthetic CYSKP, or of any oUgopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • “Complementary” describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3 * pairs with its complement,
  • composition comprising a given polynucleotide sequence and a “composition comprising a given amino acid sequence” refer broadly to any composition containing the given polynucleotide or amino acid sequence.
  • the composition may comprise a dry formulation or an aqueous solution.
  • compositions comprising polynucleotide sequences encoding CYSKP or fragments of CYSKP may be employed as hybridization probes.
  • the probes may be stored in freeze-dried form and may be associated with a stabiUzing agent such as a carbohydrate.
  • the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate;
  • SDS Styrene-maleic anhydride copolymer
  • other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
  • Consensus sequence refers to a nucleic acid sequence which has been subjected to repeated
  • Constant amino acid substitutions are those substitutions that are predicted to least interfere with the properties of the original protein, i. e. , the structure and especially the function of the protein is conserved and not significantly changed by such substitutions.
  • the table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
  • Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha heUcal conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
  • a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • derivative refers to a chemically modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group.
  • a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
  • a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
  • a “detectable label” refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
  • “Differential expression” refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
  • a “fragment” is a unique portion of CYSKP or the polynucleotide encoding CYSKP which is identical in sequence to but shorter in length than the parent sequence.
  • a fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue.
  • a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues.
  • a fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule.
  • a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence.
  • these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
  • a fragment of SEQ ID NO:35-68 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ID NO:35-68, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
  • a fragment of SEQ ID NO: 35 -68 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO:35-68 from related polynucleotide sequences.
  • the precise length of a fragment of SEQ ID NO:35-68 and the region of SEQ ID NO:35-68 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
  • a fragment of SEQ ID NO: 1-34 is encoded by a fragment of SEQ ID NO:35-68.
  • a fragment of SEQ ID NO:l-34 comprises a region of unique amino acid sequence that specifically identifies SEQ ID NO:l-34.
  • a fragment of SEQ ID NO: 1-34 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:l-34.
  • the precise length of a fragment of SEQ ID NO:l-34 and the region of SEQ ID NO:l-34 to which the fragment corresponds are routinely determinable by one of ordinary skUl in the art based on the intended purpose for the fragment.
  • a “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon.
  • a “full length” polynucleotide sequence encodes a "full length” polypeptide sequence.
  • Homology refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
  • percent identity and % identity refer to the percentage of residue matches between at least two polynucleotide sequences aUgned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize aUgnment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local AUgnment Search Tool
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local AUgnment Search Tool
  • the BLAST software suite includes various sequence analysis programs including "blastn,” that is used to aUgn a known polynucleotide sequence with other polynucleotide sequences from a variety of databases.
  • BLAST 2 Sequences are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) set at default parameters. Such default parameters may be, for example: Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch: -2 Open Gap: 5 and Extension Gap: 2 penalties
  • Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
  • percent identity and % identity refer to the percentage of residue matches between at least two polypeptide sequences aUgned using a standardized algorithm.
  • Methods of polypeptide sequence aUgnment are well-known. Some aUgnment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and_hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
  • Gap x drop-off 50
  • Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • HACs Human artificial chromosomes
  • HACs are Unear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain all of the elements required for chromosome repUcation, segregation and maintenance.
  • humanized antibody refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding abiUty.
  • Hybridization refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive anneaUng conditions and remain hybridized after the "washing" step(s). The washing step(s) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
  • Permissive conditions for anneaUng of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity.
  • Permissive anneaUng conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 ⁇ g/ml sheared, denatured salmon sperm DNA.
  • stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out.
  • Such wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (T for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1% SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1 %.
  • blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ⁇ g/ml.
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • RNA:DNA hybridizations Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art.
  • Hybridization particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
  • hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex may be formed in solution (e.g., C 0 t or R 0 t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobiUzed on a soUd support (e.g., paper, membranes, filters, chips, pins or glass sUdes, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
  • soUd support e.g., paper, membranes, filters, chips, pins or glass sUdes, or any other appropriate substrate to which cells or their nucleic acids have been fixed.
  • insertion and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
  • Immuno response can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokmes, chemokines, and other signaUng molecules, which may affect cellular and systemic defense systems.
  • an "immunogenic fragment” is a polypeptide or oUgopeptide fragment of CYSKP which is capable of eUciting an immune response when introduced into a Uving organism, for example, a mammal.
  • the term "immunogenic fragment” also includes any polypeptide or oUgopeptide fragment of CYSKP which is useful in any of the antibody production methods disclosed herein or known in the art.
  • microarray refers to an arrangement of a plurahty of polynucleotides, polypeptides, or other chemical compounds on a substrate.
  • element and “array element” refer to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray.
  • modulate refers to a change in the activity of CYSKP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of CYSKP.
  • nucleic acid and nucleic acid sequence refer to a nucleotide, oUgonucleoti.de, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-Uke or RNA-Uke material.
  • PNA peptide nucleic acid
  • operably linked refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably Unked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an oUgonucleoti.de of at least about 5 nucleotides in length Unked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubiUty to the composition.
  • PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their Ufespan in the cell.
  • Post-translational modification of an CYSKP may involve Upidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cell type depending on the enzymatic miUeu of CYSKP.
  • Probe refers to nucleic acid sequences encoding CYSKP, their complements, or fragments thereof which are used to detect identical, aUelic or related nucleic acid sequences.
  • Probes are isolated oUgonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, Ugands, chemiluminescent agents, and enzymes.
  • Probes are short nucleic acids, usually DNA oUgonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme.
  • Primer pairs can be used for ampUfication (and identification) of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR).
  • Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
  • OUgonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oUgonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have incorporated additional features for expanded capabiUties. For example, the PrimOU primer selection program (available to the pubUc from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome- wide scope.
  • Primer3 primer selection program (available to the pubUc from the Whitehead Institute MIT Center for Genome Research, Cambridge MA) allows the user to input a "mispriming Ubrary," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays.
  • the source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.
  • the PrimeGen program (available to the pubUc from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence aUgnments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aUgned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oUgonucleotides and polynucleotide fragments.
  • oUgonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of ohgonucleotide selection are not Umited to those described above.
  • a "recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accompUshed by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra.
  • the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
  • a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence.
  • Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
  • such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
  • a “regulatory element” refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stabiUty.
  • Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionucUdes; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.
  • RNA equivalent in reference to a DNA sequence, is composed of the same Unear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
  • sample is used in its broadest sense.
  • a sample suspected of containing CYSKP, nucleic acids encoding CYSKP, or fragments thereof may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
  • binding and “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
  • substantially purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.
  • substitution refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
  • Substrate refers to any suitable rigid or semi-rigid support including membranes, filters, chips, sUdes, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
  • the substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
  • a “transcript image” refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.
  • Transformation describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not Umited to, bacteriophage or viral infection, electroporation, heat shock, Upofection, and particle bombardment.
  • transformed cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously repUcating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for Umited periods of time.
  • a "transgenic organism,” as used herein, is any organism, including but not Umited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques weU known in the art.
  • the nucleic acid is introduced into the ceU, directly or indirectly by introduction into a precursor of the cell, by way of deUberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
  • the transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals.
  • the isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. (1989), supra.
  • a "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07-1999) set at default parameters.
  • Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length.
  • a variant may be described as, for example, an "alleUc” (as defined above), "splice,” “species,” or “polymorphic” variant.
  • a spUce variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternative sphcing of exons during mRNA processing.
  • the corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
  • Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides will generaUy have significant amino acid identity relative to each other.
  • a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
  • Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
  • SNPs single nucleotide polymorphisms
  • a "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07-1999) set at default parameters.
  • Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length of one of the polypeptides.
  • the invention is based on the discovery of new human cytoskeleton-associated proteins (CYSKP), the polynucleotides encoding CYSKP, and the use of these compositions for the diagnosis, treatment, or prevention of cell proUferative, autoimmune/inflammatory, vesicle trafficking, neurological, cell motiUty, reproductive, and muscle disorders.
  • CYSKP cytoskeleton-associated proteins
  • Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ID). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide ID) as shown.
  • polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as shown.
  • Table 2 shows sequences with homology to the polypeptides of the invention as identified by
  • FIG. 1 shows the polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention.
  • Column 3 shows the GenBank identification number (GenbankID NO:) of the nearest GenBank homolog.
  • Column 4 shows the probabiUty score for the match between each polypeptide and its GenBank homolog.
  • Column 5 shows the annotation of the GenBank homolog along with relevant citations where appUcable, all of which are expressly incorporated by reference herein.
  • Table 3 shows various structural features of the polypeptides of the invention.
  • Columns 1 and 2 show the polypeptide sequence identification number (SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of the invention.
  • Column 3 shows the number of amino acid residues in each polypeptide.
  • Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS program of the GCG sequence analysis software package (Genetics Computer Group, Madison WI).
  • Column 6 shows amino acid residues comprising signature sequences, domains, and motifs.
  • Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were appUed.
  • SEQ ID NO:31 is 34% identical to a Caenorhabditis elegans protein similar to mouse ankyrin (GenBank ID g3879121) as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.)
  • the BLAST probabiUty score is 1. le-146, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance.
  • SEQ ID NO:31 also contains Ank repeats as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • SEQ ID NO:34 is 96% identical over 97 amino acids to human Intermediate Filament Associated Protein (GenBank ID 1333846) as determined by the Basic Local AUgnment Search Tool (BLAST).
  • BLAST Basic Local AUgnment Search Tool
  • the BLAST probabiUty score is 8.2e-45, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance.
  • Data from BLAST analyses using the PRODOM database provide further corroborative evidence that SEQ ID NO:34 is a cytoskeleton protein.
  • SEQ ID NO:1-30 and SEQ ID NO:32-33 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ ID NO: 1-34 are described in Table 7.
  • the full length polynucleotide sequences of the present invention were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences.
  • Columns 1 and 2 Ust the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and the corresponding Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) for each polynucleotide of the invention.
  • Column 3 shows the length of each polynucleotide sequence in basepairs.
  • Column 4 Usts fragments of the polynucleotide sequences which are useful, for example, in hybridization or amplification technologies that identify SEQ ID NO:35-68 or that distinguish between SEQ ID NO:35-68 and related polynucleotide sequences.
  • Column 5 shows identification numbers corresponding to cDNA sequences, coding sequences (exons) predicted from genomic DNA, and/or sequence assemblages comprised of both cDNA and genomic DNA. These sequences were used to assemble the full length polynucleotide sequences of the invention.
  • Columns 6 and 7 of Table 4 show the nucleotide start (5') and stop (3') positions of the cDNA and genomic sequences in column 5 relative to their respective full length sequences.
  • the identification numbers in Column 5 of Table 4 may refer specifically, for example, to Incyte cDNAs along with their corresponding cDNA Ubraries.
  • 3824958H1 is the identification number of an Incyte cDNA sequence
  • BRAXNOT01 is the cDNA Ubrary from which it is derived.
  • Incyte cDNAs for which cDNA Ubraries are not indicated were derived from pooled cDNA Ubraries (e.g., 71263527V1).
  • the identification numbers in column 5 may refer to GenBank cDNAs or ESTs (e.g., g2276318) which contributed to the assembly of the full length polynucleotide sequences.
  • the identification numbers in column 5 may refer to coding regions predicted by Genscan analysis of genomic DNA. The Genscan-predicted coding sequences may have been edited prior to assembly. (See Example IV.) Alternatively, the identification numbers in column 5 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm. (See Example V.) Alternatively, the identification numbers in column 5 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon- stretching" algorithm. (See Example V.) In some cases, Incyte cDNA coverage redundant with the sequence coverage shown in column 5 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
  • Table 5 shows the representative cDNA Ubraries for those full length polynucleotide sequences which were assembled using Incyte cDNA sequences.
  • the representative cDNA Ubrary is the Incyte cDNA Ubrary which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotide sequences.
  • the tissues and vectors which were used to construct the cDNA Ubraries shown in Table 5 are described in Table 6.
  • the invention also encompasses CYSKP variants.
  • a preferred CYSKP variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the CYSKP amino acid sequence, and which contains at least one functional or structural characteristic of CYSKP.
  • the invention also encompasses polynucleotides which encode CYSKP.
  • the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:35-68, which encodes CYSKP.
  • the polynucleotide sequences of SEQ ID NO:35-68, as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base fhymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
  • the invention also encompasses a variant of a polynucleotide sequence encoding CYSKP.
  • a variant polynucleotide sequence will have at least about 70%, or alternatively at least about 85 %, or even at least about 95 % polynucleotide sequence identity to the polynucleotide sequence encoding CYSKP.
  • a particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:35-68 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:35-68.
  • Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of CYSKP.
  • nucleotide sequences which encode CYSKP and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring CYSKP under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding CYSKP or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-Ufe, than transcripts produced from the naturally occurring sequence.
  • the invention also encompasses production of DNA sequences which encode CYSKP and
  • CYSKP derivatives, or fragments thereof, entirely by synthetic chemistry After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding CYSKP or any fragment thereof.
  • polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NO:35-68 and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol. 152:507-511.) Hybridization conditions, including anneaUng and wash conditions, are described in "Definitions.”
  • Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention.
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (AppUed Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE ampUfication system (Life Technologies, Gaithersburg MD).
  • sequence preparation is automated with machines such as the MICROLAB 2200 Uquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (AppUed Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (AppUed Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997) Short Protocols in Molecular Biology. John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biology and Biotechnology. Wiley VCH, New York NY, pp. 856-853.)
  • the nucleic acid sequences encoding CYSKP may be extended utiUzing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • restriction-site PCR uses universal and nested primers to ampUfy unknown sequence from genomic DNA within a cloning vector.
  • inverse PCR uses primers that extend in divergent directions to ampUfy unknown sequence from a circularized template.
  • the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences.
  • a third method, capture PCR involves PCR ampUfication of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
  • capture PCR involves PCR ampUfication of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
  • multiple restriction enzyme digestions and Ugations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
  • Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al.
  • primers may be designed using commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
  • Ubraries When screening for full length cDNAs, it is preferable to use Ubraries that have been size-selected to include larger cDNAs. In addition, random-primed Ubraries, which often include sequences containing the 5' regions of genes, are preferable for situations in which an oUgo d(T) Ubrary does not yield a full-length cDNA. Genomic Ubraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
  • capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
  • Output/tight intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, AppUed Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled.
  • Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in Umited amounts in.a particular sample.
  • polynucleotide sequences or fragments thereof which encode CYSKP may be cloned in recombinant DNA molecules that direct expression of CYSKP, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express CYSKP.
  • nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter CYSKP-encoding sequences for a variety of purposes including, but not Umited to, modification of the cloning, processing, and or expression of the gene product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oUgonucleotides may be used to engineer the nucleotide sequences.
  • oUgonucleotide- mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce spUce variants, and so forth.
  • the nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent Number 5,837,458; Chang, C.-C et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of CYSKP, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds.
  • MOLECULARBREEDING Maxygen Inc., Santa Clara CA; described in U.S. Patent Number 5,837,458; Chang, C.-C et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C et al. (19
  • DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening.
  • genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized.
  • sequences encoding CYSKP may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232.)
  • CYSKP itself or a fragment thereof may be synthesized using chemical methods. For example, peptide synthesis can be performed using various solution-phase or soUd-phase techniques. (See, e.g.,
  • the peptide may be substantially purified by preparative high performance Uquid chromatography. (See, e.g., Chiez, R.M. andF.Z. Regnier (1990) Methods Enzymol. 182:392-421.)
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, supra, pp. 28-53.)
  • the nucleotide sequences encoding CYSKP or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host.
  • these elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5 ' and 3 ' untranslated regions in the vector and in polynucleotide sequences encoding CYSKP.
  • Such elements may vary in their strength and specificity.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding CYSKP. Such signals include the ATG initiation codon and adjacent sequences, e.g.
  • a variety of expression vector/host systems may be utilized to contain and express sequences encoding CYSKP. These include, but are not Umited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauUflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauUflower mosaic
  • Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population.
  • cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding CYSKP.
  • routine cloning, subcloning, and propagation of polynucleotide sequences encoding CYSKP can be achieved using a multifunctional E. coU vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORTl plasmid (Life Technologies). Ligation of sequences encoding CYSKP into the vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules.
  • these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
  • vectors which direct high level expression of CYSKP may be used.
  • vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
  • Yeast expression systems may be used for production of CYSKP.
  • a number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomvces cerevisiae or Pichia pastoris.
  • such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
  • Plant systems may also be used for expression of CYSKP. Transcription of sequences encoding CYSKP may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:17-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; BrogUe, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl.
  • a number of viral-based expression systems may be utilized.
  • sequences encoding CYSKP may be Ugated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses CYSKP in host cells.
  • transcription enhancers such as theRous sarcoma virus (RSV) enhancer, may be used to increase expression in mammaUan host cells.
  • SV40 or EBV- based vectors may also be used for high-level protein expression.
  • HACs Human artificial chromosomes
  • HACs may also be employed to deUver larger fragments of DNA than can be contained in and expressed from a plasmid.
  • HACs of about 6 kb to 10 Mb are constructed and deUvered via conventional deUvery methods (Uposomes, polycationic amino polymers, or vesicles) for therapeutic purposes.
  • deUvery methods Uposomes, polycationic amino polymers, or vesicles
  • sequences encoding CYSKP can be transformed into cell Unes using expression vectors which may contain viral origins of repUcation and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
  • the purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
  • Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
  • Any number of selection systems may be used to recover transformed cell Unes. These include, but are not Umited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk ' and apr cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetaboUte, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methofrexate
  • neo confers resistance to the aminoglycosides neomycin and G-418
  • als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
  • Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metaboUtes.
  • Visible markers e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), ⁇ glucuronidase and its substrate ⁇ -glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, CA. (1995) Methods Mol. Biol. 55:121-131.)
  • marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
  • sequence encoding CYSKP is inserted within a marker gene sequence
  • transformed cells containing sequences encoding CYSKP can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding CYSKP under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
  • host cells that contain the nucleic acid sequence encoding CYSKP and that express
  • CYSKP may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not Umited to, DNA-DNA or DNA-RNA hybridizations, PCR ampUfication, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences. Immunological methods for detecting and measuring the expression of CYSKP using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-Unked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS).
  • ELISAs enzyme-Unked immunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated cell sorting
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on CYSKP is preferred, but a competitive binding assay may be employed.
  • assays are well known in the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual. APS Press, St. Paul MN, Sect. IV; CoUgan, J.E. et al. (1997) Current Protocols in Immunology, Greene Pub. Associates and Wiley-Interscience, New York NY; and Pound, J.D.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding CYSKP include oUgolabeUng, nick translation, end-labeUng, or PCR ampUfication using a labeled nucleotide.
  • the sequences encoding CYSKP, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
  • T7, T3, or SP6 RNA polymerase
  • reporter molecules or labels which may be used for ease of detection include radionucUdes, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the Uke.
  • Host cells transformed with nucleotide sequences encoding CYSKP may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode CYSKP may be designed to contain signal sequences which direct secretion of CYSKP through a prokaryotic or eukaryotic cell membrane.
  • a host cell strain may be chosen for its abiUty to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not Umited to, acetylation, carboxylation, glycosylation, phosphorylation, Upidation, and acylation.
  • Post-translational processing which cleaves a "prepro” or "pro” form of the protein may also be used to specify protein targeting, folding, and/or activity.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture Collection
  • natural, modified, or recombinant nucleic acid sequences encoding CYSKP may be Ugated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric CYSKP protein containing a heterologous moiety that can be recognized by a commercially available antibody may faciUtate the screening of peptide Ubraries for inhibitors of CYSKP activity.
  • Heterologous protein and peptide moieties may also faciUtate purification of fusion proteins using commercially available affinity matrices.
  • Such moieties include, but are not Umited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmoduUn binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA).
  • GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmoduUn, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags.
  • a fusion protein may also be engineered to contain a proteolytic cleavage site located between the CYSKP encoding sequence and the heterologous protein sequence, so that CYSKP may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10). A variety of commercially available kits may also be used to faciUtate expression and purification of fusion proteins.
  • synthesis of radio-labeled CYSKP may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35 S-methionine.
  • CYSKP of the present invention or fragments thereof may be used to screen for compounds that specifically bind to CYSKP. At least one and up to a plurahty of test compounds may be screened for specific binding to CYSKP. Examples of test compounds include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
  • the compound thus identified is closely related to the natural ligand of CYSKP, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner.
  • the compound can be closely related to the natural receptor to which CYSKP binds, or to at least a fragment of the receptor, e.g., the ligand binding site. In either case, the compound can be rationally designed using known techniques. In one embodiment, screening for these compounds involves producing appropriate ceUs which express CYSKP, either as a secreted protein or on the cell membrane.
  • Preferred ceUs include cells from mammals, yeast, Drosophila. or R coli. Cells expressing CYSKP or cell membrane fractions which contain CYSKP are then contacted with a test compound and binding, stimulation, or inhibition of activity of either CYSKP or the compound is analyzed.
  • An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label.
  • the assay may comprise the steps of combining at least one test compound with CYSKP, either in solution or affixed to a solid support, and detecting the binding of CYSKP to the compound.
  • the assay may detect or measure binding of a test compound in the presence of a labeled competitor.
  • the assay may be carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a soUd support.
  • CYSKP of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of CYSKP.
  • Such compounds may include agonists, antagonists, or partial or inverse agonists.
  • an assay is performed under conditions permissive for CYSKP activity, wherein CYSKP is combined with at least one test compound, and the activity of CYSKP in the presence of a test compound is compared with the activity of CYSKP in the absence of the test compound. A change in the activity of CYSKP in the presence of the test compound is indicative of a compound that modulates the activity of CYSKP.
  • a test compound is combined with an in vitro or cell-free system comprising CYSKP under conditions suitable for CYSKP activity, and the assay is performed.
  • a test compound which modulates the activity of CYSKP may do so indirectly and need not come in direct contact with the test compound.
  • At least one and up to a plurahty of test compounds may be screened.
  • polynucleotides encoding CYSKP or their mammaUan homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells. Such techniques are well known in the art and are useful for the generation of animal models of human disease.
  • mouse ES cells such as the mouse 129/SvJ cell Une
  • the ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292).
  • 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) CUn. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
  • Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains. Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
  • Polynucleotides encoding CYSKP may also be manipulated in vitro in ES cells derived from human blastocysts.
  • Human ES cells have the potential to differentiate into at least eight separate cell Uneages including endoderm, mesoderm, and ectodermal cell types. These cell Uneages differentiate into, for example, neural cells, hematopoietic Uneages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147).
  • Polynucleotides encoding CYSKP can also be used to create "knockin” humanized animals (pigs) or transgenic animals (mice or rats) to model human disease.
  • knockin technology a region of a polynucleotide encoding CYSKP 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 Unes are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
  • a mammal inbred to overexpress CYSKP may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).
  • THERAPEUTICS Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of CYSKP and cytoskeleton-associated proteins.
  • the expression of CYSKP is closely associated with lung, reproductive (including placenta), neural (including brain), adrenal, endothelial, kidney, and spleen tissue, as well as with ovarian, breast, and testicular tumor tissue.
  • CYSKP appears to play a role in cell proUferative, autoimmune/inflammatory, vesicle trafficking, neurological, cell motiUty, reproductive, and muscle disorders.
  • CYSKP appears to play a role in cell proUferative, autoimmune/inflammatory, vesicle trafficking, neurological, cell motiUty, reproductive, and muscle disorders.
  • CYSKP or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of CYSKP.
  • disorders include, but are not limited to, a cell proUferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, gangUa, gastrointestinal tract, heart, kidney, U
  • a reproductive disorder such as a disorder of prolactin production, infertility, including tubal disease, ovulatory defects, endometriosis, a disruption of the estrous cycle, a disruption of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, an endomefrial or ovarian tumor, a uterine fibroid, autoimmune disorders, ectopic pregnancy, teratogenesis, cancer of the breast, fibrocystic breast disease, galactorrhea, a disruption of spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, carcinoma of the male breast, gynecomastia, hypergonadotropic and hypogonadotropic hypogonadism, pseudohermaphrodit
  • composition comprising a substantially purified CYSKP in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of CYSKP including, but not Umited to, those provided above.
  • an agonist which modulates the activity of CYSKP may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of CYSKP including, but not Umited to, those Usted above.
  • an antagonist of CYSKP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of CYSKP. Examples of such disorders include, but are not Umited to, those cell proUferative, autoimmune/inflainmatory, vesicle trafficking, neurological, cell motiUty, reproductive, and muscle disorders described above.
  • an antibody which specifically binds CYSKP may be used directly as an antagonist or indirectly as a targeting or deUvery mechanism for bringing a pharmaceutical agent to cells or tissues which express CYSKP.
  • a vector expressing the complement of the polynucleotide encoding CYSKP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of CYSKP including, but not Umited to, those described above.
  • any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • An antagonist of CYSKP may be produced using methods which are generally known in the art.
  • purified CYSKP may be used to produce antibodies or to screen Ubraries of pharmaceutical agents to identify those which specifically bind CYSKP.
  • Antibodies to CYSKP may also be generated using methods that are well known in the art.
  • Such antibodies may include, but are not Umited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression Ubrary.
  • NeutraUzing antibodies i.e., those which inhibit dimer formation
  • various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with CYSKP or with any fragment or oUgopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include, but are not Umited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinifrophenol.
  • BCG BacilU Calmette-Guerin
  • Corynebacterium parvum are especially preferable.
  • the oUgopeptides, peptides, or fragments used to induce antibodies to CYSKP have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these oUgopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein. Short stretches of CYSKP amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
  • Monoclonal antibodies to CYSKP may be prepared using any technique which provides for the production of antibody molecules by continuous cell Unes in culture. These include, but are not Umited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique.
  • the hybridoma technique the human B-cell hybridoma technique
  • the EBV-hybridoma technique See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol.
  • chimeric antibodies such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used.
  • techniques developed for the production of single chain antibodies may be adapted, using methods known in the art, to produce CYSKP-specific single chain antibodies.
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobuUn Ubraries. (See, e.g., Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobuUn Ubraries or panels of highly specific binding reagents as disclosed in theUterature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
  • Antibody fragments which contain specific binding sites for CYSKP may also be generated.
  • such fragments include, but are not Umited to, F(ab') 2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression Ubraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.)
  • immunoassays may be used for screening to identify antibodies having the desired specificity.
  • Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with estabUshed specificities are well known in the art.
  • Such immunoassays typically involve the measurement of complex formation between CYSKP and its specific antibody.
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering CYSKP epitopes is generally used, but a competitive binding assay may also be employed (Pound, supra).
  • K ⁇ is defined as the molar concentration of CYSKP-antibody complex divided by the molar concentrations of free antigen and free antibody under equiUbrium conditions.
  • the L determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple CYSKP epitopes, represents the average affinity, or avidity, of the antibodies for CYSKP.
  • the K ⁇ determined for a preparation of monoclonal antibodies, which are monospecific for a particular CYSKP epitope, represents a true measure of affinity.
  • High-affinity antibody preparations with K ⁇ ranging from about 10 9 to 10 12 L/mole are preferred for use in immunoassays in which the CYSKP-antibody complex must withstand rigorous manipulations.
  • Low-affinity antibody preparations with K-. ranging from about 10 ⁇ to 10 7 L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of CYSKP, preferably in active form, from the antibody (Catty, D. (1988) Antibodies. Volume I: A Practical Approach, IRL Press, Washington DC; Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
  • polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream appUcations.
  • a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is generally employed in procedures requiring precipitation of CYSKP-antibody complexes.
  • Procedures for evaluating antibody specificity, titer, and avidity, and guideUnes for antibody quaUty and usage in various appUcations, are generally available. (See, e.g., Catty, supra, and CoUgan et al. supra.)
  • the polynucleotides encoding CYSKP may be used for therapeutic purposes.
  • modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified oUgonucleotides) to the coding or regulatory regions of the gene encoding CYSKP.
  • complementary sequences or antisense molecules DNA, RNA, PNA, or modified oUgonucleotides
  • antisense oUgonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding CYSKP.
  • Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein.
  • Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors.
  • viral vectors such as retrovirus and adeno-associated virus vectors.
  • Other gene deUvery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art.
  • polynucleotides encoding CYSKP may be used for somatic or germUne 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-Unked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C et al.
  • SCID severe combined immunodeficiency
  • ADA adenosine deaminase
  • CYSKP hepatitis B or C virus
  • fungal parasites such as Candida albicans and Paracoccidioides brasiUensis
  • protozoan parasites such as Plasmodium falciparum and Trypanosoma cruzi
  • diseases or disorders caused by deficiencies in CYSKP are treated by constructing mammaUan expression vectors encoding CYSKP and introducing these vectors by mechanical means into CYSKP-deficient cells.
  • Mechanical transfer technologies for use with cells in vivo or ex vifro include (i) direct DNA microinjection into individual cells, (U) balUstic gold particle deUvery, (in) Uposome-mediated fransfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev. Biochem. 62:191- 217; Ivies, Z. (1997) Cell 91:501-510; Boulay, J-L.
  • Expression vectors that may be effective for the expression of CYSKP include, but are not Umited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invifrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG. PEGSH PERV (Stratagene, La Jolla CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
  • CYSKP 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), (U) an inducible promoter (e.g., the tefracycUne-regulated promoter (Gossen, M. andH. Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V. andH.M. Blau (1998) Curr. Opin. Biotechnol.
  • a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -act
  • FK506/rapamycin inducible promoter or the RU486/mifepristone inducible promoter (Rossi, F.M.V. and Blau, H.M. supra)), or (Ui) a tissue-specific promoter or the native promoter of the endogenous gene encoding CYSKP from a normal individual.
  • Uposome transformation kits allow one with ordinary skill in the art to deUver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters.
  • transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J. 1 :841-845).
  • the introduction of DNA to primary cells requires modification of these standardized mammaUan tr ansf ection protocols .
  • diseases or disorders caused by genetic defects with respect to CYSKP expression are treated by constructing a refrovirus vector consisting of (i) the polynucleotide encoding CYSKP under the control of an independent promoter or the refrovirus long terminal repeat (LTR) promoter, (U) appropriate RNA packaging signals, and (Ui) a Rev-responsive element (RRE) along with additional refrovirus ds-acting RNA sequences and coding sequences required for efficient vector propagation.
  • Refrovirus vectors e.g., PFB and PFBNEO
  • PFB and PFBNEO are commercially available (Stratagene) and are based onpubUshed data (Riviere, I. et al. (1995) Proc. Natl.
  • the vector is propagated in an appropriate vector producing cell Une (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al. (1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey, R.
  • VSVg vector producing cell Une
  • U.S. Patent Number 5,910,434 to Rigg discloses a method for obtaining refrovirus packaging cell Unes and is hereby incorporated by reference. Propagation of refrovirus vectors, transduction of a population of cells (e.g., CD4 + T-cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. Virol. 71:7020-7029; Bauer, G.
  • an adenovirus-based gene therapy deUvery system is used to deUver polynucleotides encoding CYSKP to cells which have one or more genetic abnormaUties with respect to the expression of CYSKP.
  • the construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art.
  • RepUcation defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful adenoviral vectors are described in U.S.
  • Patent Number 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby incorporated by reference.
  • adenoviral vectors see also Antinozzi, P.A. et al. (1999) Annu. Rev. Nutr. 19:511-544 and Verma, I.M. and N. Somia (1997) Nature 18:389:239-242, both incorporated by reference herein.
  • a herpes-based, gene therapy deUvery system is used to deliver polynucleotides encoding CYSKP to target cells which have one or more genetic abnormaUties with respect to the expression of CYSKP.
  • the use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing CYSKP to cells of the central nervous system, for which HSV has a fropism.
  • the construction and packaging of herpes-based vectors are well known to those with ordinary skill in the art.
  • a repUcation-competent herpes simplex virus (HSV) type 1 -based vector has been used to deUver a reporter gene to the eyes of primates (Liu, X. et al.
  • HSV-1 virus vector has also been disclosed in detail in U.S. Patent Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference.
  • U.S. Patent Number 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the control of the appropriate promoter for purposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22.
  • HSV vectors see also Goins, W.F. et al.
  • an alphavirus (positive, single-stranded RNA virus) vector is used to deUver polynucleotides encoding CYSKP to target cells.
  • SFV SemUki Forest Virus
  • alphavirus RNA repUcation a subgenomic RNA is generated that normally encodes the viral capsid proteins.
  • This subgenomic RNA repUcates to higher levels than the full length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase).
  • enzymatic activity e.g., protease and polymerase.
  • inserting the coding sequence for CYSKP into the alphavirus genome in place of the capsid-coding region results in the production of a large number of CYSKP- coding RNAs and the synthesis of high levels of CYSKP in vector transduced cells.
  • alphavirus infection is typically associated with cell lysis within a few days
  • the abiUty to estabUsh a persistent infection in hamster normal kidney cells (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic repUcation of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S.A. et al. (1997) Virology 228:74-83).
  • the wide host range of alphaviruses will allow the introduction of CYSKP into a variety of cell types. The specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction.
  • a complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules
  • Ribozymes may also be used to catalyze the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding CYSKP.
  • RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oUgonucleotide inoperable.
  • the suitabiUty of candidate targets may also be evaluated by testing accessibiUty to hybridization with complementary oUgonucleotides using ribonuclease protection assays.
  • RNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oUgonucleotides such as soUd phase phosphoramidite chemical synthesis.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding CYSKP. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6.
  • these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell Unes, cells, or tissues.
  • RNA molecules may be modified to increase intracellular stabiUty and half-Ufe. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5 ' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase Urikages within the backbone of the molecule.
  • An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding CYSKP.
  • Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oUgonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, transcription factors and other polypeptide transcriptional regulators, and non- macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression.
  • a compound which specifically inhibits expression of the polynucleotide encoding CYSKP may be therapeutically useful, and in the treament of disorders associated with decreased CYSKP expression or activity, a compound which specifically promotes expression of the polynucleotide encoding CYSKP may be therapeutically useful.
  • test compounds may be screened for effectiveness in altering expression of a specific polynucleotide.
  • a test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commercially-available or proprietary library of naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide; and selection from a library of chemical compounds created combinatorially or randomly.
  • a sample comprising a polynucleotide encoding CYSKP is exposed to at least one test compound thus obtained.
  • the sample may comprise, for example, an intact or permeabilized cell, or an in vifro cell-free or reconstituted biochemical system.
  • Alterations in the expression of a polynucleotide encoding CYSKP are assayed by any method commonly known in the art.
  • the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding CYSKP.
  • the amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds.
  • a screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem. Biophys. Res. Commun.
  • a particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No. 6,022,691). Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vifro. and ex vivo.
  • oligonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides
  • vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient.
  • DeUvery by fransfection, by Uposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:462-466.)
  • any of the therapeutic methods described above may be appUed to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
  • An additional embodiment of the invention relates to the administration of a composition which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient.
  • Excipients may include, for example, sugars, starches, celluloses, gums, and proteins.
  • Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack PubUshing, Easton PA).
  • Such compositions may consist of CYSKP, antibodies to CYSKP, and mimetics, agonists, antagonists, or inhibitors of CYSKP.
  • compositions utilized in this invention may be administered by any number of routes including, but not Umited to, oral, intravenous, intramuscular, infra-arterial, inframedullary, infrathecal, infravenfricular, pulmonary, transdermal* subcutaneous, infraperitoneal, infranasal, enteral, topical, subUngual, or rectal means.
  • compositions for pulmonary administration may be prepared in tiquid or dry powder form. These compositions are generally aerosoUzed immediately prior to inhalation by the patient.
  • aerosol deUvery of fast-acting formulations is well-known in the art.
  • macromolecules e.g. larger peptides and proteins
  • Pulmonary deUvery has the advantage of administration without needle injection, and obviates the need for potentially toxic penetration enhancers.
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • the determination of an effective dose is well within the capabiUty of those skilled in the art. SpeciaUzed forms of compositions may be prepared for direct intracellular deUvery of macromolecules comprising CYSKP or fragments thereof.
  • Uposome preparations containing a cell-impermeable macro-molecule may promote cell fusion and intracellular deUvery of the macro-molecule.
  • CYSKP or a fragment thereof may be joined to a short cationic N- terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to fransduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs.
  • An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of active ingredient, for example CYSKP or fragments thereof, antibodies of CYSKP, and agonists, antagonists or inhibitors of CYSKP, which ameUorates the symptoms or condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeutically effective in 50% of the population) or LD 50 (the dose lethal to 50% of the population) statistics.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD 50 ED 50 ratio. Compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED 50 with Uttle or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration. The exact dosage will be determined by the practitioner, in Ught of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combinations), reaction sensitivities, and response to therapy. Long-acting compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-Ufe and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration.
  • Guidance as to particular dosages and methods of deUvery is provided in the Uterature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. DIAGNOSTICS
  • antibodies which specifically bind CYSKP may be used for the diagnosis of disorders characterized by expression of CYSKP, or in assays to monitor patients being treated with CYSKP or agonists, antagonists, or inhibitors of CYSKP.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for CYSKP include methods which utihze the antibody and a label to detect CYSKP in human body fluids or in extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
  • a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
  • a variety of protocols for measuring CYSKP including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of CYSKP expression.
  • Normal or standard values for CYSKP expression are estabUshed by combining body fluids or cell extracts taken from normal mammaUan subjects, for example, human subjects, with antibodies to CYSKP under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of CYSKP expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values estabUshes the parameters for diagnosing disease.
  • the polynucleotides encoding CYSKP may be used for diagnostic purposes.
  • the polynucleotides which may be used include oUgonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of CYSKP may be correlated with disease.
  • the diagnostic assay may be used to determine absence, presence, and excess expression of CYSKP, and to monitor regulation of CYSKP levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding CYSKP or closely related molecules may be used to identify nucleic acid sequences which encode CYSKP.
  • the specificity of the probe whether it is made from a highly specific region, e.g., the 5 'regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or ampUfication will determine whether the probe identifies only naturally occurring sequences encoding CYSKP, alleUc variants, or related sequences.
  • Probes may also be used for the detection of related sequences, and may have at least 50% sequence identity to any of the CYSKP encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO:35-68 or from genomic sequences including promoters, enhancers, and introns of the CYSKP gene.
  • Means for producing specific hybridization probes for DNAs encoding CYSKP include the cloning of polynucleotide sequences encoding CYSKP or CYSKP derivatives into vectors for the production of mRNA probes.
  • Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
  • Hybridization probes may be labeled by a variety of reporter groups, for example, by radionucUdes such as 32 P or 35 S, or by enzymatic labels, such as alkaUne phosphatase coupled to the probe via avidin/biotin coupUng systems, and the Uke.
  • Polynucleotide sequences encoding CYSKP may be used for the diagnosis of disorders associated with expression of CYSKP.
  • disorders include, but are not limited to, a cell proUferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, gangUa, gastrointestinal tract, heart, kidney, Uver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, saUvary glands, skin, spleen, testis,
  • the polynucleotide sequences encoding CYSKP may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-Uke assays; and in microarrays utilizing fluids or tissues from patients to detect altered CYSKP expression. Such quaUtative or quantitative methods are well known in the art.
  • the nucleotide sequences encoding CYSKP may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
  • the nucleotide sequences encoding CYSKP may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a confrol sample then the presence of altered levels of nucleotide sequences encoding CYSKP in the sample indicates the presence of the associated disorder.
  • Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in cUnical trials, or to monitor the treatment of an individual patient. In order to provide a basis for the diagnosis of a disorder associated with expression of
  • CYSKP a normal or standard profile for expression is estabUshed. This may be accompUshed by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding CYSKP, under conditions suitable for hybridization or ampUfication. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to estabUsh the presence of a disorder.
  • hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
  • the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual cUnical symptoms.
  • a more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment eariier thereby preventing the development or further progression of the cancer.
  • Additional diagnostic uses for oUgonucleotides designed from the sequences encoding CYSKP may involve the use of PCR. These oUgomers may be chemically synthesized, generated enzymatically, or produced in vitro.
  • OUgomers will preferably contain a fragment of a polynucleotide encoding CYSKP, or a fragment of a polynucleotide complementary to the polynucleotide encoding CYSKP, and will be employed under optimized conditions for identification of a specific gene or condition. OUgomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
  • oUgonucleotide primers derived from the polynucleotide sequences encoding CYSKP may be used to detect single nucleotide polymorphisms (SNPs).
  • SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans.
  • Methods of SNP detection include, but are not Umited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods.
  • SSCP single-stranded conformation polymorphism
  • fSSCP fluorescent SSCP
  • oUgonucleotide primers derived from the polynucleotide sequences encoding CYSKP are used to ampUfy 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 Uke.
  • SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels.
  • the oligonucleotide primers are fluorescently labeled, which allows detection of the ampUmers in high-throughput equipment such as DNA sequencing machines.
  • sequence database analysis methods termed in siUco SNP (isSNP) are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence.
  • SNPs may be detected and characterized by mass spectromefry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
  • Methods which may also be used to quantify the expression of CYSKP include radiolabeUng or biotinylating nucleotides, coampUfication of a confrol nucleic acid, and interpolating results from standard curves. (See, e.g., Melby, P.C et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.
  • the speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the oUgomer or polynucleotide of interest is presented in various dilutions and a specfrophotomefric or colorimefric response gives rapid quantitation.
  • oUgonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as elements on a microarray.
  • the microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below.
  • the microarray may also be used to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease.
  • this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient.
  • therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
  • CYSKP, fragments of CYSKP, or antibodies specific for CYSKP may be used as elements on a microarray.
  • the microarray may be used to monitor or measure protein- protein interactions, drug-target interactions, and gene expression profiles, as described above.
  • a particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or cell type.
  • a franscript image represents the global pattern of gene expression by a particular tissue or cell type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time. (See Seilhamer et al, "Comparative Gene Transcript Analysis," U.S. Patent Number 5,840,484, expressly incorporated by reference herein.)
  • a franscript image may be generated by hybridizing the polynucleotides of the present invention or their complements to thetotaUty of transcripts or reverse transcripts of a particular tissue or cell type.
  • the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray.
  • the resultant transcript image would provide a profile of gene activity.
  • Transcript images may be generated using transcripts isolated from tissues, cell Unes, biopsies, or other biological samples.
  • the transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vifro, as in the case of a cell Une.
  • Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vifro model systems and precUnical evaluation of pharmaceuticals, as well as toxicological testing of industrial and naturally-occurring environmental compounds. All compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L. Anderson (2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein). If a test compound has a signature similar to that of a compound with known toxicity, it is Ukely to share those toxic properties.
  • the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound.
  • Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified.
  • the transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the franscript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
  • Another particular embodiment relates to the use of the polypeptide sequences of the present invention to analyze the proteome of a tissue or cell type.
  • proteome refers to the global pattern of protein expression in a particular tissue or cell type. Each protein component of a proteome can be subjected individually to further analysis. Proteome expression patterns, or profiles, are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time. A profile of a cell' s proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type.
  • the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelecfric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel elecfrophoresis in the second dimension (Steiner and Anderson, supra).
  • the proteins are visuaUzed in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains.
  • the optical density of each protein spot is generally proportional to the level of the protein in the sample.
  • the optical densities of equivalently positioned protein spots from different samples are compared to identify any changes in protein spot density related to the treatment.
  • the proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass specfromefry.
  • the identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of the present invention. In some cases, further sequence data may be obtained for definitive protein identification.
  • a proteomic profile may also be generated using antibodies specific for CYSKP to quantify the levels of CYSKP expression.
  • the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino- reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.
  • Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in parallel with toxicant signatures at the franscript level.
  • There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the franscript image, but which alter the proteomic profile.
  • the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiting may be more reUable and informative in such cases.
  • the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound.
  • Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified.
  • the amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
  • Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
  • the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the freated sample.
  • Microarrays may be prepared, used, and analyzed using methods known in the art.
  • methods known in the art See, e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT appUcation W095/251116; Shalon, D. et al. (1995) PCT appUcation WO95/35505; Heller, R.A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150- 2155; and Heller, M.J.
  • nucleic acid sequences encoding CYSKP may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping.
  • sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA Ubraries.
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PI constructions or single chromosome cDNA Ubraries.
  • the nucleic acid sequences of the invention may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP).
  • RFLP restriction fragment length polymorphism
  • FISH Fluorescent in situ hybridization
  • Examples of genetic map data can be found in various scientific journals or at the OnUne MendeUan Inheritance in Man (OMIM) World Wide Web site. Correlation between the location of the gene encoding CYSKP on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional cloning efforts.
  • OMIM OnUne MendeUan Inheritance in Man
  • In situ hybridization of chromosomal preparations and physical mapping techniques such as Unkage analysis using estabUshed chromosomal markers, may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammaUan species, such as mouse, may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the gene or genes responsible for a disease or syndrome have been crudely locaUzed by genetic Unkage to a particular genomic region, e.g., ataxia-telangiectasia to 1 lq22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • Unkage analysis using estabUshed chromosomal markers may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammaUan species, such as mouse, may reveal associated markers even if the exact
  • the nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to fr anslocation, inversion, etc. , among normal, carrier, or affected individuals.
  • CYSKP its catalytic or immunogenic fragments, or oUgopeptides thereof can be used for screening Ubraries of compounds in any of a variety of drug screening techniques.
  • the fragment employed in such screening may be free in solution, affixed to a soUd support, borne on a cell surface, or located infracellularly. The formation of binding complexes between CYSKP and the agent being tested may be measured.
  • Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest.
  • This method large numbers of different small test compounds are synthesized on a sohd substrate. The test compounds are reacted with CYSKP, or fragments thereof, and washed. Bound CYSKP is then detected by methods well known in the art. Purified CYSKP can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutraUzing antibodies can be used to capture the peptide and immobiUze it on a soUd support.
  • the nucleotide sequences which encode CYSKP may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not Umited to, such properties as the triplet genetic code and specific basepair interactions.
  • Incyte cDNAs were derived from cDNA Ubraries described in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
  • poly(A)+ RNA was isolated using oUgo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
  • Stratagene was provided with RNA and constructed the corresponding cDNA Ubraries. Otherwise, cDNA was synthesized and cDNA Ubraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6.) Reverse transcription was initiated using oUgo d(T) or random primers. Synthetic oUgonucleotide adapters were Ugated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes.
  • cDNA was size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis.
  • cDNAs were Ugated into compatible restriction enzyme sites of the polyUnker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORTl plasmid (Life Technologies), PCDNA2.1 plasmid (Invifrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), or pINCY (Incyte Genomics, Palo Alto CA), or derivatives thereof.
  • PBLUESCRIPT plasmid (Stratagene)
  • PSPORTl plasmid (Life Technologies)
  • PCDNA2.1 plasmid Invifrogen, Carlsbad CA
  • PBK-CMV plasmid (Stratagene)
  • Recombinant plasmids were transformed into competent E. coU cells including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Life Technologies. II. Isolation of cDNA Clones
  • Plasmids obtained as described in Example I were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E. A.L. PREP 96 plasmid purification kit from QIAGEN.
  • a Magic or WIZARD Minipreps DNA purification system Promega
  • an AGTC Miniprep purification kit Edge Biosystems, Gaithersburg MD
  • Plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophiUzation, at 4°C Alternatively, plasmid DNA was ampUfied from host cell lysates using direct tink PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycUng steps were carried out in a single reaction mixture.
  • Incyte cDNA recovered in plasmids as described in Example II were sequenced as follows. Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (AppUed Biosystems) thermal cycler or the PTC -200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) Uquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or suppUed in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (AppUed Biosystems).
  • Elecfrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (AppUed Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VIII.
  • the polynucleotide sequences derived from Incyte cDNAs were vaUdated by removing vector, tinker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleoti.de nearest neighbor analysis.
  • the Incyte cDNA sequences or franslations thereof were then queried against a selection of pubUc databases such as the GenBank primate, rodent, mammaUan, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and hidden Markov model (HMM)-based protein family databases such as PFAM.
  • PubUc databases such as the GenBank primate, rodent, mammaUan, vertebrate, and eukaryote databases
  • BLOCKS, PRINTS, DOMO, PRODOM, and hidden Markov model (HMM)-based protein family databases such as PFAM.
  • HMM is a probabilistic approach which analyzes consensus primary structures of gene families. See, for example, Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.)
  • the queries were performed using programs based on BLAST, FASTA, BLIMPS, and HMMER.
  • the Incyte cDNA sequences were assembled to produce full length polynucleotide sequences.
  • GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences were used to extend Incyte cDNA assemblages to full length.
  • Polynucleotide and polypeptide sequence aUgnments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence aUgnment program (DNASTAR), which also calculates the percent identity between aUgned sequences.
  • Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and full length sequences and provides appUcable descriptions, references, and threshold parameters.
  • the first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where appUcable, the scores, probabiUty values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probabiUty value, the greater the identity between two sequences).
  • Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (SeeBurge, C and S. KarUn (1997) J. Mol. Biol. 268:78-94, an Burge, C and S. Kariin (1998) Curr. Opia Struct. Biol. 8:346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a mefhionine to a stop codon.
  • Genscan is a FASTA database of polynucleotide and polypeptide sequences.
  • the maximum range of sequence for Genscan to analyze at once was set to 30 kb.
  • the encoded polypeptides were analyzed by querying against PFAM models for cytoskeleton-associated proteins. Potential cytoskeleton-associated proteins were also identified by homology to Incyte cDNA sequences that had been annotated as cytoskeleton- associated proteins. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri pubUc databases.
  • Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct errors in the sequence predicted by Genscan, such as extra or omitted exons.
  • BLAST analysis was also used to find any Incyte cDNA or pubUc cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription. When Incyte cDNA coverage was available, this information was used to correct or confirm the Genscan predicted sequence.
  • Full length polynucleotide sequences were obtained by assembUng Genscan-predicted coding sequences with Incyte cDNA sequences and/or pubUc cDNA sequences using the assembly process described in Example III. Alternatively, full length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences. V. Assembly of Genomic Sequence Data with cDNA Sequence Data "Stitched" Sequences
  • Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example III were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible spUce variants that were subsequently confirmed, edited, or extended to create a full length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by transitivity.
  • Partial DNA sequences were extended to full length with an algorithm based on BLAST analysis.
  • the nearest GenBank protein homolog was then compared by BLAST analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV.
  • a chimeric protein was generated by using the resultant high-scoring segment pairs (HSPs) to map the translated sequences onto the GenBank protein homolog. Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog.
  • HSPs high-scoring segment pairs
  • GenBank protein homolog The GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the pubUc human genome databases. Partial DNA sequences were therefore "stretched” or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene. VI. Chromosomal Mapping of CYSKP Encoding Polynucleotides The sequences which were used to assemble SEQ ID NO:35-68 were compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith- Waterman algorithm.
  • pubUc resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of all sequences of that cluster, including its particular SEQ ID NO:, to that map location.
  • Map locations are represented by ranges, or intervals, of human chromosomes.
  • the map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome' s p- arm.
  • centiMorgan cM
  • centiMorgan is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.
  • the cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters.
  • SEQ ID NO:44 was mapped to chromosome 17 within the interval from 62.90 to 64.20 centiMorgans
  • SEQ ID NO:49 was mapped to chromosome 14 within the interval from 73.70 to 76.40 centiMorgans
  • SEQ ID NO:50 was mapped to chromosome 8 within the interval from 25.80 to 40.30 centiMorgans
  • SEQ ID NO:54 was mapped to chromosome 1 within the interval from 117.6 to 132.4 centiMorgans
  • SEQ ID NO:64 was mapped to chromosome 4 within the interval from 56.7 to 60.5 centiMorgans
  • SEQ ID NO:65 was mapped to chromosome 5 within the interval from 141.40 to 142.60 centiMorgans.
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; Ausubel (1995) supra, ch. 4 and 16.)
  • the product score takes into account both the degree of similarity between two sequences and the length of the sequence match.
  • the product score is a normaUzed value between 0 and 100, and is calculated as follows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences).
  • the BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP (separated by gaps). If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score.
  • the product score represents a balance between fractional overlap and quatity in a BLAST aUgnment. For example, a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap.
  • polynucleotide sequences encoding CYSKP are analyzed with respect to the tissue sources from which they were derived. For example, some full length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example III).
  • Each cDNA sequence is derived from a cDNA Ubrary constructed from a human tissue.
  • Each human tissue is classified into one of the following organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitatia*. female; genitaUa, male; germ cells; hemic and immune system; Uver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified mixed; or urinary tract.
  • the number of Ubraries in each category is counted and divided by the total number of Ubraries across all categories.
  • each human tissue is classified into one of the following disease/condition categories: cancer, cell Une, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of Ubraries in each category is counted and divided by the total number of Ubraries across all categories. The resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding CYSKP. cDNA sequences and cDNA Ubrary/tissue information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA). VIII.
  • the initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72°C Any stretch of nucleotides which would result in hairpin structures and primer-primer dirnerizations was avoided.
  • Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed. High fideUty ampUfication was obtained by PCR using methods well known in the art. PCR was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.).
  • the reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg 2 " , (NH 4 ) 2 S0 4 , and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1 : 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 68 °C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 °C, 5 min; Step 7: storage at 4°C
  • the parameters for primer pair T7 and SK+ were as follows: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68
  • the plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
  • a 5 ⁇ l to 10 ⁇ l atiquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to determine which reactions were successful in extending the sequence.
  • the extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to reUgation into pUC 18 vector (Amersham Pharmacia Biotech).
  • CviJI cholera virus endonuclease Molecular Biology Research, Madison WI
  • sonicated or sheared prior to reUgation into pUC 18 vector
  • the digested nucleotides were separated on low concenfration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega).
  • Extended clones were reUgated using T4 Ugase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), freated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coU cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37 °C in 384-well plates in LB/2x carb tiquid media.
  • the cells were lysed, and DNA was ampUfied by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters: Step 1 : 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 72°C, 2 min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reampUfied using the same conditions as described above.
  • Hybridization probes derived from SEQ ID NO:35-68 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oUgonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. OUgonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oUgomer, 250 ⁇ Ci of [ ⁇ - 32 P] adenosine friphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
  • the labeled oUgonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech). An atiquot containing 10 7 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
  • the DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nyfran Plus, Schleicher & Schuell, Durham NH). Hybridization is carried out for 16 hours at 40 °C. To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visuaUzed using autoradiography or an alternative imaging means and compared. X.
  • Microarrays The Unkage or synthesis of array elements upon a microarray can be achieved utilizing photoUthography, piezoelectric printing (ink-jet printing, See, e.g., Baldeschweiler, supra.), mechanical microspottmg technologies, and derivatives thereof.
  • the substrate in each of the aforementioned technologies should be uniform and soUd with a non-porous surface (Schena (1999), supra).
  • Suggested subsfrates include siUcon, siUca, glass sUdes, glass chips, and silicon wafers.
  • a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
  • a typical array may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropriate number of elements. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)
  • Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oUgomers thereof may comprise the elements of the microarray. Fragments or oUgomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR).
  • the array elements are hybridized with polynucleotides in a biological sample.
  • the polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
  • a fluorescence scanner is used to detect hybridization at each array element.
  • laser desorbtion and mass specfromefry may be used for detection of hybridization.
  • the degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microarray may be assessed.
  • microarray preparation and usage is described in detail below.
  • Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A) + RNA is purified using the oligo-(dT) cellulose method.
  • Each poly(A) + RNA sample is reverse transcribed using MMLV reverse-franscriptase, 0.05 pg/ ⁇ l oUgo-(dT) primer (21mer), IX first strand buffer, 0.03 units/ ⁇ l RNase inhibitor, 500 ⁇ M dATP, 500 ⁇ M dGTP, 500 ⁇ M dTTP, 40 ⁇ M dCTP, 40 ⁇ M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech).
  • the reverse franscription reaction is performed in a 25 ml volume containing 200 ng poly (A) + RNA with GEMB RIGHT kits (Incyte).
  • Specific control poly(A) + RNAs are synthesized by in vifro franscription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.
  • Microarray Preparation Sequences of the present invention are used to generate array elements. Each array element is amplified from bacterial cells containing vectors with cloned cDNA inserts. PCR ampUfication uses primers complementary to the vector sequences flanking the cDNA insert. Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g. Amplified array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech).
  • Purified array elements are immobilized on polymer-coated glass slides.
  • Glass microscope sUdes (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments.
  • Glass slides are etched in 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are cured in a 110°C oven.
  • Array elements are appUed to the coated glass substrate using a procedure described in US Patent No. 5,807,522, incorporated herein by reference.
  • 1 ⁇ l of the array element DNA is loaded into the open capillary printing element by a high-speed robotic apparatus.
  • the apparatus then deposits about 5 nl of array element sample per slide.
  • Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene). Microarrays are washed at room temperature once in 0.2% SDS and three times in distflled water.
  • Non-specific binding sites are blocked by incubation of microarrays in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60° C followed by washes in 0.2% SDS and distilled water as before.
  • PBS phosphate buffered saline
  • Hybridization reactions contain 9 ⁇ l of sample mixture consisting of 0.2 ⁇ g each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
  • the sample mixture is heated to 65° C for 5 minutes and is aliquoted onto the microarray surface and covered with an 1.8 cm 2 coverslip.
  • the arrays are fransferred to a waterproof chamber having a cavity just sUghtiy larger than a microscope sUde.
  • the chamber is kept at 100% humidity internally by the addition of 140 ⁇ l of 5X SSC in a corner of the chamber.
  • the chamber containing the arrays is incubated for about 6.5 hours at 60° C
  • the arrays are washed for 10 min at 45° C in a first wash buffer (IX SSC, 0.1% SDS), three times for 10 minutes each at 45° C in a second wash buffer (0.1X SSC), and dried. Detection
  • Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral Unes at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5.
  • the excitation laser light is focused on the array using a 20X microscope objective (Nikon, Inc., Melville NY).
  • the slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster- scanned past the objective.
  • the 1.8 cm x 1.8 cm array used in the present example is scanned with a resolution of 20 micrometers.
  • a mixed gas multiline laser excites the two fluorophores sequentially. Emitted light is split, based on wavelength, into two photomultipUer tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultipUer tubes are used to filter the signals.
  • the emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5.
  • Each array is typically scanned twice, one scan per fiuorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
  • the sensitivity of the scans is typically calibrated using the signal intensity generated by a cDNA confrol species added to the sample mixture at a known concentration.
  • a specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1 : 100,000.
  • the calibration is done by labeling samples of the caUbrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
  • the output of the photomultiplier tube is digitized using a 12-bit RTT-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-compatible PC computer.
  • the digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
  • the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fiuorophore' s emission spectrum.
  • a grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid.
  • the fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal.
  • the software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
  • XI Complementary Polynucleotides Sequences complementary to the CYSKP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring CYSKP. Although use of oUgonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of CYSKP. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5 ' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the CYSKP-encoding franscript.
  • CYSKP is achieved using bacterial or virus-based expression systems.
  • cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
  • promoters include, but are not Umited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
  • Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
  • Antibiotic resistant bacteria express CYSKP upon induction with isopropyl beta-D- thiogalactopyranoside (IPTG).
  • CYSKP in eukaryotic cells is achieved by infecting insect or mammaUan cell Unes with recombinant Autographiea californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus.
  • AcMNPV Autographiea californica nuclear polyhedrosis virus
  • the nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding CYSKP by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA franscription.
  • Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases.
  • CYSKP 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 immobiUzed glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech). Following purification, the GST moiety can be proteolytically cleaved from CYSKP at specifically engineered sites.
  • FLAG an 8-amino acid peptide
  • 6- His a stretch of six consecutive histidine residues, enables purification on metal-chelate resins
  • CYSKP function is assessed by expressing the sequences encoding CYSKP at physiologically elevated levels in mammaUan cell culture systems.
  • cDNA is subcloned into a mammaUan expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include PCMV SPORT (Life Technologies) and PCR3.1 (Invifrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter.
  • recombinant vector 5-10 g of recombinant vector are transiently transfected into a human cell Une, for example, an endotheUal or hematopoietic cell Une, using either Uposome formulations or electroporation.
  • 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-fransfected.
  • Expression of a marker protein provides a means to distinguish transfected cells from nonfransfected cells and is a reUable predictor of cDNA expression from the recombinant vector.
  • Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein.
  • FCM Flow cytomefry
  • CYSKP The influence of CYSKP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding CYSKP and either CD64 or CD64-GFP.
  • CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobuUn G (IgG).
  • Transfected cells are efficiently separated from nonfransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
  • mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding CYSKP and other genes of interest can be analyzed by northern analysis or microarray techniques.
  • PAGE polyacrylamide gel electrophoresis
  • CYSKP amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oUgopeptide is synthesized and used to raise antibodies by means known to those of skill in the art.
  • LASERGENE software DNASTAR
  • Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophiUc regions are well described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
  • oUgopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (AppUed Biosystems) using FMOC chemistry and coupled to KLH (Sigma-
  • Naturally occurring or recombinant CYSKP is substantially purified by immunoaffinity chromatography using antibodies specific for CYSKP.
  • An immunoaffinity column is constructed by covalently coupUng anti-CYSKP antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupUng, the resin is blocked and washed according to the manufacturer's instructions.
  • Media containing CYSKP are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of CYSKP (e.g. , high ionic strength buffers in the presence of detergent).
  • the column is eluted under conditions that disrupt antibody/CYSKP binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaofrcpe, such as urea or thiocyanate ion), and CYSKP is collected.
  • CYSKP or biologically active fragments thereof, are labeled with 125 I Bolton-Hunter reagent.
  • Bolton-Hunter reagent See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.
  • Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled CYSKP, washed, and any wells with labeled CYSKP complex are assayed. Data obtained using different concentrations of CYSKP are used to calculate values for the number, affinity, and association of CYSKP with the candidate molecules.
  • molecules interacting with CYSKP are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
  • CYSKP may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large Ubraries of genes (Nandabalan, K. et al. (2000) U.S. Patent
  • a microtubule motiUty assay for CYSKP measures motor protein activity.
  • recombinant CYSKP is immobilized onto a glass slide or similar substrate.
  • Taxol-stabilized bovine brain microtubules (commercially available) in a solution containing ATP and cytosolic exfract are perfused onto the sUde. Movement of microtubules as driven by CYSKP motor activity can be visualized and quantified using video-enhanced light microscopy and image analysis techniques.
  • CYSKP activity is directly proportional to the frequency and velocity of microtubule movement.
  • an assay for CYSKP measures the formation of protein filaments in vitro.
  • a solution of CYSKP at a concenfration greater than the "critical concentration" for polymer assembly is applied to carbon-coated grids. Appropriate nucleation sites may be suppUed in the solution.
  • the grids are negative stained with 0.7% (w/v) aqueous uranyl acetate and examined by electron microscopy. The appearance of filaments of approximately 25 nm (microtubules), 8 nm (actin), or 10 nm (intermediate filaments) is a demonstration of protein activity.
  • an assay for CYSKP measures the binding affinity of CYSKP for actin as described by Hammell, R.L.
  • CYSKP and actin are prepared from in vifro recombinant cDNA expression systems and the N- terminus of CYSKP is acetylated using methods well known in the art. Binding of N-terminal acetyl- CYSKP to actin is measured by cosedimentation at 25 °C in a Beckman model TL-100 centrifuge as described. The bound and free CYSKP are determined by quantitative densitomefry of SDS- polyacrylamide gels stained with Coomassie Blue.
  • Apparent binding constants (K ⁇ ,) and Hill coefficients (H) are determined by using methods well known in the art to fit the data to the equation as described by Hammell and Hitchcock-DeGregori (1997, supra).
  • the CYSKP:actin ratio determined using densitomefry, is normaUzed.
  • Hammell and Hitchcock-DeGregori (1997, supra) have shown that saturation of binding corresponds to a CYSKP:actin molar ratio of 0.14, a stoichiomefry of 1 CYSKP:7 actin.
  • the binding of CYSKP to actin is proportional to the CYSKP activity.
  • CYSKP activity is measured as abiUty to bind to microtubules.
  • Microtubules are purified from adult rat brain by reversible assembly (Vallee, R. B. (1982) Methods Enzymol. 134:89-104) or the taxol method (Vallee, R. B. (1982) J. Cell Biol. 92:435-442) using PEM buffer
  • CYSKP 250 mg/ml
  • 80 ml of whole microtubules 450 mg/ml
  • tubutin 300 mg/ml
  • the suspension is centrifuged, the supernatant is removed, and the microtubule pellet is resuspended to the original reaction volume in PEM buffer.
  • CYSKP activity is associated with its abiUty to form protein-protein complexes and is measured by its abiUty to regulate growth characteristics of NIH3T3 mouse fibroblast cells.
  • a cDNA encoding CYSKP is subcloned into an appropriate eukaryotic expression vector. This vector is transfected into NIH3T3 cells using methods known in the art. Transfected cells are compared with non-fransfected cells for the following quantifiable properties: growth in culture to high density, reduced attachment of cells to the substrate, altered cell morphology, and abiUty to induce tumors when injected into immunodeficient mice.
  • the activity of CYSKP is proportional to the extent of increased growth or frequency of altered cell morphology in NIH3T3 cells transfected with CYSKP.
  • ABI FACTURA A program that removes vector sequences and Applied Biosystems, Foster City, CA. masks ambiguous bases in nucleic acid sequences.
  • ABI/PARACEL FDF A Fast Data Finder useful in comparing and Applied Biosystems, Foster City, CA; Mismatch ⁇ 50% annotating amino acid or nucleic acid sequences. Paracel Inc., Pasadena, CA.
  • ABI AutoAssembler A program that assembles nucleic acid sequences. Applied Biosystems, Foster City, CA.
  • fastx score 100 or greater
  • HMM hidden Markov model
  • Phred A base-calling algorithm that examines automated Ewing, B. et al. (1998) Genome Res. sequencer traces with high sensitivity and probability. 8:175-185; Ewing, B. and P. Green (1998) Genome Res. 8:186-194.
  • TMHMMER A program that uses a hidden Markov model (HMM) to Sonnhammer, E.L. et al. (1998) Proc. Sixth Intl. delineate transmembrane segments on protein sequences Conf. on Intelligent Systems for Mol. Biol., and determine orientation. Glasgow et al., eds., The Am. Assoc. for Artificial Intelligence Press, Menlo Park, CA, pp. 175-182.
  • HMM hidden Markov model

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Abstract

L'invention concerne des protéines associées au cytosquelette (CYSKP) humain et des polynucléotides qui identifient et codent pour CYSKP. L'invention porte également sur des vecteurs d'expression, des cellules hôtes, des anticorps, des agonistes et des antagonistes. Elle porte encore sur des méthodes de diagnostic, de traitement ou de prévention de troubles associés à l'expression aberrante de CYSKP.
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